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Background: Tyrosine-rich amelogenin peptide (TRAP) is the main amelogenin digestion product in the developmental enamel matrix. It has been shown to promote remineralization of demineralized enamel in our previous study. However, direct evidence of the effect of TRAP on the morphology and nanostructure of crystal growth on an enamel surface has not been reported. This study aimed to examine the effect of TRAP on the morphology of calcium phosphate crystals grown on early enamel erosion using a pH-cycling model., Methods: Eroded lesions were produced in human premolars by 30-second immersion in 37% phosphoric acid. Forty-five samples of eroded human premolar enamel blocks were selected and randomly divided into 3 groups: deionized water (DDW, negative control); 100 µg/mL TRAP, and 2 ppm sodium fluoride (NaF, positive control group). For 14 days, the specimens were exposed to a pH-cycling model. Using scanning electron microscopy (SEM) and atomic force microscopy (AFM) methods, the surface morphology, calcium-phosphorus ratio, and enamel surface roughness were examined. X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FT-IR) were used to assess crystal characteristics., Results: After pH-cycling, compared to the two control groups, the surface of the eroded enamel of the peptide TRAP group shows a large number of new, densely arranged rod-like crystals, parallel to each other, regularly arranged, forming an ordered structure, with crystal morphology similar to that of natural enamel. The crystals are mostly hydroxyapatite (HA)., Conclusion: This study demonstrates that the peptide TRAP modulates the formation of hydroxyapatite in eroded enamel and that the newly formed crystals resemble natural enamel crystals and promote the remineralization of enamel, providing a promising biomaterial for remineralization treatment of enamel lesions., (© 2024. The Author(s).)

Background: Several methods were introduced for enamel biomimetic remineralization that utilize a biomimetic analogue to interact and absorb bioavailable calcium and phosphate ions and induce crystal nucleation on demineralized enamel. Amelogenin is the most predominant enamel matrix protein that is involved in enamel biomineralization. It plays a major role in developing the enamel's hierarchical microstructure. Therefore, this study was conducted to evaluate the ability of an amelogenin-inspired peptide to promote the remineralization potential of fluoride and a supersaturated calcium phosphate solution in treating artificially induced enamel carious lesions under pH-cycling regimen., Methods: Fifty enamel slices were prepared with a window (4*4 mm 2 ) on the surface. Five samples were set as control healthy enamel and 45 samples were subjected to demineralization for 3 days. Another 5 samples were set as control demineralized enamel and 40 enamel samples were assigned into 8 experimental groups (n=5) (P/I, P/II, P/III, P/AS, NP/I, NP/II, NP/III and NP/AS) according to peptide treatment (peptide P or non-peptide NP) and remineralizing solution used (I; calcium phosphate solution, II; calcium phosphate fluoride solution, III; fluoride solution and AS; artificial saliva). Samples were then subjected to demineralization/remineralization cycles for 9 days. Samples in all experimental groups were evaluated using Raman spectroscopy for mineral content recovery percentage, microhardness and nanoindentation as healthy, demineralized enamel and after pH-cycling. Data were statistically analysed using two-way repeated measures Anova followed by Bonferroni-corrected post hoc test for pairwise multiple comparisons between groups. Statistical significance was set at p= 0.05. Additionally, XRD, FESEM and EDXS were used for crystal orientation, surface morphology and elemental analysis after pH-cycling., Results: Nanocrystals clumped in a directional manner were detected in peptide-treated groups. P/II showed the highest significant mean values in mineral content recovery (63.31%), microhardness (268.81±6.52 VHN), elastic modulus (88.74±2.71 GPa), nanohardness (3.08±0.59 GPa) and the best crystal orientation with I 002 / I300 (1.87±0.08)., Conclusion: Despite pH changes, the tested peptide was capable of remineralizing enamel with ordered crystals. Moreover, the supplementary use of calcium phosphate fluoride solution with peptide granted an enhancement in enamel mechanical properties after remineralization., (© 2024. The Author(s).)

