Amelogenin-inspired peptide, calcium phosphate solution, fluoride and their synergistic effect on enamel biomimetic remineralization: an in vitro pH-cycling model.

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 mm2 ) 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 I002/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.

Triple Function of Amelogenin Peptide-Chitosan Hydrogel for Dentin Repair.

Biomimetic strategies like peptide-guided collagen mineralization promise to enhance the effectiveness of dentin remineralization. We recently reported that rationally designed amelogenin-derived peptides P26 and P32 promoted apatite nucleation, mineralized collagen, and showed potential in enamel regrowth and dentin remineralization. To facilitate the clinical application of amelogenin-derived peptides and to uncover their effectiveness in repairing dentin, we have now implemented a chitosan (CS) hydrogel for peptide delivery and have investigated the effects of P26-CS and P32-CS hydrogels on dentin remineralization using 2 in situ experimental models that exhibited different levels of demineralization. The efficacy of the peptide-CS hydrogels in dentin repair was evaluated by characterizing the microstructure, mineral density, mineral phase, and nanomechanical properties of the remineralized samples. The new strategy of atomic force microscopy PeakForce quantitative nanomechanical mapping was used for direct visualization and nanomechanical analysis of repaired dentin lesions across the lesion depth. Results from the 2 models indicated the potential triple functions of peptide-CS hydrogels for dentin repair: building a highly organized protective mineralized layer on dentin, occluding dentinal tubules by peptide-guided in situ mineralization, and promoting biomimetic dentinal collagen remineralization. Importantly, peptides released from the CS hydrogel could diffuse into the dentinal matrix and penetrate the dentinal tubules, leading to both surface and subsurface remineralization and tubule occlusion. Given our previous findings on peptide-CS hydrogels' potential for remineralizing enamel, we see further promise for hydrogels to treat tooth defects involving multiple hard tissues, as in the case of noncarious cervical lesions.

Unveiling the mechanism of an amelogenin-derived peptide in promoting enamel biomimetic remineralization.

Amelogenin and its derived peptides have exhibited excellent efficacy in promoting enamel biomimetic remineralization. However, little is known about their specific action mechanisms. Herein, by combining experiments and computer simulation, the mechanism of an amelogenin-derived peptide QP5 in regulating enamel biomimetic remineralization is unveiled for the first time. In experiments, peptide QP5 was separated into (QPX)5 and C-tail domains, the interactions of peptide-minerals in nucleation solution and the regulation of peptide on enamel biomimetic remineralization were explored. QP5 exhibited an unordered conformation when mineral ions existed, and it could adsorb on minerals through its two domains, thereby inhibiting spontaneous nucleation. The remineralized enamel regulated by C-tail showed better mechanical properties and formed more biomimetic crystals than that of (QPX)5, indicating the C-tail domain of QP5 played an important role in forming enamel-like crystals. The simulation results showed that the conformation of QP5 changed greatly, mainly exhibiting ß-bend, ß-turn, and coil structures, and it eventually adsorbed on enamel through negatively charged residues of the C-tail domain, then captured Ca2+ from solution to promote enamel remineralization. This study improved the evaluation methods of the mechanism of biomimetic peptides, and laid a theoretical basis for the amelioration and clinical transformation of peptide QP5.

Evaluation of the biological effects of amelogenin on human oral keratinocytes.

OBJECTIVES: Amelogenins are clinically used in periodontal regeneration as main components of root surface modifying agents, even without specifically preventing the premature colonization of the healing tissue defect by means of a physical barrier membrane. The objective of this study was to investigate the effects of human amelogenin on the proliferation, migration, and morphology of Immortalized Human Oral Keratinocytes (iHOKs). METHODS: Immortalized Human Oral Keratinocytes were expanded in Keratinocyte Growth Medium-2 (KGM-2). Full-length recombinant amelogenin protein was diluted in KGM-2 in five concentrations (10 ng/ml, 100 ng/ml, 1.000 ng/ml, 5.000 ng/ml and 10.000 ng/ml). iHOKs were cultured in medium supplemented with the amelogenin dilutions. Samples without amelogenin served as control. Cell metabolism and cell proliferation together with cell migration were evaluated at day 7, 14, 21. RESULTS: At day 7, iHOKs treated with 10,000 ng/ml showed a significant decrease in keratinocytes´ proliferation. The metabolic activity at this timepoint was significantly lower for concentrations ≥ 1000 ng/ml. At days 14 and 21, both the addition of 5000 ng/ml and even more 10,000 ng/ml amelogenin reduced significantly the proliferation of keratinocytes. The effects on the metabolic activity for these timepoints were visible already with 100 ng/ml. Treatment of iHOKs with amelogenin of ≥ 1000 ng/ml led to inhibitory effects on cell migration already after 24 h. CONCLUSIONS: The full-length recombinant amelogenin has a significant biological impact on iHOKs. The increasing dose dependent inhibitory effects of amelogenin shown on iHOKs might explain the disruption of the apical migration of the junctional epithelium during regenerative healing. CLINICAL SIGNIFICANCE: Amelogenin, presents time- and dose-dependent inhibitory effects on the growth of keratinocytes, which might explain the biological rationale behind its application in periodontal regeneration.

