Enamel Matrix Derivative Research Articles

Posttranslational Amelogenin Processing and Changes in Matrix Assembly during Enamel Development.

The extracellular tooth enamel matrix is a unique, protein-rich environment that provides the structural basis for the growth of long and parallel oriented enamel crystals. Here we have conducted a series of in vivo and in vitro studies to characterize the changes in matrix shape and organization that take place during the transition from ameloblast intravesicular matrices to extracellular subunit compartments and pericrystalline sheath proteins, and correlated these changes with stages of amelogenin matrix protein posttranslational processing. Our transmission electron microscopic studies revealed a 2.5-fold difference in matrix subunit compartment dimensions between secretory vesicle and extracellular enamel protein matrix as well as conformational changes in matrix structure between vesicles, stippled materials, and pericrystalline matrix. Enamel crystal growth in organ culture demonstrated granular mineral deposits associated with the enamel matrix framework, dot-like mineral deposits along elongating initial enamel crystallites, and dramatic changes in enamel matrix configuration following the onset of enamel crystal formation. Atomic force micrographs provided evidence for the presence of both linear and hexagonal/ring-shaped full-length recombinant amelogenin protein assemblies on mica surfaces, while nickel-staining of the N-terminal amelogenin N92 His-tag revealed 20 nm diameter oval and globular amelogenin assemblies in N92 amelogenin matrices. Western blot analysis comparing loosely bound and mineral-associated protein fractions of developing porcine enamel organs, superficial and deep enamel layers demonstrated (i) a single, full-length amelogenin band in the enamel organ followed by 3 kDa cleavage upon entry into the enamel layer, (ii) a close association of 8–16 kDa C-terminal amelogenin cleavage products with the growing enamel apatite crystal surface, and (iii) a remaining pool of N-terminal amelogenin fragments loosely retained between the crystalline phases of the deep enamel layer. Together, our data establish a temporo-spatial correlation between amelogenin protein processing and the changes in enamel matrix configuration that take place during the transition from intracellular vesicle compartments to extracellular matrix assemblies and the formation of protein coats along elongating apatite crystal surfaces. In conclusion, our study suggests that enzymatic cleavage of the amelogenin enamel matrix protein plays a key role in the patterning of the organic matrix framework as it affects enamel apatite crystal growth and habit.

Bioinductive biomaterials for periodontal regeneration

Periodontal regeneration is a great challenge faced by clinical treatment, and traditional therapies are not effective in regenerating periodontal tissues. Bioinductive biomaterials are materials that are able to induce the regeneration of damaged tissues, and they can induce the migration of stem cells or precursor cells to the defect site to proliferate, differentiate for tissue regeneration. For example, the osteoinductive biomaterials are able to induce regeneration of alveolar bone. In this review, the writer combined the recent advances of both clinical and scientific field, and summarize the application of bioinductive biomaterials includingautogenous bone, human derived growth factors, enamel matrix proteins and platelet-rich fibrin.

Three years outcome following surgical treatment of peri‐implantitis with and without enamel matrix derivative

Three years outcome following surgical treatment of peri‐implantitis with and without enamel matrix derivative

Histological Effects of Enamel Matrix Derivative Proteins (Emdogain®) on the Healing of Rats Wounds

Histological Effects of Enamel Matrix Derivative Proteins (Emdogain®) on the Healing of Rats Wounds

Overexpression of constitutively active MAP3K7 in ameloblasts causes enamel defects of mouse teeth

ObjectiveCompelling evidence suggests that mitogen-activated protein kinases (Mapks) play an important role in amelogenesis. However, the role of transforming growth factor (TGF)-β-activating kinase 1 (Tak1, Map3k7), which is a known upstream kinase of Mapks, during amelogenesis remains to be determined. The aim of this study was to investigate the possible involvement of Map3k7 in amelogenesis. DesignWe generated transgenic mice that produced constitutively active human MAP3K7 (caMAP3K7) under the control of amelogenin (Amelx) gene promoter. Radiography and micro-computed tomography (μCT) analysis was used to detect the radio-opacity and density of the teeth. The enamel microstructure was observed with a scanning electron microscope. Histological analysis was used to observe the adhesion between ameloblasts and residual organic matrix of the enamel. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to analyze the expression of enamel matrix protein. ResultsThe enamel of mandibular molars in caMAP3K7-overexpressing mice displayed pigmentation and a highly irregular structure compared with the wild type littermates. Teeth of transgenic animals underwent rapid attrition due to the brittleness of the enamel layer. The microstructure of enamel, normally a highly ordered arrangement of hydroxyapatite crystals, was completely disorganized. The gross histological appearances of ameloblasts and supporting cellular structures, as well as the expression of the enamel protein amelotin (Amtn) were altered by the overexpression of caMAP3K7. ConclusionsOur data demonstrated that protein expression, processing and secretion occurred abnormally in transgenic mice overexpressing caMAP3K7. The overexpression of caMAP3K7 had a profound effect on enamel structure by disrupting the orderly growth of enamel prisms.

