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.

Amelogenin (Amel) accelerates the nucleation of hydroxyapatite (HAP) in supersaturated solutions of calcium phosphate (Ca-P), shortening the induction time (delay period), under near-physiological conditions of pH, temperature, and ionic strength. Hierarchically organized Amel and amorphous calcium phosphate (ACP) nanorod microstructures are formed involving co-assembly of Amel-ACP particles at low supersaturations and low protein concentrations in a slow, well-controlled, constant composition (CC) crystallization system. At the earliest nucleation stages, the CC method allows the capture of prenucleation clusters and intermediate nanoclusers, spherical nanoparticles, and nanochains prior to enamel-like nanorod microstructure formations at later maturation stages. Amel-ACP nanoscaled building blocks are formed spontaneously by synergistic interactions between flexible Amel protein molecules and Ca-P prenucleation clusters, and these spherical nanoparticles evolve by orientated aggregation to form nanochains. Our results suggest that, in vivo, Amel may determine the structure of enamel by controlling prenucleation cluster aggregation at the earliest stages by forming stable Amel-ACP microstructures prior to subsequent crystal growth and mineral maturation.

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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.

Enamel matrix proteins, including the most abundant amelogenin and lesser amounts of enamelin, ameloblastin, and proteinases, play vital roles in controlling crystal nucleation and growth during enamel formation. The cooperative action between amelogenin and the 32-kDa enamelin is critical to regulating the growth morphology of octacalcium phosphate crystals. Using biophysical methods, we investigated the interaction between the 32-kDa enamelin and recombinant pig amelogenin 148 (rP148) at pH 6.5 in phosphate-buffered saline (PBS). Dynamic light scattering results showed a trend of increasing particle size in the mixture with the addition of enamelin to amelogenin. Upon addition of the 32-kDa enamelin, the shift and intensity decrease in the ellipticity minima of rP148 in the circular dichroism spectra of rP148 illustrated a direct interaction between the 2 proteins. In the fluorescence spectra, the maximum emission of rP148 was blue shifted from 335 to 333 nm in the presence of enamelin as a result of complexation of the 2 proteins. Our results demonstrate that the 32-kDa enamelin has a close association with amelogenin at pH 6.5 in PBS buffer. Our present study provides novel insights into the possible cooperation between enamelin and amelogenin in macromolecular coassembly and in controlling enamel mineral formation

Tooth formation is tightly regulated by epithelial-mesenchymal interactions via hierarchic cascades of signaling molecules. The glycosaminoglycan (GAG) chains covalently attached to the core protein of proteoglycans (PGs) provide docking sites for signaling molecules and their receptors during the morphogenesis of tissues and organs. While PGs are believed to play important roles in tooth formation, little is known about their exact functions in this developmental process and the relevant molecular basis. Family with sequence similarity member 20-B (FAM20B) is a newly identified kinase phosphorylating the xylose in the common linkage region connecting the GAG with the protein core of PGs. The phosphorylation of xylose is essential to the common linkage elongation and the subsequent GAG assembly. In this study, we generated Fam20B-floxed allele in mice and found that inactivating Fam20B in the dental epithelium leads to supernumerary maxillary and mandibular incisors. This finding highlights the pivotal role of PGs in tooth morphogenesis and opens a new window for understanding the regulatory mechanism of PG-mediated signaling cascades during tooth formation.