Histological evaluation following treatment of recession-type defects with coronally advanced flap and a novel human recombinant amelogenin.

OBJECTIVES: To histologically evaluate the effects of a novel human recombinant amelogenin (rAmelX) on periodontal wound healing / regeneration in recession-type defects. MATERIALS AND METHODS: A total of 17 gingival recession-type defects were surgically created in the maxilla of three minipigs. The defects were randomly treated with a coronally advanced flap (CAF) and either rAmelX (test), or a CAF and placebo (control). At three months following reconstructive surgery, the animals were euthanized, and the healing outcomes histologically evaluated. RESULTS: The test group yielded statistically significantly (p = 0.047) greater formation of cementum with inserting collagen fibers compared with the control group (i.e., 4.38 mm ± 0.36 mm vs. 3.48 mm ± 1.13 mm). Bone formation measured 2.15 mm ± 0.8 mm in the test group and 2.24 mm ± 1.23 mm in the control group, respectively, without a statistically significant difference (p = 0.94). CONCLUSIONS: The present data have provided for the first-time evidence for the potential of rAmelX to promote regeneration of periodontal ligament and root cementum in recession-type defects, thus warranting further preclinical and clinical testing. CLINICAL RELEVANCE: The present results set the basis for the potential clinical application of rAmelX in reconstructive periodontal surgery.

Healing of intrabony defects using a novel human recombinant amelogenin: a preclinical study.

OBJECTIVE: To histologically evaluate the effects of a novel human recombinant amelogenin (rAmelX) on periodontal wound healing/regeneration in intrabony defects. METHOD AND MATERIALS: Intrabony defects were surgically created in the mandible of three minipigs. Twelve defects were randomly treated with either rAmelX and carrier (test group) or with the carrier only (control group). At 3 months following reconstructive surgery, the animals were euthanized, and the tissues histologically processed. Thereafter, descriptive histology, histometry, and statistical analyses were performed. RESULTS: Postoperative clinical healing was uneventful. At the defect level, no adverse reactions (eg, suppuration, abscess formation, unusual inflammatory reaction) were observed with a good biocompatibility of the tested products. The test group yielded higher values for new cementum formation (4.81 ± 1.17 mm) compared to the control group (4.39 ± 1.71 mm) without reaching statistical significance (P = .937). Moreover, regrowth of new bone was greater in the test compared to the control group (3.51 mm and 2.97 mm, respectively, P = .309). CONCLUSIONS: The present results provided for the first-time histologic evidence for periodontal regeneration following the use of rAmelX in intrabony defects, thus pointing to the potential of this novel recombinant amelogenin as a possible alternative to regenerative materials from animal origins.

The power of weak ion-exchange resins assisted by amelogenin for natural remineralization of dental enamel: an in vitro study.

This study aims to develop an innovative dental product to remineralize dental enamel by a proper combination of ion-exchange resins as controlled release of mineral ions that form dental enamel, in the presence of amelogenin to guide the appropriate crystal growth. The novel product proposed consists of a combination of ion-exchange resins (weak acid and weak base) individually loaded with the remineralizing ions: Ca2+, PO43- and F-, also including Zn2+ in a minor amount as antibacterial, together with the protein amelogenin. Such cocktail provides onsite controlled release of the ions necessary for enamel remineralization due to the weak character of the resins and at the same time, a guiding tool for related crystal growth by the indicated protein. Amelogenin protein is involved in the structural development of natural enamel and takes a key role in controlling the crystal growth morphology and alignment at the enamel surface. Bovine teeth were treated by applying the resins and protein together with artificial saliva. Treated teeth were evaluated with nanoindentation, scanning electron microscopy and energy-dispersive X-ray spectroscopy. The innovative material induces the dental remineralization creating a fluorapatite layer with a hardness equivalent to sound enamel, with the appropriate alignment of corresponding nanocrystals, being the fluorapatite more acid resistant than the original mineral. Our results suggest that the new product shows potential for promoting long-term remineralization leading to the inhibition of caries and protection of dental structures.