Caries and dental erosion are common oral diseases. Traditional treatments involve the mechanical removal of decay and filling but these methods are not suitable for cases involving large-scale enamel erosion, such as hypoplasia. To develop a noninvasive treatment, promoting remineralisation in the early stage of caries is of considerable clinical significance. Therefore, biomimetic mineralisation is an ideal approach for restoring enamel. Biomimetic mineralisation forms a new mineral layer that is tightly attached to the surface of the enamel. This review details the state-of-art achievements on the application of amelogenin and non-amelogenin, amorphous calcium phosphate, ions flow and other techniques in the biomimetic mineralisation of enamel. The ultimate goal of this review was to shed light on the requirements for enamel biomineralisation. Hence, herein, we summarise two strategies of biological minimisation systems for in situ enamel restoration inspired by amelogenesis that have been developed in recent years and compare their advantages and disadvantages., (© 2021. The Author(s).)

The recovery of impaired periodontium is still a challenge to the treatment of periodontitis. This study was the first to apply the mesoporous hydroxyapatites/chitosan (mHA/CS) composite scaffold to periodontal regeneration. The aim of our study is to evaluate the biological effects of mesoporous hydroxyapatite/chitosan (mHA/CS) loaded with recombinant human amelogenin (rhAm) on periodontal regeneration. The physicochemical properties of mHA/CS scaffolds were examined by Fourier transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM), and Brunauer-Emmett-Teller (BET) analysis. Then, the biological effects of the mHA/CS loaded with rhAm were evaluated, including antibacterial effect, controlled-release capacity, osteogenic and cementogenic effects in vitro and in vivo . The antibacterial effect was tested on 1.5 mg/mL CS; 3 mg/mL mHA; 2.25 mg/mL mHA/CS; 4.5 mg/mL mHA/CS and 20 μg/mL rhAm. Tryptic Soy Broth culture medium was used as a baseline control. Osteogenic effect of rhAm (20 μg/mL rhAm), mHA/CS (4.5 mg/mL mHA/CS), and mHA/CS-rhAm (4.5 mg/mL mHA/CS and 20 μg/mL rhAm) on human periodontal ligament cells (hPDLCs) was evaluated in osteogenic media. The hPDLCs treated either with osteogenic media or Dulbecco's modified Eagle's medium (DMEM) alone were used as the baseline control. In the animal model, 4-week-old nude mice (BALB/c) ( n = 6) implanted with root slices subcutaneously were used to observe the cementogenic effect in vivo . The root slices were treated with rhAm (20 μg/mL rhAm), mHA/CS (4.5 mg/mL mHA/CS), and mHA/CS-rhAm (4.5 mg/mL mHA/CS and 20 μg/mL rhAm). The root slices treated with osteogenic medium alone were used as the baseline control. The analyses showed that the mHA/CS particles were 2 μm in diameter and had a uniform pore size. The mesoporous structure was 7 nm in diameter and its surface area was 33.95 m 2 /g. The scaffold exhibited antibacterial effects against Fusobacterium nucleatum and Porphyromonas gingivalis . The mHA/CS scaffold sustainably released rhAm. The mHA/CS loaded with 20 μg/mL rhAm upregulated ALP activity, the expression levels of osteogenesis-related genes and proteins in vitro . Additionally, it promoted the formation of cementum-like tissue in vivo . Our findings suggest that mHA/CS loaded with 20 μg/mL rhAm could inhibit the growth of periodontal pathogens and promote the formation of bone and cementum-like tissue., (Copyright © 2020 Liao, Li, Shu, Chen, Zhao, Song and Zhou.)

