Effects Of Amelogenin Research Articles

Regeneration of injured articular cartilage using the recombinant human amelogenin protein.

Cartilage injuries rarely heal spontaneously and often require surgical intervention, leading to the formation of biomechanically inferior fibrous tissue. This study aimed to evaluate the possible effect of amelogenin on the healing process of a large osteochondral injury (OCI) in a rat model. A reproducible large OCI was created in the right leg femoral trochlea of 93 rats. The OCIs were treated with 0.1, 0.5, 1.0, 2.5, or 5.0 μg/μl recombinant human amelogenin protein (rHAM+) dissolved in propylene glycol alginate (PGA) carrier, or with PGA carrier alone. The degree of healing was evaluated 12 weeks after treatment by morphometric analysis and histological evaluation. Cell recruitment to the site of injury as well as the origin of the migrating cells were assessed four days after treatment with 0.5 μg/μl rHAM+ using immunohistochemistry and immunofluorescence. A total of 12 weeks after treatment, 0.5 μg/μl rHAM+ brought about significant repair of the subchondral bone and cartilage. Increased expression of proteoglycan and type II collagen and decreased expression of type I collagen were revealed at the surface of the defect, and an elevated level of type X collagen at the newly developed tide mark region. Conversely, the control group showed osteoarthritic alterations. Recruitment of cells expressing the mesenchymal stem cell (MSC) markers CD105 and STRO-1, from adjacent bone marrow toward the OCI, was noted four days after treatment. We found that 0.5 μg/μl rHAM+ induced in vivo healing of injured articular cartilage and subchondral bone in a rat model, preventing the destructive post-traumatic osteoarthritic changes seen in control OCIs, through paracrine recruitment of cells a few days after treatment.

Microarray Analysis of the Effects of Amelogenin on U937 Monocytic Cells

Periodontal diseases are chronic inflammation caused by particular types of bacteria and have been recognized as a cause of tooth loss in adults. These bacteria which invade periodontal tissue are phagocytosed mainly by monocytes and macrophages in this immune response, and will be presented to lymphocytes. Recently, therapies for regenerating periodontal tissues have been used extensively to treat periodontal disease, and in particular, enamel matrix derivative (EMD) is commonly used for such therapies in Japan. Amelogenin is a type of the extracellular matrix protein that accounts for 90% of the constituents of EMD. In this study, we carried out a detailed microarray analysis in order to evaluate a gene group involved in amelogenin stimuli in the human monocytic cell line U-937. Microarray analysis revealed that statistically significant changes were apparent in 273 genes (163 up-regulated and 110 down-regulated) subsequent to 4 h of amelogenin stimulation. The most highly enriched categories included “cell cycle”, “DNA replication”, and “DNA repair” in up-regulated annotation terms. On the other hand, “type I diabetes mellitus”, “allograft rejection”, and “graft versus host disease” were observed in down-regulated annotation terms. Specifically, the gene expression of major to compatibility complex (MHC) class I/II and CD80/86 was impaired in U937 cells after stimulation with amelogenin. In addition, the results of heat-map showed that the gene expression of inflammatory cytokine such as tumor necrosis factor (TFN), interleukin-18 (IL-18), and CXCL16 was markedly decreased after stimulation of monocytes with amelogenin. In conclusion, the findings of our study showed that by inducing monocyte growth through the suppression of the antigen-presenting ability of U937 cells, amelogenin may affect the immune responses of periodontal tissues originating from monocytes. Examining the effects of amelogenin on the transformation of macrophages differentiating from monocytes may establish a molecular basis for the anti-inflammatory effect of amelogenin in periodontal tissues.

Amelogenin Affects Brushite Crystal Morphology and Promotes Its Phase Transformation to Monetite.

