Amyloid-like amelogenin nanoribbons template mineralization via a low-energy interface of ion binding sites.

Protein scaffolds direct the organization of amorphous precursors that transform into mineralized tissues, but the templating mechanism remains elusive. Motivated by models for the biomineralization of tooth enamel, wherein amyloid-like amelogenin nanoribbons guide the mineralization of apatite filaments, we investigated the impact of nanoribbon structure, sequence, and chemistry on amorphous calcium phosphate (ACP) nucleation. Using full-length human amelogenin and peptide analogs with an amyloid-like domain, films of ß-sheet nanoribbons were self-assembled on graphite and characterized by in situ atomic force microscopy and molecular dynamics simulations. All sequences substantially reduce nucleation barriers for ACP by creating low-energy interfaces, while phosphoserines along the length of the nanoribbons dramatically enhance kinetic factors associated with ion binding. Furthermore, the distribution of negatively charged residues along the nanoribbons presents a potential match to the Ca­Ca distances of the multi-ion complexes that constitute ACP. These findings show that amyloid-like amelogenin nanoribbons provide potent scaffolds for ACP mineralization by presenting energetically and stereochemically favorable templates of calcium phosphate ion binding and suggest enhanced surface wetting toward calcium phosphates in general.

DENTAL ENAMEL FORMATION AND IMPLICATIONS FOR ORAL HEALTH AND DISEASE.

Dental enamel is the hardest and most mineralized tissue in extinct and extant vertebrate species and provides maximum durability that allows teeth to function as weapons and/or tools as well as for food processing. Enamel development and mineralization is an intricate process tightly regulated by cells of the enamel organ called ameloblasts. These heavily polarized cells form a monolayer around the developing enamel tissue and move as a single forming front in specified directions as they lay down a proteinaceous matrix that serves as a template for crystal growth. Ameloblasts maintain intercellular connections creating a semi-permeable barrier that at one end (basal/proximal) receives nutrients and ions from blood vessels, and at the opposite end (secretory/apical/distal) forms extracellular crystals within specified pH conditions. In this unique environment, ameloblasts orchestrate crystal growth via multiple cellular activities including modulating the transport of minerals and ions, pH regulation, proteolysis, and endocytosis. In many vertebrates, the bulk of the enamel tissue volume is first formed and subsequently mineralized by these same cells as they retransform their morphology and function. Cell death by apoptosis and regression are the fates of many ameloblasts following enamel maturation, and what cells remain of the enamel organ are shed during tooth eruption, or are incorporated into the tooth's epithelial attachment to the oral gingiva. In this review, we examine key aspects of dental enamel formation, from its developmental genesis to the ever-increasing wealth of data on the mechanisms mediating ionic transport, as well as the clinical outcomes resulting from abnormal ameloblast function.

Biomimetic Mineral Synthesis by Nanopatterned Supramolecular-Block Copolymer Templates.

Supramolecular structures of matrix proteins in mineralizing tissues are known to direct the crystallization of inorganic materials. Here we demonstrate how such structures can be synthetically directed into predetermined patterns for which functionality is maintained. The study employs block copolymer lamellar patterns with alternating hydrophilic and hydrophobic regions to direct the assembly of amelogenin-derived peptide nanoribbons that template calcium phosphate nucleation by creating a low-energy interface. Results show that the patterned nanoribbons retain their ß-sheet structure and function and direct the formation of filamentous and plate-shaped calcium phosphate with high fidelity, where the phase, amorphous or crystalline, depends on the choice of mineral precursor and the fidelity depends on peptide sequence. The common ability of supramolecular systems to assemble on surfaces with appropriate chemistry combined with the tendency of many templates to mineralize multiple inorganic materials implies this approach defines a general platform for bottom-up-patterning of hybrid organic-inorganic materials.

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.

SATB1 establishes ameloblast cell polarity and regulates directional amelogenin secretion for enamel formation.