Amelogenin is cleaved by enamelysin (Mmp-20) soon after its secretion, and the cleavage products accumulate in specific locations during enamel formation, suggesting that parent amelogenin proteolysis is necessary for activating its functions. To investigate the precise roles of Mmp-20 and its influence on the assembly of amelogenin, an in vitro enzymatic digestion process mimicking the initial stages of amelogenin proteolysis was investigated at near-physiological conditions using recombinant porcine amelogenin (rP172) and enamelysin. Hierarchically organized nanorod structures formed during different digestion stages were detected by TEM. At the earliest stage, uniformly dispersed parent amelogenin spherical particles, mixed with some darker stained smaller spheres, and accompanying elongated chain-like nanostructures were observed. Cylindrical nanorods, which appeared to be the result of tight assembly of thin subunit cylindrical discs with thicknesses ranging from ~2.5 nm to ~6.0 nm, were formed after an hour of proteolysis. These subunit building blocks stacked to form nanorods with maximum length of ~100 nm. With the production of more cleavage products, additional morphologies spontaneously evolved from the cylindrical nanorods. Larger ball-like aggregates ultimately formed at the end of proteolysis. The uniform spherical particles, nanorods, morphological patterns evolved from nanorods, and globular aggregated microstructures were successively formed by means of co-assembly of amelogenin and its cleavage products during a comparatively slow proteolysis process. We propose that, following the C-terminal cleavage of amelogenin, co-assembly with its fragments leads to formation of nanorod structures whose properties eventually dictate the super-structural organization of enamel matrix, controlling the elongated growth of enamel apatite crystals.

Recent studies have identified family with sequence similarity member 20C (FAM20C) as a kinase that phosphorylates the Ser in Ser-X-Glu/phospho-Ser (pSer) motifs in the small-integrin-binding ligand N-linked glycoproteins (SIBLINGs). There is no in vivo evidence that validates this finding, and it is unclear whether FAM20C is the only kinase for SIBLINGs. We extracted bone noncollagenous proteins (NCPs) from Fam20C-knockout (KO) mice and analyzed the phosphorylation levels. The total NCPs were separated into osteopontin-, bone sialoprotein-, and dentin matrix protein-1-enriched fractions by anion-exchange chromatography and analyzed by SDS-PAGE, native PAGE, and Western immunoblot analysis. The NCP phosphorylation level in the KO mice was lower than that in the wild-type (WT). On the native gel, the SIBLINGs from KO mice showed a lower migration rate (M r ) than those from the WT. Calf intestine phosphatase treatment shifted SIBLINGs from the WT mice to the level adjacent to the KO, but failed to shift the latter, suggesting a phosphorylation loss of SIBLINGs in the KO mice. Mass spectrometry identified less pSers in the SIBLINGs from the KO mice [including the region of the acidic Ser-and aspartate-rich motif (ASARM) peptides]. In an intriguing finding, several pSers in the Ser-X-Glu motifs in the KO mice maintained their phosphorylation, whereas several others in non-Ser-X-Glu motifs did not. Phospho-Tyrs and phospho-Thrs in the SIBLINGs did not appear to be associated with FAM20C. Our results indicate that FAM20C is the primary, but not the only, kinase for the SIBLINGs.-Yang, X., Yan, W., Tian, Y., Ma, P., Opperman, L. A., Wang, X. Family with sequence similarity member 20C is the primary but not the only kinase for the small-integrin-binding ligand N-linked glycoproteins in bone. FASEB J. 30, 121-128 (2016 The organic phase of the extracellular matrix (ECM) in the bone is mainly composed of type I collagen and noncollagenous proteins (NCPs) synthesized and secreted by osteoblasts. A prominent category of NCPs in bone ECM is termed the small-integrin-binding ligand N-linked glycoprotein (SIBLING) family, which includes osteopontin (OPN), bone sialoprotein (BSP), and dentin matrix protein (DMP)-1. These polyanionic proteins play pivotal roles in osteogenesis and biomineralization, and their biologic functions are closely related to posttranslational modifications, such as phosphorylation (1-7).OPN is commonly recognized as an effective inhibitor of hydroxyapatite formation and growth (8). Rat bone OPN contains 12 phospho-Sers (pSers) and 1 phospho-Thr, and has considerable heterogeneity in degree of phosphorylation (9). In contrast, bovine milk OPN has 27 pSers and 1 phospho-Thr and appears to be maximally phosphorylated (10). The extent of phosphorylation of OPN, as well as of the specific sites, may play an important role in its physiologic function. The activity of OPN in regulating biomineralization is lost after the removal of the phosphates (2, 11). BSP in bone has been reported to have 11 phosp...