Alternatives to connective tissue graft in the treatment of localized gingival recessions: A systematic review.

The aim of this Systematic Review (SR) was to assess the clinical efficacy of alternatives procedures; Acellular Dermal Matrix (ADM), Xenogeneic Collagen Matrix (XCM), Enamel Matrix Derivative (EMD) and Platelet Rich Fibrin (PRF), compared to conventional procedures in the treatment of localized gingival recessions. Electronic searches were performed to identify randomized clinical trials (RCTs) on treatment of single gingival recession with at least 6 months of follow-up. Applying guidelines of the Preferred Reporting Items for Systematic Review and Meta-Analyses statement (PRISMA). The risk of bias was assessed using the Cochrane Collaboration's Risk of Bias tool. Eighteen randomized controlled trials (RCTs) with a total of 390 treated patients (606 recessions) were included. This systematic review showed that: Coronally Advanced Flap (CAF) in conjunction with ADM was significantly better than CAF alone, while the comparison between CAF+ADM and CTG was affected by large uncertainty. The CAF+EMD was significantly better than CAF alone, whereas the comparison between CAF+EMD and CTG was affected by large uncertainty. No significant difference was recorded when comparing CAF+XCM with CAF alone, and the comparison between CAF+XCM and CTG was affected by large uncertainty. The comparison between PRF and others technique was affected by large uncertainty. ADM, XCM and EMD assisted to CAF might be considered alternatives of CTG in the treatment of Miller class I and II gingival recession.

Microvessel Density Evaluation of the Effect of Enamel Matrix Derivative on Soft Tissue After Implant Placement: A Preliminary Study.

Enamel matrix derivative (EMD) is commonly used in periodontal therapy and has been used successfully for periodontal regeneration. In addition, this material has a possible angiogenic effect that has been associated with enhanced wound healing. The aim of this study was to evaluate the effect of EMD on microvessel density (angiogenesis) on the soft tissues surrounding newly placed implants after 14 days. Five patients were selected, each requiring at least one implant on each side of the maxilla, in a split-mouth experimental design. The implants were placed in a two-stage procedure. Each side was then randomized as test or control. On the test side, 0.1 mL of EMD was topically applied to the soft tissues surrounding the implants, while the control side did not receive any treatment. Second-stage surgery was performed after 14 days. A 6-mm punch biopsy was performed for each implant, with the samples subsequently prepared for histology and immunohistochemistry. Quantitative vascularization analysis was performed, which involved counting three areas or "hotspots" containing vessels strongly positive for CD34 and CD105, a pan-endothelial and new vessel marker, respectively. There was no significant difference between test and control groups when evaluating the formation of new blood vessels. The total number of blood vessels, however, was significantly higher in the group treated with EMD (test group). Within the limits of the present study, it can be concluded that topical application of EMD on the soft tissues surrounding newly placed implants resulted in an increased number of blood vessels at 14 days, suggesting that EMD may play a beneficial role in this aspect of wound healing.

Clinical Outcomes after Regenerative Periodontal Therapy with Emdogain

Abstract Objective: Regeneration is defined as a reconstruction of a lost part of the body in such a way that the structure and function of the lost tissue are completely restored. The aim of this study is to compare the clinical outcomes of intrabony defects treatment using regenerative periodontal therapy with enamel matrix proteins (Emdogain, EMD) with a control group. Methods: Ten patients with chronic periodontitis were included in this randomized, controlled clinical study. Two groups received conservative periodontal therapy. In the test group, different teeth received regenerative treatment with EMD. In the control group teeth received solely conservative periodontal therapy. Pocket depth probing (PD) and bone reduction (based on X rays) were registered at baseline and after eight months in both groups. In the control group Results: Both groups showed a significant reduction of PD. The teeth treated with EMD showed a significant attachment gain. Within the test group, the radiographic examination of the teeth treated with EMD showed no significant change, whereas the teeth in the control group showed significant bone reduction. Conclusions: Intrabony defects in teeth treaded with EMD exhibit a substantially higher gain in clinical attachment and defect filling. The use of EMD in dental practice can prevent further bone loss.