BACKGROUND: Polarity is necessary for epithelial cells to perform distinct functions at their apical and basal surfaces. Oral epithelial cell-derived ameloblasts at secretory stage (SABs) synthesize large amounts of enamel matrix proteins (EMPs), largely amelogenins. EMPs are unidirectionally secreted into the enamel space through their apical cytoplasmic protrusions, or Tomes' processes (TPs), to guide the enamel formation. Little is known about the transcriptional regulation underlying the establishment of cell polarity and unidirectional secretion of SABs. RESULTS: The higher-order chromatin architecture of eukaryotic genome plays important roles in cell- and stage-specific transcriptional programming. A genome organizer, special AT-rich sequence-binding protein 1 (SATB1), was discovered to be significantly upregulated in ameloblasts compared to oral epithelial cells using a whole-transcript microarray analysis. The Satb1-/- mice possessed deformed ameloblasts and a thin layer of hypomineralized and non-prismatic enamel. Remarkably, Satb1-/- ameloblasts at the secretory stage lost many morphological characteristics found at the apical surface of wild-type (wt) SABs, including the loss of Tomes' processes, defective inter-ameloblastic adhesion, and filamentous actin architecture. As expected, the secretory function of Satb1-/- SABs was compromised as amelogenins were largely retained in cells. We found the expression of epidermal growth factor receptor pathway substrate 8 (Eps8), a known regulator for actin filament assembly and small intestinal epithelial cytoplasmic protrusion formation, to be SATB1 dependent. In contrast to wt SABs, EPS8 could not be detected at the apical surface of Satb1-/- SABs. Eps8 expression was greatly reduced in small intestinal epithelial cells in Satb1-/- mice as well, displaying defective intestinal microvilli. CONCLUSIONS: Our data show that SATB1 is essential for establishing secretory ameloblast cell polarity and for EMP secretion. In line with the deformed apical architecture, amelogenin transport to the apical secretory front and secretion into enamel space were impeded in Satb1-/- SABs resulting in a massive cytoplasmic accumulation of amelogenins and a thin layer of hypomineralized enamel. Our studies strongly suggest that SATB1-dependent Eps8 expression plays a critical role in cytoplasmic protrusion formation in both SABs and in small intestines. This study demonstrates the role of SATB1 in the regulation of amelogenesis and the potential application of SATB1 in ameloblast/enamel regeneration.

Remineralization of demineralized dentin using a dual analog system.

OBJECTIVE: Improved methods are needed to remineralize dentin caries in order to promote conservation of dentin tissue and minimize the surgical interventions that are currently required for clinical treatment. Here, we test the hypothesis that bulk substrates can be effectively mineralized via a dual analog system proposed by others, using a tripolyphosphate (TPP) "templating analog" and a poly(acrylic acid) (PAA) or poly(aspartic acid) (pAsp) "sequestration analog," the latter of which generates the polymer-induced liquid-precursor (PILP) mineralization process studied in our laboratory. MATERIAL & METHODS: Demineralized human dentin slices were remineralized with and without pre-treatment with TPP, using either PAA or pAsp as the PILP process-directing agent. A control experiment with no polymer present was used for comparison. RESULTS: No mineralization was observed in any of the PAA groups. In both the pAsp and no polymer groups, TPP inhibited mineralization on the surfaces of the specimens but promoted mineralization within the interiors. Pre-treatment with TPP enhanced overall mineralization of the pAsp group. However, when analysed via TEM, regions with little mineral were still present. CONCLUSION: Poly(acrylic acid) was unable to remineralize demineralized dentin slices under the conditions employed, even when pre-treated with TPP. However, pre-treatment with TPP enhanced overall mineralization of specimens that were PILP-remineralized using pAsp.

Progression of Self-Assembly of Amelogenin Protein Supramolecular Structures in Simulated Enamel Fluid.