Amelogenin protein is involved in organized apatite crystallization during enamel formation. Brushite (CaHPO4·2H2O), one of the precursors of hydroxyapatite mineralization in vitro, has been used for fabrication of biomaterials for hard tissue repair. In order to explore its potential application in biomimetic material synthesis, we studied the influence of the enamel protein amelogenin on brushite morphology and phase transformation to monetite. Our results show that amelogenin can adsorb onto the surface of brushite, leading to the formation of layered morphology on the (010) face. Amelogenin promoted the phase transformation of brushite into monetite (CaHPO4) in the dry state, presumably by interacting with crystalline water layers in brushite unit cells. Changes to the crystal morphology mediated by amelogenin continued even after the phase transformation from brushite to monetite, leading to the formation of organized platelets with an interlocked structure. This effect of amelogenin on brushite morphology and the phase transformation to monetite could provide a new approach to developing biomimetic materials.

Effects of Amelogenin on Proliferation, Differentiation, and Mineralization of Rat Bone Marrow Mesenchymal Stem CellsIn Vitro

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.

Effects of porcine 25 kDa amelogenin and its proteolytic derivatives on bone sialoprotein expression

Amelogenins are hydrophobic proteins that are the major component of developing enamel. Enamel matrix derivative has been used for periodontal regeneration. Bone sialoprotein is an early phenotypic marker of osteoblast differentiation. In this study, we examined the ability of porcine amelogenins to regulate bone sialoprotein transcription. To determine the molecular basis of the transcriptional regulation of the bone sialoprotein gene by amelogenins, we conducted northern hybridization, transient transfection analyses and gel mobility shift assays using the osteoblast-like ROS 17/2.8 cells. Amelogenins (100 ng/mL) up-regulated bone sialoprotein mRNA at 3 h, with maximal mRNA expression occurring at 12 h (25 and 20 kDa) and 6 h (13 and 6 kDa). Amelogenins (100 ng/mL, 12 h) increased luciferase activities in pLUC3 (nucleotides -116 to +60), and 6 kDa amelogenin up-regulated pLUC4 (nucleotides -425 to +60) activity. The tyrosine kinase inhibitor inhibited amelogenin-induced luciferase activities, whereas the protein kinase A inhibitor abolished 25 kDa amelogenin-induced bone sialoprotein transcription. The effects of amelogenins were abrogated by 2-bp mutations in the fibroblast growth factor 2 response element (FRE). Gel-shift assays with radiolabeled FRE, homeodomain-protein binding site (HOX) and transforming growth factor-beta1 activation element (TAE) double-strand oligonucleotides revealed increased binding of nuclear proteins from amelogenin-stimulated ROS 17/2.8 cells at 3 h (25 and 13 kDa) and 6 h (20 and 6 kDa). These results demonstrate that porcine 25 kDa amelogenin and its proteolytic derivatives stimulate bone sialoprotein transcription by targeting FRE, HOX and TAE in the bone sialoprotein gene promoter, and that full-length amelogenin and amelogenin cleavage products are able to regulate bone sialoprotein transcription via different signaling pathways.

Gingival crevicular fluid can degrade Emdogain and inhibit Emdogain-induced proliferation of periodontal ligament fibroblasts

Emdogain (EMD), consisting mostly of amelogenin, is used in periodontal therapy to regenerate lost connective tissue. Emdogain is applied onto periodontally affected root surfaces, where it becomes exposed to proteolytic enzymes. In this study, we aimed to find out whether gingival crevicular fluid or matrix metalloproteinases (MMPs) could degrade EMD, and whether this degradation has consequences for in vitro cell proliferation. We studied the effects of 156 gingival crevicular fluid samples collected from subjects with different stages of periodontal disease and from healthy control subjects and the effects of MMP-1, -2, -8, -9, -13 and -14 on the degradation of EMD using EMD-embedded zymography. The effects of gingival crevicular fluid with or without EMD and the effects of amelogenin on the proliferation of cultured periodontal ligament fibroblasts were studied by cell proliferation enzyme-linked immunosorbent assay kit. Degradation of Emdogain induced by gingival crevicular fluid was greater in samples from all stages of periodontal diseases compared with healthy control samples. Of the MMPs studied, only MMP-2 and MMP-8 showed limited EMD-degrading activities. One hundred micrograms per millilitre of EMD increased proliferation of periodontal ligament fibroblasts on average by 24% (confidence interval 0.60-0.64) and at 200 microg/mL by 30% (confidence interval 0.62-0.68) compared with control fibroblasts (confidence interval 0.48-0.52). However, gingival crevicular fluid (10 microg/mL) together with 100 microg/mL EMD induced the proliferation only by 6% (confidence interval 0.51-0.55) and with 200 microg/mL EMD by 12% (confidence interval 0.54-0.58). Amelogenin at 200 microg/mL decreased the proliferation of periodontal ligament fibroblasts by 54% (confidence interval 0.22-0.25). We suggest that diseased gingival crevicular fluid containing various proteases leads to degradation of EMD and decreased proliferation of periodontal ligament fibroblasts.