The Presence of MMP-20 Reinforces Biomimetic Enamel Regrowth

Biomimetic synthesis of artificial enamel is a promising strategy for the prevention and restoration of defective enamel. We have recently reported that a hydrogel system composed of chitosan-amelogenin (CS-AMEL) and calcium phosphate is effective in forming an enamel-like layer that has a seamless interface with natural tooth surfaces. Here, to improve the mechanical system function and to facilitate the biomimetic enamel regrowth, matrix metalloproteinase–20 (MMP-20) was introduced into the CS-AMEL hydrogel. Inspired by our recent finding that MMP-20 prevents protein occlusion inside enamel crystals, we hypothesized that addition of MMP-20 to CS-AMEL hydrogel could reinforce the newly grown layer. Recombinant human MMP-20 was added to the CS-AMEL hydrogel to cleave full-length amelogenin during the growth of enamel-like crystals on an etched enamel surface. The MMP-20 proteolysis of amelogenin was studied, and the morphology, composition, and mechanical properties of the newly grown layer were characterized. We found that amelogenin was gradually degraded by MMP-20 in the presence of chitosan. The newly grown crystals in the sample treated with MMP-20–CS-AMEL hydrogel showed more uniform orientation and greater crystallinity than the samples treated with CS-AMEL hydrogel without MMP-20. Stepwise processing of amelogenin by MMP-20 in the CS-AMEL hydrogel prevented undesirable protein occlusion within the newly formed crystals. As a result, both the modulus and hardness of the repaired enamel were significantly increased (1.8- and 2.4-fold, respectively) by the MMP-20–CS-AMEL hydrogel. Although future work is needed to further incorporate other enamel matrix proteins into the system, this study brings us one step closer to biomimetic enamel regrowth.

The Effect of Enamel Matrix Derivate Proteins EMD on Ortodontically Induced Bone Resorption Based on Mini-implants

Amelogenine protein is the major component of the continuously secreted enamel extracellular matrix that controls the mineralization of enamel crystals. EmdogainTM is an extract of porcine fetal tooth material, a product based on the high degree of homology between porcine and human enamel proteins, composed primarily of amelogenine protein. It was created to promote the regeneration of periodontal tissues such as cementum, periodontal ligament and alveolar bone by stimulating normal development of these tissues, it is used to treat deep intraosseus defects.

Amelogenin induces M2 macrophage polarisation via PGE2/cAMP signalling pathway

ObjectivesAmelogenin, 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. DesignHuman 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. ResultsThe 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. ConclusionAmelogenin 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.

Bicarbonate Transport During Enamel Maturation.

Amelogenesis (tooth enamel formation) is a biomineralization process consisting primarily of two stages (secretory stage and maturation stage) with unique features. During the secretory stage, the inner epithelium of the enamel organ (i.e., the ameloblast cells) synthesizes and secretes enamel matrix proteins (EMPs) into the enamel space. The protein-rich enamel matrix forms a highly organized architecture in a pH-neutral microenvironment. As amelogenesis transitions to maturation stage, EMPs are degraded and internalized by ameloblasts through endosomal-lysosomal pathways. Enamel crystallite formation is initiated early in the secretory stage, however, during maturation stage the more rapid deposition of calcium and phosphate into the enamel space results in a rapid expansion of crystallite length and mineral volume. During maturation-stage amelogenesis, the pH value of enamel varies considerably from slightly above neutral to acidic. Extracellular acid-base balance during enamel maturation is tightly controlled by ameloblast-mediated regulatory networks, which include significant synthesis and movement of bicarbonate ions from both the enamel papillary layer cells and ameloblasts. In this review we summarize the carbonic anhydrases and the carbonate transporters/exchangers involved in pH regulation in maturation-stage amelogenesis. Proteins that have been shown to be instrumental in this process include CA2, CA6, CFTR, AE2, NBCe1, SLC26A1/SAT1, SLC26A3/DRA, SLC26A4/PDS, SLC26A6/PAT1, and SLC26A7/SUT2. In addition, we discuss the association of miRNA regulation with bicarbonate transport in tooth enamel formation.

Inconclusive evidence about whether the addition of enamel matrix derivatives to bone substitutes yields better clinical outcomes for treatment of intrabony defects

Troiano G, Laino L, Zhurakivska K, Cicciú M, Lo Muzio L, Lo Russo L. Addition of enamel matrix derivatives to bone substitutes for the treatment of intrabony defects: a systematic review, meta-analysis and trial sequential analysis [published online ahead of print June 23, 2017]. J Clin Periodontol. http://dx.doi.org/10.1111/jcpe.12742.