Mechanisms of protein-guided mineralization in enamel, leading to organized fibrillar apatite nanocrystals, remain elusive. In vitro studies reveal recombinant human amelogenin (rH174), a matrix protein templating this process, self-assembles into a variety of structures. This study endeavors to clarify the self-assembly of rH174 in physiologically relevant conditions. Self-assembly in simulated enamel fluid was monitored up to 2 months. At alkali (7.3-8.7) and acidic (5.5-6.1) pH ranges, a distinct progression in formation was observed from nanospheres (17-23 nm) to intermediate-length nanorods, concluding with the formation of long 17-18 nm wide nanoribbons decorated with nanospheres. Assembly in acidic condition progressed quicker to nanoribbons with fewer persistent nanospheres. X-ray diffraction exhibited reflections characteristic of antiparallel ß-sheets (4.7 and 9.65 Å), supporting the model of amyloid-like nanoribbon formation. This is the first observation of rH174 nanoribbons at alkaline pH as well as concurrent nanosphere formation, indicating both supramolecular structures are stable together under physiological conditions.

The proteolytic processing of amelogenin by enamel matrix metalloproteinase (MMP-20) is controlled by mineral ions.

BACKGROUND: Enamel synthesis is a highly dynamic process characterized by simultaneity of matrix secretion, assembly and processing during apatite mineralization. MMP-20 is the first protease to hydrolyze amelogenin, resulting in specific cleavage products that self-assemble into nanostructures at specific mineral compositions and pH. In this investigation, enzyme kinetics of MMP-20 proteolysis of recombinant full-length human amelogenin (rH174) under different mineral compositions is elucidated. METHODS: Recombinant amelogenin was cleaved by MMP-20 under various physicochemical conditions and the products were analyzed by SDS-PAGE and MALDI-TOF MS. RESULTS: It was observed that mineral ions largely affect cleavage pattern, and enzyme kinetics of rH174 hydrolysis. Out of the five selected mineral ion compositions, MMP-20 was most efficient at high calcium concentration, whereas it was slowest at high phosphate, and at high calcium and phosphate concentrations. In most of the compositions, N- and C-termini were cleaved rapidly at several places but the central region of amelogenin was protected up to some extent in solutions with high calcium and phosphate contents. CONCLUSION: These in vitro studies showed that the chemistry of the protein solutions can significantly alter the processing of amelogenin by MMP-20, which may have significant effects in vivo matrix assembly and subsequent calcium phosphate mineralization. GENERAL SIGNIFICANCE: This study elaborates the possibilities of the processing of the organic matrix into mineralized tissue during enamel development.

Corrigendum: Calcium interactions in amelogenin-derived peptide assembly.

[This corrects the article DOI: 10.3389/fphys.2022.1063970.].

Poly(Aspartic Acid) Promotes Odontoblast-like Cell Differentiation in Rat Molars with Exposed Pulp.

In recent years, alternative pulpal therapies targeting dentinogenesis signaling pathways using different peptides have been investigated. The aim of this study was to verify the effectiveness of poly(aspartic acid), pAsp, in dentin regeneration using an animal model. METHODS: Mechanical pulp exposure was performed in the upper molars of 56 Wistar rats, randomly divided as follows (n = 14): control (no treatment); MTA group-pulp capping with mineral trioxide aggregate (MTA Angelus); pAsp group-application of 20 µL of pAsp solution (25 mg·mL-1); MTA+pAsp group-application of MTA mixed with pAsp (5:1 by mass). Animals were euthanized after 7 or 21 days. Histological sections were submitted to hematoxylin-eosin and Brown and Brenn staining and immunohistochemical analysis for osteopontin (OPN) and dentin matrix protein 1 (DMP 1). RESULTS: At 7 days, an acute inflammatory infiltrate and the presence of disorganized mineralized tissue were observed in all groups. At 21 days, the quality and thickness of the reparative dentin in treated groups were superior to the control, and bacterial contamination was observed in two MTA-pAsp specimens. While all treated groups showed intense immunostaining for OPN at 21 days, only the pAsp group expressed DMP 1, indicating the presence of fully differentiated odontoblast-like cells. CONCLUSION: Poly(aspartic) acid promoted dentin regeneration in rat molars in the absence of an additional calcium source and may be an alternative to MTA as a pulp-capping agent.