Evaluation of a near-senescent human dermal fibroblast cell line and effect of amelogenin

Fibroblast senescence may delay healing of chronic wounds. To characterize a chronic human dermal fibroblast cell line (CRL-7815) with near-senescent properties, cell proliferation and production of wound-healing modulating cytokines, and biosynthesis and remodelling of collagen were compared with normal human dermal fibroblasts. Also, the response of CRL-7815 fibroblasts to the extracellular matrix protein amelogenin that is beneficial in the treatment of stalled chronic wounds was studied. Fibroblast proliferation was monitored by time-resolved growth curves and factors secreted into the culture medium containing 10% fetal bovine serum were measured by enzyme-linked immunosorbent assays. Fibroblast-mediated reorganization was examined in three-dimensional type I collagen matrices. Cell proliferation over 9 days was significantly (P < 0.01) slower for CRL-7815 than for normal fibroblasts. Amelogenin at 1 mg mL(-1) increased (P < 0.01) CRL-7815 proliferation to the level of the normal fibroblasts. The neutrophil chemoattractant interleukin (IL)-8 was low while the constitutive production of monocyte chemoattractant protein (MCP)-1 was highly elevated in medium from cultured CRL-7815 fibroblasts. Amelogenin augmented IL-8 but attenuated MCP-1 secretion in CRL-7815 fibroblasts. The elevated vascular endothelial growth factor production in CRL-7815 fibroblasts was further increased with amelogenin while increased type I collagen synthesis by CRL-7815 was reduced with 0.1 mg mL(-1) amelogenin. The dramatically impaired collagen matrix remodelling with CRL-7815 fibroblasts (P < 0.001) was slightly improved with amelogenin (P = 0.0011). The near-senescent cell line CRL-7815 shares functional anomalies with fibroblasts isolated from nonhealing chronic cutaneous wounds. Amelogenin has the capacity to switch chronic fibroblasts into an acute-like phenotype.

The effect of amelogenins (Xelma™) on hard‐to‐heal venous leg ulcers

With an aging population venous ulceration is likely to become an increasing problem. Despite improvements in care and the widespread introduction of compression bandaging, the mainstay of current management, a significant proportion of venous leg ulcers remain hard to heal. Therefore, a single-blinded, randomized multicenter study was performed to compare wound size reduction using amelogenin proteins (Xelma) formulated into a solution which forms a temporary extracellular matrix on contact with the wound bed. Propylene glycol alginate 7% served as a control. Patients were randomized to receive either amelogenin protein or control treatment. The investigational products were applied weekly under soft silicone secondary dressings for up to a maximum of 12 weeks. Compression therapy was maintained throughout the investigation. Wound size reduction was measured by tracing and all wounds were photographed. In total 123 patients were recruited, 62 patients in the amelogenin group, and 61 in the control group, respectively. Subgroup analyses were performed for ulcers with a size>10 cm2 at baseline and for ulcers of duration of >12 months. The wound size reduction was greatest in the group treated with amelogenin (33.8 vs. 25.6%, n=117), this difference being greatest for larger ulcers (25 vs. 7.9% for ulcers>10 cm(2), n=61) and those of long duration (29.3 vs. 10.9% for ulcers>12-month duration, n=61). We conclude that this product may be clinically useful in the treatment of these venous leg ulcers.