Mediator 1 contributes to enamel mineralization as a coactivator for Notch1 signaling and stimulates transcription of the alkaline phosphatase gene

Tooth enamel is mineralized through the differentiation of multiple dental epithelia including ameloblasts and the stratum intermedium (SI), and this differentiation is controlled by several signaling pathways. Previously, we demonstrated that the transcriptional coactivator Mediator 1 (MED1) plays a critical role in enamel formation. For instance, conditional ablation of Med1 in dental epithelia causes functional changes in incisor-specific dental epithelial stem cells, resulting in mineralization defects in the adult incisors. However, the molecular mechanism by which Med1 deficiency causes these abnormalities is not clear. Here, we demonstrated that Med1 ablation causes early SI differentiation defects resulting in enamel hypoplasia of the Med1-deficient molars. Med1 deletion prevented Notch1-mediated differentiation of the SI cells resulting in decreased alkaline phosphatase (ALPL), which is essential for mineralization. However, it does not affect the ability of ameloblasts to produce enamel matrix proteins. Using the dental epithelial SF2 cell line, we demonstrated that MED1 directly activates transcription of the Alpl gene through the stimulation of Notch1 signaling by forming a complex with cleaved Notch1-RBP-Jk on the Alpl promoter. These results suggest that MED1 may be essential for enamel matrix mineralization by serving as a coactivator for Notch1 signaling regulating transcription of the Alpl gene.

Periodontal plastic surgery of gingival recessions at single and multiple teeth

This manuscript aims to review periodontal plastic surgery for root coverage at single and multiple gingival recessions. Techniques are assessed based on biological principles, surgical procedures, prognosticative factors and expected clinical and esthetic outcomes. The use of coronally advanced flap, laterally sliding flap, free gingival graft, the tunnel grafting technique, barrier membranes, enamel matrix derivative, collagen matrix and acellular dermal matrix are evaluated. The clinical scenario and practical implications are analyzed according to a modern evidence-based approach.

Effects of the incorporation of ε-aminocaproic acid/chitosan particles to fibrin on cementoblast differentiation and cementum regeneration.

Cementum, the mineralized layer on root dentin surfaces, functions to anchor fibrous connective tissues on tooth-root surfaces with the collagenous Sharpey's fibers integration, of which are essential for periodontal functioning restoration in the complex. Through the cementum-responsible fiber insertions on tooth-root surfaces, PDLs transmit various mechanical responses to periodontal complexes against masticatory/occlusal stimulations to support teeth. In this study, periodontal tissue regeneration was enhanced by use of modified fibrin biomaterial which significantly promoted cementogenesis within the periodontal complex with structural integration by collagenous Sharpey's fiber insertions in vivo by controlling fibrin degradation and consequent cementoblast apoptosis. Furthermore, the modified fibrin could improve repair and regeneration of tooth roof-of-furcation defects, which has spatial curvatures and geometrical difficulties and hardly regenerates periodontal tissues.

Stim1 Regulates Enamel Mineralization and Ameloblast Modulation

Loss-of-function mutations in the Ca2+ release-activated Ca2+ channel genes ORAI1 and STIM1 abolish store-operated Ca2+ entry (SOCE) and result in ectodermal dysplasia with amelogenesis imperfecta. However, because of the limited availability of patient tissue, analyses of enamel mineralization or possible changes in ameloblast function or morphology have not been possible. Here, we generated mice with ectodermal tissue-specific deletion of Stim1 (Stim1 cKO [conditional knockout]), Stim2 (Stim2 cKO), and Stim1 and Stim2 (Stim1/2 cKO) and analyzed their enamel phenotypes as compared with those of control (Stim1/2fl/fl) animals. Ablation of Stim1 and Stim1/2 but not Stim2 expression resulted in chalky enamel and severe attrition at the incisor tips and molar cusps. Stim1 and Stim1/2 cKO, but not Stim2 cKO, demonstrated inferior enamel mineralization with impaired structural integrity, whereas the shape of the teeth and enamel thickness appeared to be normal in all animals. The gene expression levels of the enamel matrix proteins Amelx and Ambn and the enamel matrix proteases Mmp20 and Klk4 were not altered by the abrogation of SOCE in Stim1/2 cKO mice. The morphology of ameloblasts during the secretory and maturation stages was not significantly altered in either the incisors or molars of the cKO animals. However, in Stim1 and Stim1/2 cKO incisors, the alternating modulation of maturation-stage ameloblasts between the smooth- and ruffle-ended cell types continued beyond the regular cycle and extended to the areas corresponding to the zone of postmodulation ameloblasts in the teeth of control animals. These results indicate that SOCE is essential for proper enamel mineralization, in which Stim1 plays a critical role during the maturation process.

