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The first barrier of the human body is the skin, and more serious harm may occur when skin wound healing is delayed. One of the components of enamel matrix proteins is amelogenin, which inhibits inflammation and promotes periodontal tissue regeneration. However, its role in skin wound healing and angiogenesis is inconclusive. Thus, this study aimed to assess the therapeutic effect of recombinant human amelogenin (rhAM) on mouse skin wounds and to determine its effect on angiogenesis and its underlying mechanism. rhAM was expressed in Escherichia coli and purified using the optimized acetic acid method. A skin injury mouse model was established to explore the effects of rhAM on skin wound healing. After treatment with rhAM for 7 days, the wound healing rate was calculated, and the therapeutic effect of rhAM on skin wounds was assessed using hematoxylin & eosin (HE), Masson, and CD31 immunofluorescence staining. The expression of growth and inflammatory factors in wound tissues were detected using Western Blot. In addition, the rhAM effects on the proliferation and migration of human umbilical vein endothelial cells (HUVEC) and mouse fibroblasts (NIH 3T3) were studied in vitro using the Cell Counting Kit-8, cell scratch, cytoskeleton staining, and qPCR. The rhAM effect on HUVEC angiogenesis and its potential mechanism was studied using tube formation and Western Blot. The results showed that the purity of the obtained rhAM was more than 90 % using the optimized acetic acid method, and high-dose rhAM treatment could improve wound healing rate in mice. Additionally, more blood vessels and collagen were produced in the skin wound, and the expression of angiopoietin-related protein 2 (ANGPTL2) and transforming growth factor (TGF)-β1 was upregulated; however, that of interleukin-6 was down-regulated. We also found that rhAM promoted the proliferation and migration of HUVEC and NIH 3T3, the mRNA levels of vascular endothelial growth factor (VEGF), fibroblast growth factor, TGF-β1 and ANGPTL2 in HUVEC cells were upregulated, and expression of VEGF and phosphorylation of the p38 mitogen-activated protein kinase were activated. Therefore, rhAM could promote skin wound healing by upregulating angiogenesis and inhibiting inflammation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

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).)

The regeneration of demineralized enamel holds great significance in the treatment of dental caries. Amelogenin (Ame), an essential protein for mediating natural enamel growth, is no longer secreted after enamel has fully matured in childhood. Although biomimetic mineralization based on peptides or proteins has made significant progress, easily accessible, low-cost, biocompatible and highly effective Ame mimics are still lacking. Herein, we construct a series of amphiphilic branched polypeptides (CAMPs) by facile coupling of the Ame's C-terminal segment and poly(γ-benzyl-L-glutamate), which serves to simulate the Ame's hydrophobic N-terminal segment. Among them, CAMP 15 is the best biomimetic mineralization template with great self-assembly performance to guide the oriented crystallization of hydroxyapatite and is capable of inhibiting the adhesion of Streptococcus mutans and Staphylococcus aureus on the enamel surfaces. This work highlights the potential application of amphiphilic branched polypeptide as Ame mimics in repairing defected enamel, providing a promising strategy for prevention and treatment of dental caries.

Objectives: In order to evaluate the effect of methacrylated hyaluronic acid (HAMA) hydrogels containing the recombinant human amelogenin (rhAm) in vitro and in vivo., Background: The ultimate goal in treating periodontal disease is to control inflammation and achieve regeneration of periodontal tissues. In recent years, methacrylated hyaluronic acid (HAMA) containing recombinant human amyloid protein (rhAm) has been widely used as a new type of biomaterial in tissue engineering and regenerative medicine. However, there is a lack of comprehensive research on the periodontal regeneration effects of this hydrogel. This experiment aims to explore the application of photoresponsive recombinant human amelogenin-loaded hyaluronic acid hydrogel for periodontal tissue regeneration and provide valuable insights into its potential use in this field., Materials and Methods: The effects of rhAm-HAMA hydrogel on the proliferation of human periodontal ligament cells (hPDLCs) were assessed using the CCK-8 kit. The osteogenic differentiation of hPDLCs was evaluated through ALP staining and real-time PCR. Calvarial parietal defects were created in 4-week-old Sprague Dawley rats and implanted with deproteinized bovine bone matrix in different treatment groups. The animals were euthanized after 4 and 8 weeks of healing. The bone volume of the defect was observed by micro-CT and histological analysis., Results: Stimulating hPDLCs with rhAm-HAMA hydrogel did not significantly affect their proliferation (p > .05). ALP staining and real-time PCR results demonstrated that the rhAm-HAMA group exhibited a significant upregulation of osteoclastic gene expression (p < .05). Micro-CT results revealed a significant increase in mineralized tissue volume fraction (MTV/TV%), trabecular bone number (Tb.N), and mineralized tissue density (MTD) of the bone defect area in the rhAm-HAMA group compared to the other groups (p < .05). The results of hematoxylin and eosin staining and Masson staining at 8 weeks post-surgery further supported the results of the micro-CT., Conclusions: The results of this study indicate that rhAm-HAMA hydrogel could effectively promote the osteogenic differentiation of hPDLCs and stabilize bone substitutes in the defects that enhance the bone regeneration in vivo., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

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).)