Enamel matrix derivatives (EMDs)-based periodontal tissue regenerative therapy is known to promote healing with minimal inflammatory response after periodontal surgery, i. e., it promotes wound healing with reduced pain and swelling. It has also been reported that macrophages stimulated with amelogenin, a major component of EMD, produce various anti-inflammatory cytokines and growth factors. We previously found that stimulation of monocytes with murine recombinant M180 (rM180) amelogenin suppresses major histocompatibility complex class II (MHC II) gene expression using microarray analysis. However, the detailed molecular mechanisms for this process remain unclear. In the present study, we demonstrated that rM180 amelogenin selectively downmodulates the interferon gamma (IFNγ)-induced cell surface expression of MHC II molecules in macrophages and this mechanism mediated by rM180 appeared to be widely conserved across species. Furthermore, rM180 accumulated in the nucleus of macrophages at 15 min after stimulation and inhibited the protein expression of class II transactivator (CIITA) which controls the transcription of MHC II by IFNγ. In addition, reduced MHC II expression on macrophages pretreated with rM180 impaired the expression of T cell activation markers CD25 and CD69, T cell proliferation ability, and IL-2 production by allogenic CD4 + T lymphocytes in mixed lymphocyte reaction assay. The chromatin immunoprecipitation assay showed that IFNγ stimulation increased the acetylation of histone H3 lysine 27, which is important for conversion to euchromatin, as well as the trimethylation of histone H3 lysine 4 levels in the CIITA promoter IV (p-IV) region, but both were suppressed in the group stimulated with IFNγ after rM180 treatment. In conclusion, the present study shows that amelogenin suppresses MHC II expression by altering chromatin structure and inhibiting CIITA p-IV transcription activity, and attenuates subsequent T cell activation. Clinically observed acceleration of wound healing after periodontal surgery by amelogenin may be partially mediated by the mechanism elucidated in this study. In addition, the use of recombinant amelogenin is safe because it is biologically derived protein. Therefore, amelogenin may also be used in future as an immunosuppressant with minimal side effects for organ transplantation or MHC II-linked autoimmune diseases such as type I diabetes, multiple sclerosis, and rheumatoid arthritis, among others., (Copyright © 2020 Yotsumoto, Sanui, Tanaka, Yamato, Alshargabi, Shinjo, Nakao, Watanabe, Hayashi, Taketomi, Fukuda and Nishimura.)

Background: Recombinant amelogenin protein (RAP) is reported to induce complete root apex formation in dog model when used as apexification therapy. It also induces pulp regeneration in 85% of the treated group. Thus, the aim of this study was to investigate the nature of the remaining regenerated calcified tissues of the RAP group that showed no pulp regeneration compared to the calcium hydroxide treated group (CH)., Methods: A total of 240 dogs' open apex root canals were used, after establishment of canals contamination. Canals were cleaned, irrigated, and filled with RAP as an apexification material and compared with CH. Treated teeth were assessed by H&E, trichrome staining, and/or immunohistochemistry technique, at 1, 3, and 6 months., Results: A time-dependent increase in the calcified tissue barrier was observed in the apex of the RAP-treated group compared to the CH-treated group. The newly formed dentin in this RAP group was mainly tubular dentin and was functionally attached to the bone by periodontal ligament, while the CH group showed dentin-associated mineralized tissue (DAMT) associated with the newly formed apical barrier., Conclusions: Out results suggest that RAP can be used as novel apexification material, resulting in a thickening and strengthening of the canal walls, and achieving apical closure.