Electromechanical Coupling in Collagen Measured under Increasing Relative Humidity.

The functional role of collagen piezoelectricity has been under debate since the discovery of piezoelectricity in bone in 1957. The possibility that piezoelectricity plays a role in bone remodeling has generated interest in the investigation of this effect in relevant physiological conditions; however, there are conflicting reports as to whether collagen is piezoelectric in a humid environment. In macroscale measurements, the piezoelectricity in hydrated tendon has been shown to be insignificant compared to dehydrated tendon, whereas, at the nanoscale, the piezoelectric effect has been observed in both dry and wet bone using piezoresponse force microscopy (PFM). In this work, the electromechanical properties of type I collagen from a rat tail tendon have been investigated at the nanoscale as a function of humidity using lateral PFM (LPFM) for the first time. The relative humidity (RH) was varied from 10% to 70%, allowing the piezoelectric behavior to be studied dry, humid, as well as in the hydrated range for collagen in physiological bone (12% moisture content, corresponding to 40-50% RH). The results show that collagen piezoresponse can be measured across the humidity range studied, suggesting that piezoelectricity remains a property of collagen at a biologically relevant humidity.

Visualizing molecular polar order in tissues via electromechanical coupling.

Electron microscopy (EM) and atomic force microscopy (AFM) techniques have long been used to characterize collagen fibril ordering and alignment in connective tissues. These techniques, however, are unable to map collagen fibril polarity, i.e., the polar orientation that is directed from the amine to the carboxyl termini. Using a voltage modulated AFM-based technique called piezoresponse force microscopy (PFM), we show it is possible to visualize both the alignment of collagen fibrils within a tissue and the polar orientation of the fibrils with minimal sample preparation. We demonstrate the technique on rat tail tendon and porcine eye tissues in ambient conditions. In each sample, fibrils are arranged into domains whereby neighboring domains exhibit opposite polarizations, which in some cases extend to the individual fibrillar level. Uniform polarity has not been observed in any of the tissues studied. Evidence of anti-parallel ordering of the amine to carboxyl polarity in bundles of fibrils or in individual fibrils is found in all tissues, which has relevance for understanding mechanical and biofunctional properties and the formation of connective tissues. The technique can be applied to any biological material containing piezoelectric biopolymers or polysaccharides.

Self-assembly of filamentous amelogenin requires calcium and phosphate: from dimers via nanoribbons to fibrils.

Enamel matrix self-assembly has long been suggested as the driving force behind aligned nanofibrous hydroxyapatite formation. We tested if amelogenin, the main enamel matrix protein, can self-assemble into ribbon-like structures in physiologic solutions. Ribbons 17 nm wide were observed to grow several micrometers in length, requiring calcium, phosphate, and pH 4.0-6.0. The pH range suggests that the formation of ion bridges through protonated histidine residues is essential to self-assembly, supported by a statistical analysis of 212 phosphate-binding proteins predicting 12 phosphate-binding histidines. Thermophoretic analysis verified the importance of calcium and phosphate in self-assembly. X-ray scattering characterized amelogenin dimers with dimensions fitting the cross-section of the amelogenin ribbon, leading to the hypothesis that antiparallel dimers are the building blocks of the ribbons. Over 5-7 days, ribbons self-organized into bundles composed of aligned ribbons mimicking the structure of enamel crystallites in enamel rods. These observations confirm reports of filamentous organic components in developing enamel and provide a new model for matrix-templated enamel mineralization.

Biophysical characterization of synthetic amelogenin C-terminal peptides.