Amelogenin Stimulates Bone Sialoprotein (BSP) Expression Through Fibroblast Growth Factor 2 Response Element and Transforming Growth Factor‐β1 Activation Element in the Promoter of the BSP Gene

Amelogenins are a complex mixture of hydrophobic proteins that are the major organic component of developing enamel. The principal function of the amelogenins and their degradation products has been assigned to structural roles in creating the space and milieu for promoting enamel mineralization. Enamel matrix derivative (EMD) has been used clinically for periodontal regeneration and its therapeutic effectiveness has been attributed to amelogenin, non-amelogenin enamel matrix proteins, and growth factors. While EMD is believed to induce periodontal regeneration, the precise mechanism is not known. Bone sialoprotein (BSP), an early phenotypic marker of osteoblast and cementoblast differentiation, has been implicated in the nucleation of hydroxyapatite during bone formation. In this study, we examined the ability of amelogenin to regulate BSP gene transcription in osteoblast like cells. We conducted Northern hybridization, transient transfection analyses, and gel mobility shift assays using full-length recombinant amelogenin to determine the molecular basis of the transcriptional regulation of BSP gene by amelogenin. Recombinant amelogenin (1 microg/ml, 12 hours) increased BSP mRNA levels approximately 2.4-fold. In transient transfection analyses, amelogenin (1 microg/ml, 12 hours) increased luciferase activity approximately 1.5-fold in pLUC3 (nucleotides -116 to +60) and further increased pLUC5 (nucleotides -801 to +60) activity approximately 2.3-fold transfected into ROS 17/2.8 cells. Amelogenin also increased luciferase activities in rat stromal bone marrow cells. The effect of amelogenin was inhibited by the tyrosine kinase inhibitor herbimycin A. Transcriptional stimulation by amelogenin was almost completely abrogated in cells expressing a BSP promoter construct with a mutation in the fibroblast growth factor 2 (FGF2) response element (FRE). Gel mobility shift assays with radiolabeled FRE and transforming growth factor-beta1 (TGF-beta1) activation element (TAE) ds-oligonucleotides revealed increased binding of nuclear proteins from amelogenin-stimulated ROS 17/2.8 cells. Amelogenin stimulation alters BSP gene transcription by inducing nuclear proteins that bind to the FRE and TAE in the rat BSP gene promoter.

Control of octacalcium phosphate and apatite crystal growth by amelogenin matrices

Tooth enamel, the hardest bioceramic composite in the vertebrate body, is the result of a cascade of intra- and extracellular events. Amelogenins, the principal extracellular matrix protein component of mineralizing enamel, have been considered to play substantial roles in controlling the growth and organization of enamel crystals. Considering octacalcium phosphate (OCP) as a precursor phase of enamel apatite crystallites, we have developed in vitro systems to grow OCP and apatite crystals in amelogenin matrices and therefore to investigate amelogenin–OCP and amelogenin–apatite interactions. This paper reviews our current findings on the effect of amelogenin on the morphology, size, phase and orientation of such crystals.

Modification of calcium-phosphate coatings on titanium by recombinant amelogenin.