Biomimetic mineralisation systems for in situ enamel restoration inspired by amelogenesis.

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.

Combined application of geranylgeranylacetone and amelogenin promotes angiogenesis and wound healing in human periodontal ligament cells.

Amelogenin directly binds to glucose-regulated protein 78 (Grp78). Cell migration activity is expected to increase when human periodontal ligament cells (hPDLCs) overexpressing Grp78 are treated with amelogenin. Geranylgeranylacetone (GGA) is a drug that induces the expression of heat shock protein and is routinely used to treat gastric ulcers. Here, we investigated the changes in the properties and behavior of hPDLCs in response to treatment with GGA and the synergistic effects of amelogenin stimulation in hPDLCs pretreated with GGA for the establishment of a novel periodontal tissue regenerative therapy. We observed that GGA treatment increased Grp78 protein expression in hPDLCs and enhanced cell migration. Microarray analysis demonstrated that increased Grp78 expression triggered the production of angiopoietin-like 4 and amphiregulin, which are involved in the enhancement of angiogenesis and subsequent wound healing via the activation of hypoxia-inducible factor 1α and peroxisome proliferator-activated receptors as well as the phosphorylation of cAMP response element-binding protein and protein kinase A. Moreover, the addition of recombinant murine amelogenin (rM180) further accelerated hPDLC migration and tube formation of human umbilical vein endothelial cells due to the upregulation of interleukin-8 (IL-8), monocyte chemotactic protein 1, and IL-6, which are also known as angiogenesis-inducing factors. These findings suggest that the application of GGA to gingival tissue and alveolar bone damaged by periodontal disease would facilitate the wound healing process by inducing periodontal ligament cells to migrate to the root surface and release cytokines involved in tissue repair. Additionally, supplementation with amelogenin synergistically enhanced the migratory capacity of these cells while actively promoting angiogenesis. Therefore, the combined application of GGA and amelogenin may establish a suitable environment for periodontal wound healing and further drive the development of novel therapeutics for periodontal tissue regeneration.

Regeneration of grade 3 ankle sprain, using the recombinant human amelogenin protein (rHAM+ ) in a rat model.

Lateral ligament tears, also known as high-grade ankle sprains, are common, debilitating, and usually heal slowly. Ten to thirty percent of patients continue to suffer from chronic pain and ankle instability even after 3 to 9 months. Previously, we showed that the recombinant human amelogenin (rHAM+ ) induced regeneration of fully transected rat medial collateral ligament, a common proof-of-concept model. Our aim was to evaluate whether rHAM+ can regenerate torn ankle calcaneofibular ligament (CFL), an important component of the lateral ankle stabilizers. Right CFLs of Sabra rats were transected and treated with 0, 0.5, or 1 µg/µL rHAM+ dissolved in propylene glycol alginate (PGA). Results were compared with the normal group, without surgery. Healing was evaluated 12 weeks after treatment by mechanical testing (ratio between the right and left, untransected ligaments of the same rat), and histology including immunohistochemical staining of collagen I and S100. The mechanical properties, structure, and composition of transected ligaments treated with 0.5 µg/µL rHAM+ (experimental) were similar to untransected ligaments. PGA (control) treated ligaments were much weaker, lax, and unorganized compared with untransected ligaments. Treatment with 1 µg/µL rHAM+ was not as efficient as 0.5 µg/µL rHAM+ . Normal arrangement of collagen I fibers and of proprioceptive nerve endings, parallel to the direction of the force, was detected in ligaments treated with 0.5 µg/µL rHAM+ , and scattered arrangement, resembling scar tissue, in control ligaments. In conclusion, we showed that rHAM+ induced significant mechanical and structural regeneration of torn rat CFLs, which might be translated into treatment for grades 2 and 3 ankle sprain injuries.