Amelogenin plays a key role in the formation of the highly mineralized structure of tooth enamel. During the secretory stage, amelogenin is cleaved gradually by a protease, matrix metalloproteinase-20 (MMP-20), releasing hydrophilic C-terminal peptides. In this study, the biophysical properties of synthetic C-terminal peptides (of 28, 17, and 11 residues), mimicking native peptides, were explored in vitro. A sudden decrease was observed in the zeta (ζ)-potential upon the addition of calcium or phosphates, which was also accompanied by an increased aggregation propensity of the peptides. Under most of the experimental conditions, the particle size increased at a pH 2-3 units higher than the isoelectric point (pI) of the peptides, while the peptides existed as smaller particles (<2 nm) near their pI values and in the acidic range. They showed poor affinity for calcium and phosphates, comparable to full-length amelogenin and variants. The secondary structure determination showed that the 11-amino-acid peptide contained defined secondary structure comprising beta-sheets and turns. Atomic force microscopy analysis revealed the presence of thin, disk-like nanostructures of 54.4 nm diameter for the 28-amino-acid peptide and 54.9 nm diameter for the 11-amino acid peptide, whereas no definite structures were observed for the 17-amino-acid peptide. It is concluded that the amelogenin C-terminal peptides are capable of interacting with calcium and phosphate ions, of self-assembly into nanostructures, and may have some secondary structure, and hence may have some role in enamel synthesis.

Calcium interactions in amelogenin-derived peptide assembly.

Phosphorylation of serine residues has been recognized as a pivotal event in the evolution of mineralized tissues in many biological systems. During enamel development, the extracellular matrix protein amelogenin is most abundant and appears to be critical to the extreme high aspect ratios (length:width) of apatite mineral fibers reaching several millimeters in larger mammalian teeth. A 14-residue peptide (14P2, residues Gly8 to Thr21) was previously identified as a key sequence mediating amelogenin assembly formation, the domain also contains the native single phosphoserine residue (Ser16) of the full-length amelogenin. In this research, 14P2 and its phosphorylated form (p14P2) were investigated at pH 6.0 with various calcium and phosphate ion concentrations, indicating that both peptides could self-assemble into amyloid-like conformation but with differences in structural details. With calcium, the distance between 31P within the p14P2 self-assemblies is averaged to be 4.4 ± 0.2Å, determined by solid-state NMR 31P PITHIRDS-CT experiments. Combining with other experimental results, solid-state Nuclear Magnetic Resonance (SSNMR) suggests that the p14P2 self-assemblies are in parallel in-register ß-sheet conformation and divalent calcium ions most likely connect two adjacent peptide chains by binding to the phosphate group of Ser16 and the carboxylate of Glu18 side-chain. This study on the interactions between calcium ions and amelogenin-derived peptides provides insights on how amelogenin may self-assemble in the presence of calcium ions in early enamel development.

Self-aligning amelogenin nanoribbons in oil-water system.

The highly organized microstructure of dental enamel is a result of protein-guided anisotropic growth of apatite nanofibers. It is established that amelogenin proteins, the main constituent of the developing enamel matrix, form nanospheres in vitro, but the amphiphilic nature of the full-length protein conveys the possibility of generating more complex structures as observed with other surfactant-like molecules. This study tested if the use of metastable oil-water emulsions can induce supramolecular assemblies of amelogenin. Recombinant full-length amelogenin, rH174, was mixed into octanol/ethyl acetate preparations of different ratios to form emulsions at pH 4.5 and 7.4. Atomic force and electron microscopy showed the formation of 16.7±1.0nm wide nanoribbons which grew to several micrometer length over a period of days. Nanoribbons formed from reverse micelles by enabling hydrophobic tails of the molecules to interact while preventing the formation of amelogenin nanospheres. Ribbon formation required the presence of calcium and phosphate ions and may be localized at a dark central line along the amelogenin ribbons. The ribbons have a strong tendency to align in parallel maintaining 5-20nm space between each other. The growth rates and number of ribbons were significantly higher at pH 4.5 and related to the metastability of the emulsion. A model for ribbon extension proposes the addition of short segments or amelogenin dimers to the ends of the ribbon. The formation of self-aligning and uniaxially elongating amelogenin structures triggered by the presence of calcium and phosphate may represent a suitable new model for protein controlled mineralization in enamel.