The Fam50a positively regulates ameloblast differentiation via interacting with Runx2.

Differentiated ameloblasts secret enamel matrix proteins such as amelogenin, ameloblastin, and enamelin. Expression levels of these proteins are regulated by various factors. To find a new regulatory factor for ameloblast differentiation, we performed 2D-PAGE analysis using mouse ameloblast lineage cell line (mALCs) cultured with mineralizing medium. Of identified proteins, family with sequence similarity 50 member A (Fam50a) was significantly increased during differentiation of mALCs. Fam50a protein was also highly expressed in secretory ameloblasts of mouse tooth germs. In mALCs cultures, forced expression of Fam50a up-regulated the expression of enamel matrix protein genes such as amelogenin, ameloblastin, and enamelin. In addition, up-regulation of Fam50a also increased ALP activity and mineralized nodule formation in a dose-dependent manner. In contrast, knockdown of Fam50a decreased expression levels of enamel matrix protein genes, ALP activity, and mineralized nodule formation. By fluorescence microscopy, endogenous Fam50a protein was found to be localized to the nucleus of ameloblasts. In addition, Fam50a synergistically increased Ambn transactivation by Runx2. Moreover, Fam50a increased binding affinity of Runx2 to Ambn promoter by physically interacting with Runx2. Taken together, these results suggest Fam50a might be a new positive regulator of ameloblast differentiation.

Biofunctionalization of porcine-derived collagen matrix using enamel matrix derivative and platelet-rich fibrin: influence on mature endothelial cell characteristics in vitro.

The present study evaluated the effect of an enamel matrix derivative (EMD) and platelet-rich fibrin (PRF)-modified porcine-derived collagen matrix (PDCM) on human umbilical vein endothelial cells (HUVEC) in vitro. PDCM (mucoderm®) was prepared to 6mm (±0.1mm) diameter discs. PDCM samples were incubated with either EMD, PRF, or control solutions for 100min at 4°C before the experiments. Cell-inducing properties of test materials on HUVEC cells were tested with cell proliferation assays (MTT, PrestoBlue®), a cytotoxicity assay (ToxiLight®), a Boyden chamber migration assay, and a cell attachment assay. Scanning electron microscopy (SEM) imaging was performed to determine the surface and the architecture of the modified matrices. Cell proliferation was elevated in the EMD and PRF groups compared with control (p each ≤0.046). PRF modification increased HUVEC migration ability by 8-fold compared with both control and EMD groups (p each <0.001). Both treatments significantly promoted the cell attachment of HUVEC to PDCM, as assessed by direct cell counts on the matrices (p each <0.001). HUVEC cell characteristics were overall improved by EMD- and PRF- modified PDCM. Adsorbed bioactive molecules to the PDCM surface may have contributed to a more preferable environment to surrounding cells. The results may give evidence that PDCM modification with EMD or PRF, respectively, might be a useful approach to improve clinical outcomes, to prevent inflammatory reactions and wound-healing disturbances, and to expand the clinical application area of PDCM.

Periodontal regenerative therapy with enamel matrix derivative in the treatment of intrabony defects: a prospective 2-year study

ObjectiveTo date, enamel matrix derivative (EMD) has been considered to be one of the few biomaterials for clinical use capable of demonstrating true periodontal regeneration. The aim of this two-center prospective clinical study was to evaluate 2-year outcome of periodontal regenerative therapy using EMD in the treatment of intrabony defects, performed as an ‘advanced medical treatment’ under the national healthcare system in Japan.ResultsPatients with chronic periodontitis who have completed initial periodontal therapy at either of the two dental school clinics were enrolled. Each contributed at least one intrabony defect of ≥3 mm in depth. During surgery, EMD was applied to the defect following debridement. Twenty-two participants (mean age 55.2 years old, 9 men and 13 women) completed 2-year reevaluation, and a total of 42 defects were subjected to data analysis. Mean gains in clinical attachment level (CAL) at 1 and 2 years were 2.9 mm (38% of baseline CAL) and 3.1 mm (41%), respectively, both showing a significant improvement from baseline. There was also a significant reduction in probing depth (PD): mean reductions at 1 and 2 years were 3.2 and 3.3 mm, respectively. There was a progressive improvement in the mean percentages of bone fill from 26% at 1 year to 36% at 2 years. No significant difference in CAL gain at 2 years was found between 3-wall bone defects and other defect types combined. In multiple regression analysis, the baseline PD was significantly associated with CAL gain at 2 years. In this population of patients, the treatment of intrabony defects with EMD yielded clinically favorable outcomes, as assessed by periodontal and radiographical parameters, over a period of 2 years.