Injuries to ligaments are common, painful and debilitating, causing joint instability and impaired protective proprioception sensation around the joint. Healing of torn ligaments usually fails to take place, and surgical replacement or reconstruction is required. Previously, we showed that in vivo application of the recombinant human amelogenin protein (rHAM(+)) resulted in enhanced healing of the tooth-supporting tissues. The aim of this study was to evaluate whether amelogenin might also enhance repair of skeletal ligaments. The rat knee medial collateral ligament (MCL) was chosen to prove the concept. Full thickness tear was created and various concentrations of rHAM(+), dissolved in propylene glycol alginate (PGA) carrier, were applied to the transected MCL. 12 weeks after transection, the mechanical properties, structure and composition of transected ligaments treated with 0.5 μg/μl rHAM(+) were similar to the normal un-transected ligaments, and were much stronger, stiffer and organized than control ligaments, treated with PGA only. Furthermore, the proprioceptive free nerve endings, in the 0.5 μg/μl rHAM(+) treated group, were parallel to the collagen fibres similar to their arrangement in normal ligament, while in the control ligaments the free nerve endings were entrapped in the scar tissue at different directions, not parallel to the axis of the force. Four days after transection, treatment with 0.5 μg/μl rHAM(+) increased the amount of cells expressing mesenchymal stem cell markers at the injured site. In conclusion application of rHAM(+) dose dependently induced mechanical, structural and sensory healing of torn skeletal ligament. Initially the process involved recruitment and proliferation of cells expressing mesenchymal stem cell markers., (© 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Porcine enamel matrix derivative (pEMD), a complex mixture of proteins and peptides including full-length amelogenin protein, splice variants, and proteolytic peptides, is used clinically with a carrier to regenerate supportive tissue around teeth. During application, pEMD self-assembles as nanospheres and precipitates as a three-dimensional matrix to facilitate cell migration and differentiation. Amelogenin, the primary constituent of pEMD, stimulates osteoblast differentiation, but it is unclear what specific roles other components of pEMD play in determining biological response. This study examined the potential of one constituent of pEMD, the N-terminal amelogenin peptide (NTAP), to promote osteoblastic differentiation of human mesenchymal stem cells (MSCs) and to elucidate possible signaling pathways involved. Effects of porcine NTAP on MSC cultures were compared to those of recombinant human amelogenin. While amelogenin induced MSC osteoblastic differentiation, a more robust osteoblastic response was seen after NTAP treatment. A phospho-kinase proteasome array measuring phosphorylation of 35 proteins indicated that protein kinase C (PKC), extracellular signal-regulated kinase 1/2 (ERK1/2), and β-catenin were highly phosphorylated by NTAP. This was confirmed by measuring PKC activity and levels of phospho-ERK1/2 and β-catenin. Both amelogenin and NTAP increased PKC, but NTAP induced higher phosho-ERK1/2 and phospho-β-catenin than amelogenin. ERK1/2 inhibition blocked both amelogenin- and NTAP-induced increases in RUNX2, ALP, OCN, COL1, and BMP2. The results demonstrate that NTAP induces osteogenic differentiation of MSCs via PKC and ERK1/2 activation and β-catenin degradation. NTAP may be an active bone regeneration component of amelogenin, and may play this role in pEMD-stimulated periodontal regeneration.

The formation of new blood vessels has been shown to be fundamental in the repair of many damaged tissues, and we have recently shown that the adult human periodontal ligament contains multipotent stem/progenitor cells that are capable of undergoing vasculogenic and angiogenic differentiation in vitro and ex vivo. Enamel matrix protein (EMP) is a heterogeneous mixture of mainly amelogenin-derived proteins produced during tooth development and has been reported to be sometimes effective in stimulating these processes, including in clinical regeneration of the periodontal ligament. However, the identity of the specific bioactive component of EMP remains unclear. In the present study we show that, while the high-molecular-weight Fraction A of enamel matrix derivative (a heat-treated form of EMP) is unable to stimulate the vasculogenic differentiation of human periodontal ligament cells (HPC) in vitro, the low-molecular-weight Fraction C significantly up-regulates the expression of the endothelial markers VEGFR2, Tie-1, Tie-2, VE-cadherin and vWF and markedly increases the internalization of low-density lipoprotein. Furthermore, we also demonstrate, for the first time, that the synthetic homolog of the 45-amino acid tyrosine-rich amelogenin peptide (TRAP) present in Fraction C is likely to be responsible for its vasculogenesis-inducing activity. Moreover, the chemically synthesized TRAP peptide is also shown here to be capable of up-regulating the angiogenic differentiation of the HPC, based on its marked stimulation of in vitro cell migration and tubule formation and of blood vessel formation assay in a chick embryo chorioallantoic membrane model ex vivo. This novel peptide, and modified derivatives, might thereby represent a new class of regenerative drug that has the ability to elicit new blood vessel formation and promote wound healing in vivo., (Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The aim of this study was to clarify the function of amelogenin, the major protein of enamel matrix derivative, on the proliferation, differentiation, and mineralization of cultured rat bone marrow stem cells (BMSCs), toward the establishment of future bone regenerative therapies. No differences in the morphology of BMSCs or in cell numbers were found between amelogenin addition and additive-free groups. The promotion of ALPase activity and the formation of mineralized nodules were detected at an early stage in amelogenin addition group. In quantitative real-time RT-PCR, mRNA expression of osteopontin, osteonectin, and type I collagen was promoted for 0.5 hours and 24 hours by addition of amelogenin. The mRNA expression of osteocalcin and DMP-1 was also stimulated for 24 hours and 0.5 hours, respectively, in amelogenin addition group. These findings clearly indicate that amelogenin promoted the differentiation and mineralization of rat BMSCs but did not affect cell proliferation or cell morphology.