Self-assembly of amelogenin proteins at the water-oil interface.

Self-assembly of amelogenin plays a key role in controlling enamel biomineralization. Recently, we generated self-aligning nanoribbons of amelogenin in water-in-oil emulsions stabilized by the full-length protein (rH174). Here, we tested the hypothesis that the hydrophilic C-terminus is critical for self-assembly of amelogenin into nanoribbons. The self-assembled structures of two amelogenin cleavage products, rH163 and rH146, were compared with structures of rH174 at different pH values and degrees of saturation using atomic force microscopy, electron microscopy, and dynamic light scattering. We observed that the number density of rH174 nanoribbons increased significantly when the initial pH was raised from 4.5 to 5.6. Nanoribbons, as well as unique helical nanostructures, were also readily observed when amelogenin rH146 was used, but showed little tendency for parallel alignment and did not bundle into fibrils like rH174. In contrast, rH163 rarely formed nanoribbons but predominantly assembled into nanospheres under the same conditions. We conclude that the presence of a hydrophilic C-terminus may not be a prerequisite for nanoribbon formation but may be critical for ribbon alignment and subsequent fibril formation. These results highlight the contribution of the hydrophobic domain in the self-assembly of elongated structures of amelogenins. Molecular mechanisms governing these processes based on the formation of reverse micelles are discussed.

Amelogenin as a Promoter of Nucleation and Crystal Growth of Apatite.

Human dental enamel forms over a period of 2 - 4 years by substituting the enamel matrix, a protein gel mostly composed of a single protein, amelogenin with fibrous apatite nanocrystals. Self-assembly of amelogenin and the products of its selective proteolytic digestion are presumed to direct the growth of apatite fibers and their organization into bundles that eventually comprise the mature enamel, the hardest tissue in the mammalian body. This work aimed to establish the physicochemical and biochemical conditions for the growth of apatite crystals under the control of a recombinant amelogenin matrix (rH174) in combination with a programmable titration system. The growth of apatite substrates was initiated in the presence of self-assembling amelogenin particles. A series of constant titration rate experiments was performed that allowed for a gradual increase of the calcium and/or phosphate concentrations in the protein suspensions. We observed a significant amount of apatite crystals formed on the substrates following the titration of rH174 sols that comprised the initial supersaturation ratio equal to zero. The protein layers adsorbed onto the substrate apatite crystals were shown to act as promoters of nucleation and growth of calcium phosphates subsequently grown on the substrate surface. Nucleation lag time experiments have showed that rH174 tends to accelerate precipitation from metastable calcium phosphate solutions in proportion to its concentration. Despite their mainly hydrophobic nature, amelogenin nanospheres, the size and surface charge properties of which were analyzed using dynamic light scattering, acted as a nucleating agent for the crystallization of apatite. The biomimetic experimental setting applied in this study proves as convenient for gaining insight into the fundamental nature of the process of amelogenesis.

Polymer-Induced Liquid Precursor (PILP) remineralization of artificial and natural dentin carious lesions evaluated by nanoindentation and microcomputed tomography.

