Amelogenin-Derived Peptides in Bone Regeneration: A Systematic Review

Amelogenins are enamel matrix proteins currently used to treat bone defects in periodontal surgery. Recent studies have highlighted the relevance of amelogenin-derived peptides, named LRAP, TRAP, SP, and C11, in bone tissue engineering. Interestingly, these peptides seem to maintain or even improve the biological activity of the full-length protein, which has received attention in the field of bone regeneration. In this article, the authors combined a systematic and a narrative review. The former is focused on the existing scientific evidence on LRAP, TRAP, SP, and C11’s ability to induce the production of mineralized extracellular matrix, while the latter is concentrated on the structure and function of amelogenin and amelogenin-derived peptides. Overall, the collected data suggest that LRAP and SP are able to induce stromal stem cell differentiation towards osteoblastic phenotypes; specifically, SP seems to be more reliable in bone regenerative approaches due to its osteoinduction and the absence of immunogenicity. However, even if some evidence is convincing, the limited number of studies and the scarcity of in vivo studies force us to wait for further investigations before drawing a solid final statement on the real potential of amelogenin-derived peptides in bone tissue engineering.

Co-Immunoprecipitation Reveals Interactions Between Amelogenin and Ameloblastin via Their Self-Assembly Domains

Macromolecular assembly of extracellular enamel matrix proteins (EMPs) is intimately associated with the nucleation, growth, and maturation of highly organized hydroxyapatite crystals giving rise to healthy dental enamel. Although the colocalization of two of the most abundant EMPs amelogenin (Amel) and ameloblastin (Ambn) in molar enamel has been established, the evidence toward their interaction is scarce. We used co-immunoprecipitation (co-IP) to show evidence of direct molecular interactions between recombinant and native Amel and Ambn. Ambn fragments containing Y/F-x-x-Y/L/F-x-Y/F self-assembly motif were isolated from the co-IP column and characterized by mass spectroscopy. We used recombinant Ambn (rAmbn) mutants with deletion of exons 5 and 6 as well as Ambn derived synthetic peptides to demonstrate that Ambn binds to Amel via its previously identified Y/F-x-x-Y/L/F-x-Y/F self-assembly motif at the N-terminus of its exon 5 encoded region. Using an N-terminal specific anti-Ambn antibody, we showed that Ambn N-terminal fragments colocalized with Amel from secretory to maturation stages of enamel formation in a single section of developing mouse incisor, and closely followed mineral patterns in enamel rod interrod architecture. We conclude that Ambn self-assembly motif is involved in its interaction with Amel in solution and that colocalization between the two proteins persists from secretory to maturation stages of amelogenesis. Our in vitro and in situ data support the notion that Amel and Ambn may form heteromolecular assemblies that may perform important physiological roles during enamel formation.

Epithelial loss of mitochondrial oxidative phosphorylation leads to disturbed enamel and impaired dentin matrix formation in postnatal developed mouse incisor

The formation of dentin and enamel matrix depends on reciprocal interactions between epithelial-mesenchymal cells. To assess the role of mitochondrial function in amelogenesis and dentinogenesis, we studied postnatal incisor development in K320E-TwinkleEpi mice. In these mice, a loss of mitochondrial DNA (mtDNA), followed by a severe defect in the oxidative phosphorylation system is induced specifically in Keratin 14 (K14+) expressing epithelial cells. Histochemical staining showed severe reduction of cytochrome c oxidase activity only in K14+ epithelial cells. In mutant incisors, H&E staining showed severe defects in the ameloblasts, in the epithelial cells of the stratum intermedium and the papillary cell layer, but also a disturbed odontoblast layer. The lack of amelogenin in the enamel matrix of K320E-TwinkleEpi mice indicated that defective ameloblasts are not able to form extracellular enamel matrix proteins. In comparison to control incisors, von Kossa staining showed enamel biomineralization defects and dentin matrix impairment. In mutant incisor, TUNEL staining and ultrastructural analyses revealed differentiation defects, while in hair follicle cells apoptosis is prevalent. We concluded that mitochondrial oxidative phosphorylation in epithelial cells of the developed incisor is required for Ca2+ homeostasis to regulate the formation of enamel matrix and induce the differentiation of ectomesenchymal cells into odontoblasts.

Ablation of Fam20c causes amelogenesis imperfecta via inhibiting Smad dependent BMP signaling pathway

Background Amelogenesis imperfecta (AI) is a type of hereditary diseases that manifest defects in the formation or mineralization of enamel. Recently, it is reported that inactivation of FAM20C, a well-known Golgi casein kinase, caused AI. However, the mechanism of it is still unknown. The aim of this study was to explore the molecular mechanism of AI, which caused by ablation of FAM20C. Results In the Sox2-Cre;Fam20Cfl/fl (cKO) mouse, we found abnormal differentiation of ameloblasts, improper formation and mineralization of enamel, and downregulation of both mRNA and protein level of enamel matrix proteins, including amelogenin (AMEL), ameloblastin (AMBN) and enamelin (ENAM). The levels of BMP2, BMP4 and BMP7, the ligands of BMP signaling pathway, and phosphorylation of Smad1/5/8, the key regulators of BMP signaling pathway, were all decreased in the enamel matrix and the ameloblast of the cKO mice, respectively. The expression of cyclin-dependent kinase inhibitor (P21), muscle segment homeobox genes 2 (Msx2), which are the target genes of the BMP signaling pathway, and laminin 3, the downstream factor of Msx2, were all significantly decreased in the ameloblasts of the cKO mice compared to the control mice. Conclusion the results of our study suggest that ablation of FAM20C leads to AI through inhibiting the Smad dependent BMP signaling pathway in the process of amelogenesis.

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.