Unraveling the mechanism for an amelogenin-derived peptide regulated hydroxyapatite mineralization via specific functional domain identification.

Amelogenin and its various derived peptides play important roles in promoting biomimetic mineralization of enamel. Previously, an amelogenin-derived peptide named QP5 was proved to be able to repair demineralized enamel. The objective here was to interpret the mechanism of QP5 by elucidating the specific function of each domain for further sequence and efficacy improvement. Peptide QP5 was separated into domains (QPX)5 and C-tail. (QPX)3 was also synthesized to investigate how QPX repeats affect the mineralization process. Circular dichroism spectroscopy showed that two (QPX) repeats adopted a ß-sheet structure, while C-tail exhibited a disordered structure. (QPX)5 showed more absorption in confocal laser scanning microscopy observation and a higher K value in Langmuir adsorption isotherms compared to C-tail, while (QPX)3 with better hydropathy had greater adsorption capability than (QPX)5. Meanwhile, calcium consumption kinetics, transmission electron microscopy and selected area electron diffraction indicated that (QPX)5, C-tail and (QPX)3 had similar inhibitory effects on the spontaneous calcium consumption and the morphology of their nucleation products were alike, while QP5 had a greater inhibitory effect than them and induced elongated plate-like crystals. X-Ray diffraction further showed that both C-tail and (QPX)3 had greater potential in improving the apatite crystal orientation degree. In conclusion, (QPX)5 was the major adsorption region, both (QPX)5 and C-tail inhibited the nucleation, and C-tail contributed more to improve the HAP orientation degree, so QP5 could exert a significant remineralization effect. By reducing two repeats, (QPX)3 showed higher hydropathicity than (QPX)5 and achieved higher binding affinity, and it was more potential in improving the HAP orientation degree with lower economic cost.

Amelogenin Downregulates Interferon Gamma-Induced Major Histocompatibility Complex Class II Expression Through Suppression of Euchromatin Formation in the Class II Transactivator Promoter IV Region in Macrophages.

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.

Mesoporous Hydroxyapatite/Chitosan Loaded With Recombinant-Human Amelogenin Could Enhance Antibacterial Effect and Promote Periodontal Regeneration.

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 m2/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.

Effects of full-length human amelogenin on the differentiation of dental epithelial cells and osteoblastic cells.

BACKGROUND AND OBJECTIVE: Amelogenins are major components of extracellular matrix proteins in developing teeth, and regulate the growth of enamel crystals. They also function as signaling molecules in cell differentiation. This study aimed to determine the biological effects of amelogenins on the differentiation of HAT-7 dental epithelial cells and MC3T3-E1 pre-osteoblastic cells using full-length recombinant human amelogenin (rh-AMEL). DESIGN: rh-AMEL was expressed in a mammalian cell line (Expi293F™) and was purified by DDK agarose beads. Effects of rh-AMEL on differentiation were evaluated by Mineralization and Alkaline phosphatase (ALP) activity using Alizarin Red S staining and colorimetric substrate p-nitrophenol, respectively. RESULTS: Western blotting and silver staining confirmed the successful purification of rh-AMEL. Mineralization and ALP activity in HAT-7 cells were significantly higher after treatment with 4 µg/mL rh-AMEL, but not after treatment with Emdogain® (EMD). In MC3T3-E1 cells, on the other hand, rh-AMEL showed biphasic effects on differentiation. Treatment with low concentrations of rh-AMEL (0.001-0.1 µg/mL) and EMD (0.01-1 µg/mL) increased mineralization and ALP activity in MC3T3-E1 cells, whereas treatment with high concentrations of rh-AMEL (4 µg/mL) and EMD (100 µg/mL) had the opposite effect. CONCLUSION: High concentrations of rh-AMEL and EMD decreased the differentiation of MC3T3-E1 cells. By contrast, a high concentration of rh-AMEL, but not that of EMD, promoted the differentiation of HAT-7 cells. This study demonstrates that the effects of rh-AMEL on cell differentiation differ between HAT-7 and MC3T3-E1 cells, and suggests that different regions on AMEL may induce the differentiation of these cell types.

Chitosan hydrogel containing amelogenin-derived peptide: Inhibition of cariogenic bacteria and promotion of remineralization of initial caries lesions.