OBJECTIVES: The study evaluates the efficacy to remineralize artificial and natural dentin lesions through restorative dental procedures that include the Polymer-Induced Liquid Precursor (PILP) method comprising polyaspartic acid (pAsp). METHODS: Novel ionomeric cement compositions based on bioglass 45S5 and pAsp mixtures, as well as conditioning solutions (conditioner) containing 5 mg/mL pAsp, were developed and tested on demineralized dentin blocks (3-4 mm thick) on shallow and deep lesions with the thickness of 140 µm ± 50 and 700 µm ± 50, respectively. In the first treatment group, 20 µL of conditioner was applied to demineralized shallow (n = 3) and deep (n = 3) lesion specimens for 20 s before restoration with glass ionomer cement (RMGIC). For the PILP cement treatment group, cement was applied onto the wet surface of the demineralized specimen for both shallow (n = 3) and deep (n = 3) artificial lesions after the application of the conditioner and before the final restoration. Sample groups were compared to RMGIC restoration, for both shallow and deep lesions (n = 3 each) and treatments in PILP-solution (n = 3 for deep lesions) without restoration for 4 weeks. All of the restored specimens were immersed in simulated body fluid (SBF) solution for 2 weeks and 4 weeks for shallow and deep lesions respectively to allow for remineralization. The artificial lesion specimens were evaluated for changes in the nanomechanical profile (E-modulus and hardness) using nanoindentation. Shallow lesions were analyzed by SEM under vacuum for changes in morphology caused by PILP treatments. Also, a pilot study on human third molars with moderate lesions in dentin (n = 3) was initiated to test the efficacy of treatments in natural lesions based on mineral densities using microcomputed tomography (µCT) at 0, 1, and 3 months. RESULTS: This study showed that functional remineralization of artificial lesions using PILP-releasing restoratives occurred, indicated by an increase of the elastic modulus in shallow lesions and in the middle zone of deep artificial lesions. The mechanical improvement was significant when compared to RMGIC restoration without pAsp (P < 0.05). Nonetheless, recovery across artificial lesions was most significant when specimens were immersed into PILP-solution with restorative (P < 0.01). Furthermore, natural lesions increased in mineral volume content to a higher degree when the restorative treatment included the PILP-method (P < 0.05). However, none of the natural lesions recovered to full mineral degree regardless of the treatments. CLINICAL SIGNIFICANCE/CONCLUSION: These findings indicate the benefit of PILP applications in the functional repair of dentin caries and illustrate the challenge to integrate the PILP-method into a restorative approach in minimally invasive dental procedures.

A N-Terminus Domain Determines Amelogenin's Stability to Guide the Development of Mouse Enamel Matrix.

Amelogenins, the principal proteins in the developing enamel microenvironment, self-assemble into supramolecular structures to govern the remodeling of a proteinaceous organic matrix into longitudinally ordered hydroxyapatite nanocrystal arrays. Extensive in vitro studies using purified native or recombinant proteins have revealed the potential of N-terminal amelogenin on protein self-assembly and its ability to guide the mineral deposition. We have previously identified a 14-aa domain (P2) of N-terminal amelogenin that can self-assemble into amyloid-like fibrils in vitro. Here, we investigated how this domain affects the ability of amelogenin self-assembling and stability of enamel matrix protein scaffolding in an in vivo animal model. Mice harboring mutant amelogenin lacking P2 domain had a hypoplastic, hypomineralized, and aprismatic enamel. In vitro, the mutant recombinant amelogenin without P2 had a reduced tendency to self-assemble and was prone to accelerated hydrolysis by MMP20, the prevailing metalloproteinase in early developing enamel matrix. A reduced amount of amelogenins and a lack of elongated fibrous assemblies in the development enamel matrix of mutant mice were evident compared with that in the wild-type mouse enamel matrix. Our study is the first to demonstrate that a subdomain (P2) at the N-terminus of amelogenin controls amelogenin's assembly into a transient protein scaffold that resists rapid proteolysis during enamel development in an animal model. Understanding the building blocks of fibrous scaffold that guides the longitudinal growth of hydroxyapatites in enamel matrix sheds light on protein-mediated enamel bioengineering. © 2021 American Society for Bone and Mineral Research (ASBMR).