Amelogenin proteins, the principal components of the developing dental enamel extracellular matrix, have been postulated to facilitate the elongated and oriented growth of the carbonated apatite crystals during enamel formation. We previously reported that amelogenin caused modulation of apatite crystals nucleated on a bioactive glass (Bioglass(R)) in vitro. Here, the effects of amelogenin on the growth morphology of calcium-phosphate crystals nucleated on a titanium surface were investigated in order to gain a better understanding of the role of amelogenins during enamel biomineralization and to explore their potential application in the design and development of novel biomaterials. The dose-dependent effects of a recombinant mouse amelogenin (rM179) were found to be different from those of bovine serum albumin, which significantly inhibited apatite crystal growth and caused the octacalcium phosphate (OCP) crystals to change from a plate-like shape to a curved shape, indicating a general inhibitory effect. The effects of rM179 on the crystal growth of OCP at 12.5-100 microg/mL and of apatite at 50 microg/mL were insignificant while the apatite crystals were remarkably elongated along their c-axes upon the use of 100 microg/mL of rM179. The unique modulation of the calcium-phosphate coatings on titanium by rM179 supports the view that amelogenins have a great potential for applications designed to develop novel biomimetic materials.

Effects of Ionic Flow and Amelogenins on the Lengthwise Growth of Octacalcium Phosphate Crystals in a Model System of Tooth Enamel Formation

AbstractThis paper briefly reviews our recent studies, which aimed to investigate the effects of 1) the Ca2+and PO43-ions flow and 2) amelogenins on the lengthwise growth of octacalcium phosphate (OCP), which is a potent precursor of enamel apatite crystal. OCP crystals were grown at 37°C in a dual membrane system under various amount of ionic inflow into a reaction space, using 1) 5-30mM Ca and PO4solutions as ionic sources and 2) extracted bovine amelogenin and recombinant murine amelogenins (rM179, rM166). With an increase in the amount of Ca2+and/or PO43-ions flow, the length of OCP crystal increased, while the width decreased. As a result, the length to width (L/W) ratio of crystal changed from 3 to 95, while the width to thickness (W/T) ratio from 32 to 9. The effect of amelogenins was unique, regardless of the type of amelogenins: Rod-like and prism-like OCP crystals with large L/W (61∼107) and small W/T (1.3∼2.2) ratios were formed in 10% amelogenin gels. In contrast, characteristic ribbon-like OCP crystals grew without protein and with gelatin, albumin, polyacrylamide gel and agarose gel. Specific interaction of amelogenins with OCP crystal was ascribed to the self-assembly property of amelogenin molecules and their hydrophobic nature. It was suggested that ionic flow and amelogenins play some critical roles in the elongated growth of enamel crystals.

Effects of bovine amelogenins on the crystal morphology of octacalcium phosphate in a model system of tooth enamel formation

Amelogenins are the major protein component of the extracellular matrix of developing dental enamel and are believed to play critical roles in controlling the growth of enamel crystallites by providing the appropriate structural framework within which the crystallites initiate and grow. Effects of bovine amelogenins on the growth of octacalcium phosphate (OCP) crystals were evaluated in a model system of tooth enamel formation, where ionic diffusion was controlled by a cation selective membrane and a dialysis membrane. Reactions were carried out at 37°C with and without 10% amelogenins in a small space between the membranes, using 10 mM Ca and PO 4 solutions adjusted at pH 6.5 as ionic sources. Some parallel reactions were carried out in 5% albumin and 10% gelatin for a comparison. While albumin and gelatin decreased the crystal size of OCP, the effect of amelogenins on the growth of OCP crystals was remarkable. Rod-like and prism-like OCP crystals were predominant in 10% amelogenins, while characteristic ribbon-like OCP grew without protein, and with gelatin and albumin. Moreover, protein adsorption was observed on the side faces of the crystals grown in amelogenins. The width to thickness ( W/ T) ratio of OCP crystals grown in amelogenins was significantly smaller than those of control OCP and OCP crystals grown in gelatin and albumin. Amelogenin suppressed the growth of OCP in the order, b-axis> c-axis> a-axis direction. These data suggest that interactions of amelogenins with OCP crystals are stronger on the (0 1 0) faces than those on the (1 0 0) faces. The interaction between amelogenins and OCP crystal is discussed from the view point of the crystal structure and hydrophobic nature of amelogenins. The present experiment suggests that the hydrophobic amelogenin proteins are involved in controlling biomineralization processes such as modulation of crystal morphology.