The formation of organized nanocrystals that resemble enamel is crucial for successful enamel remineralization. Calcium, phosphate and fluoride ions, and amelogenin are important ingredients for the formation of organized hydroxyapatite (HAP) crystals in vitro. However, the effects of these remineralization agents on the enamel crystal morphology have not been thoroughly studied. The objective of this study was to investigate the effects of fluoride ions, supersaturation degree and amelogenin on the crystal morphology and organization of ex vivo remineralized human enamel. Extracted third molars were sliced thin and acid-etched to provide the enamel surface for immersion in different remineralization solutions. The crystal morphology and mineral phase of the remineralized enamel surface were analyzed by field emission-scanning electron microscopy, attenuated total reflection-Fourier transformed infrared and X-ray diffraction. The concentration of fluoride and the supersaturation degree of hydroxyapatite had significant effects on the crystal morphology and crystal organization, which varied from plate-like loose crystals to rod-like densely packed nanocrystal arrays. Densely packed arrays of fluoridated hydroxyapatite nanorods were observed under the following conditions: σ(HAP)=10.2±2.0 with 1.5±0.5 mg l(-1) fluoride and 40±10 μg ml(-1) amelogenin, pH 6.8±0.4. A phase diagram summarizes the conditions that form dense or loose hydroxyapatite nanocrystal structures. This study provides the basis for the development of novel dental materials for caries management., (Copyright © 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Regeneration of mineralized tissues affected by chronic diseases comprises a major scientific and clinical challenge. Periodontitis, one such prevalent disease, involves destruction of the tooth-supporting tissues, alveolar bone, periodontal-ligament and cementum, often leading to tooth loss. In 1997, it became clear that, in addition to their function in enamel formation, the hydrophobic ectodermal enamel matrix proteins (EMPs) play a role in the regeneration of these periodontal tissues. The epithelial EMPs are a heterogeneous mixture of polypeptides encoded by several genes. It was not clear, however, which of these many EMPs induces the regeneration and what mechanisms are involved. Here we show that a single recombinant human amelogenin protein (rHAM(+)), induced in vivo regeneration of all tooth-supporting tissues after creation of experimental periodontitis in a dog model. To further understand the regeneration process, amelogenin expression was detected in normal and regenerating cells of the alveolar bone (osteocytes, osteoblasts and osteoclasts), periodontal ligament, cementum and in bone marrow stromal cells. Amelogenin expression was highest in areas of high bone turnover and activity. Further studies showed that during the first 2 weeks after application, rHAM(+) induced, directly or indirectly, significant recruitment of mesenchymal progenitor cells, which later differentiated to form the regenerated periodontal tissues. The ability of a single protein to bring about regeneration of all periodontal tissues, in the correct spatio-temporal order, through recruitment of mesenchymal progenitor cells, could pave the way for development of new therapeutic devices for treatment of periodontal, bone and ligament diseases based on rHAM(+).

Reconstructing enamel-like structures on teeth have been an important topic of study in the material sciences and dentistry. The important role of amelogenin in modulating the mineralization of organized calcium phosphate crystals has been previously reported. We used amelogenin and utilized a modified biomimetic deposition method to remineralize the surface of etched enamel to form mineral layers containing organized needle-like fluoridated hydroxyapatite crystals. The effect of a recombinant amelogenins (rP172) on the microstructure of the mineral in the coating was analyzed by SEM, XRD and FT-IR. At rP172 concentrations below 33 microg/mL, no significant effect was observed. In the presence of 1 mg/L F and at a concentration of 33 microg/mL rP172, formation of fused crystals growing from the enamel surface was initiated. Amelogenin promoted the oriented bundle formation of needle-like fluoridated hydroxyapatite in a dose dependent manner. Biomimetic synthesis of the amelogenin fluoridated hydroxyapatite nano-composite is one of the primary steps towards the development and design of novel biomaterial for future application in reparative and restorative dentistry.