OBJECTIVE: Nowadays, caries prevention focuses on controlling pathogenic bacteria, inhibiting demineralization and promoting re-mineralization. The aim of this study is to design a more clinically powerful anti-caries treatment by combining amelogenin-derived peptide QP5 with antibacterial chitosan in a hydrogel (CS-QP5 hydrogel), and characterize its effects on inhibition of cariogenic bacteria and promotion of remineralization of initial caries lesions. DESIGN: CS-QP5 interactions at different pH and chitosan concentrations were studied using UV-vis spectroscopy, fluorescence spectroscopy and circular dichroism. Antibacterial activity was measured using broth microdilution and biofilm assays. Remineralizing activity was measured using tests of surface micro-hardness(SMH), polarized light microscopy(PLM) and transverse microradiography(TMR) in a pH cycling model that simulates intra-oral pH conditions. RESULTS: The results of UV-vis spectroscopy, fluorescence spectroscopy and circular dichroism analyses suggest that the micro-environment of QP5 changes upon addition of chitosan and the interaction between QP5 and chitosan is reversible and dependent on pH. CS-QP5 hydrogel showed good antibacterial potency towards Streptococcus mutans with MIC/MBC of 5 mg/mL, reducing adhesion and biofilm formation up to 95.43% and nearly 100% respectively. According to the results of remineralizing studies, CS-QP5 hydrogel demonstrated 50.06% surface micro-hardness recovery, shallower lesion depth, significantly less mineral loss and more mineral content at different depth in the lesion body after pH cycling. CONCLUSIONS: The hydrogel showed promise as a dual-action caries control agent in vitro, whether it could present good effects in vivo still needs to be determined, which requires further study. Nonetheless, the new design of bioactive hydrogel with antibacterial and remineralizing properties has the potential to substantially benefit oral health.

Characterization of the apical bridge barrier formed following amelogenin apexification.

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.

Amelogenin induces M2 macrophage polarisation via PGE2/cAMP signalling pathway.

OBJECTIVES: Amelogenin, the major component of the enamel matrix derivative (EMD), has been suggested as a bioactive candidate for periodontal regeneration. Apart from producing a regenerative effect on periodontal tissues, amelogenin has also been reported to have an anti-inflammatory effect. However, the precise molecular mechanisms underlying these effects remain unclear. In the present study, we examined the immunomodulatory effects of amelogenin on macrophages. DESIGN: Human phorbol 12-myristate 13-acetate (PMA)-differentiated U937 macrophages and CD14+ peripheral blood-derived monocytes (PBMC)-derived macrophages were stimulated with recombinant amelogenin (rM180). After performing a detailed microarray analysis, the effects of rM180 on macrophage phenotype and signal transduction pathways were evaluated by real-time polymerase chain reaction, enzyme-linked immunosorbent assay, confocal microscopy and flow cytometry. RESULTS: The microarray analysis demonstrated that rM180 increased the expression of anti-inflammatory genes in lipopolysaccharide (LPS)-challenged macrophages after 24h, while it temporarily up-regulated inflammatory responses at 4h. rM180 significantly enhanced the expression of M2 macrophage markers (CD163 and CD206). rM180-induced M2 macrophage polarisation was associated with morphological changes as well as vascular endothelial growth factor (VEGF) production. rM180 enhanced prostaglandin E2 (PGE2) expression, and the activation of the cAMP/cAMP-responsive element binding (CREB) signaling pathway was involved in amelogenin-induced M2 macrophage polarisation. Blocking of PGE2 signaling by indomethacin specifically abrogated rM180 with or without LPS-induced M2 shift in PBMC-derived macrophages. CONCLUSION: Amelogenin could reprogram macrophages into the anti-inflammatory M2 phenotype. It could therefore contribute to the early resolution of inflammation in periodontal lesions and provide a suitable environment for remodeling-periodontal tissues.

Human gingival fibroblast response to enamel matrix derivative, porcine recombinant 21.3-kDa amelogenin and 5.3-kDa tyrosine-rich amelogenin peptide.

Enamel matrix derivative (EMD) containing a variety of protein fractions has been used for periodontal tissue regeneration. It is suggested that the proteins contained in EMD positively influence gingival fibroblasts migration and proliferation. Effects of EMD as well as of porcine recombinated 21.3-kDa amelogenin (prAMEL) and 5.3-kDa tyrosine-rich amelogenin peptide (prTRAP) on human gingival fibroblast (HGF-1, ATCC; USA) cell line were investigated. Real-time cell analysis (xCELLigence system; Roche Applied Science) was performed to determine the effects of EMD, prAMEL and prTRAP (12.5-50 µg/mL) on HGF-1 cell proliferation and migration. The effect of treatment on cell cycle was determined using flow cytometry. EMD significantly increased HGF-1 cell proliferation after 24- and 48-h incubation. Individually, prAMEL and prTRAP also increased HGF-1 cell proliferation; however, the difference was significant only for prAMEL 50 µg/mL. prAMEL and TRAP significantly increased HGF-1 cell migration after 60- and 72-h incubation. Cell cycle analysis showed significant decrease of the percentage of cells in the G0/G1 phase and a buildup of cells in the S and M phase observed after EMD and prAMEL stimulation. This process was ligand and concentration-dependent. The various molecular components in the enamel matrix derivative might contribute to the reported effects on gingival tissue regeneration; however, biologic effects of prAMEL and prTRAP individually were different from that of EMD.

The C-terminus of amelogenin enhances osteogenic differentiation of human cementoblast lineage cells.

BACKGROUND AND OBJECTIVES: Amelogenin proteins are the major constituent of developing extracellular enamel matrix and are believed to have an exclusively epithelial origin. Recent studies have suggested that amelogenins might induce the differentiation and maturation of various cells, including cementoblast lineage cells. However, the residues comprising the active site of amelogenin remain unclear. The purpose of this study was to identify the active site region of amelogenin by studying the effects of amelogenin fragments on the osteogenic differentiation of cementoblasts. MATERIAL AND METHODS: Amelogenin fragments lacking the C-terminus (rh163) and N-terminus (rh128) and a fragment consisting of the C-terminal region of rh174 (C11 peptide) were synthesized and purified. Human cementoblast lineage cells were cultured in osteogenic differentiation medium and treated with 0, 10, 100 or 1000 ng/mL of rh163, rh128 or C11 peptide. The mRNA levels of bone markers were examined by real-time polymerase chain reaction analysis. Alkaline phosphatase activity and calcium deposition were also determined. Mineralization was evaluated by alizarin red staining. RESULTS: The osteogenic differentiation of human cementoblast lineage cells was significantly enhanced by treatment with rh128 or C11 peptide, whereas rh163 had no significant effect as compared with untreated controls. CONCLUSIONS: The C-terminus of amelogenin promotes the osteogenic differentiation of human cementoblast lineage cells, indicating the possible utility of C11 peptide in periodontal tissue regeneration.

Promotion of enamel caries remineralization by an amelogenin-derived peptide in a rat model.

OBJECTIVE: An amelogenin-derived peptide has been shown to promote remineralization of demineralized enamel in an in vitro model of initial caries induced by pH cycling. The present study examines whether the peptide exerts similar effects within the complex oral environment in vivo. DESIGN: Specific pathogen-free Sprague-Dawley rats (n=36) were infected with Streptococcus mutans, given ad libitum access to Diet 2000 and drinking water supplemented with sucrose (10%, w/v), and then randomly divided into three groups treated with 25µM peptide solution, 1g/L NaF or deionized water. Molar teeth were swabbed twice daily with the respective solutions for 24days. Then animals were killed, their jaws were removed and caries lesions were analyzed using the quantitative light-induced fluorescence-digital (QLF-D) technique to measure changes in mineral content. To verify QLF-D results, caries were scored for lesion depth and size using the Keyes method, and analyzed using polarized light microscopy (PLM). RESULTS: Mineral gain was significantly higher in teeth treated with peptide or NaF than in teeth treated with water (p<0.05), based on the QLF-D results (ΔF and ΔQ). Incidence of smooth-surface and sulcal caries based on Keyes scores was similar in rats treated with peptide or NaF, and significantly lower in these groups than in rats treated with water (p<0.05). Lesions on teeth treated with peptide or NaF were shallower, based on PLM. No significant differences were observed between molar enamel caries treated with peptide or NaF. CONCLUSIONS: This amelogenin-derived peptide can promote remineralization in a rat caries model, indicating strong potential for clinical use.