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 β-bend, β-turn, and coil structures, and it eventually adsorbed on enamel through negatively charged residues of the C-tail domain, then captured Ca 2+ 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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of Conflicting InterestsThe authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

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., (Copyright © 2023 Elsevier Inc. All rights reserved.)

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.

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., (© 2023. The Author(s).)

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: Ca 2+ , PO 4 3- and F - , also including Zn 2+ 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., (© 2022. 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).)

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., (© 2021 Wiley Periodicals LLC.)

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., (© 2020 Orthopaedic Research Society. Published by Wiley Periodicals LLC.)

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 β-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.

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 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., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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.

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., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

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.

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., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

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., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Background: Extracts of enamel matrix proteins are used to regenerate periodontal tissue; amelogenin, the most abundant enamel protein, plays an important role in this regeneration. Studies have demonstrated that amelogenin fragments promote tissue regeneration, but the bioactive site of amelogenin remains unclear. This study explores the functional domain of amelogenin by investigating effects of four amelogenin species on cementoblast proliferation., Methods: Four amelogenin species based on amelogenin cleavage products were investigated: 1) recombinant human full-length amelogenin (rh174); 2) amelogenin cleavage product lacking the C-terminal (rh163); 3) amelogenin cleavage product lacking the N-terminal (rh128); and 4) the C-terminal region of rh174 (C11 peptide), which was synthesized and purified. Human cementoblast-like cell line (HCEM) cells were cultured and treated with rh174, rh163, rh128, or C11 peptide. Cell proliferation was evaluated using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H- tetrazolium assay and cell proliferation enzyme-linked immunosorbent assay. Mitogen-activated protein kinase (MAPK)-extracellular signal-regulated kinase (ERK) (MAPK-ERK) pathway was examined by Western blot analysis., Results: Proliferation of HCEM cells was significantly enhanced on treatment with rh174, rh128, or C11 peptide. However, rh163 had no effect compared with the untreated control group. Western blot analysis revealed enhanced phosphorylated ERK1/2 signaling after addition of rh128 or C11 peptide and reduced phosphorylated ERK1/2 signaling after blocking with a specific MAPK inhibitor (U0126)., Conclusion: C-terminal amelogenin cleavage product increased proliferation of HCEM through MAPK-ERK signaling pathway, indicating possible application of C11 peptide for periodontal tissue regeneration.

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.)

Introduction: Recombinant DNA-produced amelogenin protein was compared with calcium hydroxide in a study of immature apex closure conducted in 24 young mongrel dogs., Methods: Root canals of maxillary and mandibular right premolars (n = 240) were instrumented and left open for 14 days. Canals were cleansed, irrigated, and split equally for treatment with recombinant mouse amelogenin (n = 120) or calcium hydroxide (n = 120)., Results: After 1, 3, and 6 months, the animals were sacrificed and the treated teeth recovered for histologic assessment and immunodetection of protein markers associated with odontogenic cells. After 1 month, amelogenin-treated canals revealed calcified tissue formed at the apical foramen and a pulp chamber containing soft connective tissue and hard tissue; amelogenin-treated canals assessed after 3- and 6-month intervals further included apical tissue functionally attached to bone by a periodontal ligament. In contrast, calcified apical tissue was poorly formed in the calcium hydroxide group, and soft connective tissue within the pulp chamber was not observed., Conclusions: The findings from this experimental strategy suggest recombinant amelogenin protein can signal cells to enhance apex formation in nonvital immature teeth and promote soft connective tissue regeneration., (Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Objective: In this study we give a preliminary study of a rationally designed small peptide, which is based on the enamel matrix protein amelogenin, to investigate its effect on remineralization of initial enamel caries lesions., Design: A novel peptide was designed and synthesized to investigate its effects on the remineralization of initial enamel carious lesions during pH cycling that simulates intra-oral conditions. Initial lesions were created in bovine enamel blocks, which were then pH-cycled for 12 days in the presence of 25μM peptide, 1g/L NaF (positive control), 50mM HEPES buffer(negative control). Before and after pH cycling, enamel blocks were analyzed by surface microhardness testing, polarized light microscopy and transverse microradiography., Results: Percentage of surface microhardness recovery (SMHR%) after pH cycling was significantly higher in peptide group than HEPES group. Lower lesion depth and less mineral mineral loss were found in peptide or NaF treatment groups after the cycling, and were significantly different to HEPES group. No significant differences were observed between the blocks treated with peptide and those treated with NaF., Conclussion: This study provides in vitro evidence that this amelogenin based peptide promotes enamel caries remineralization, offering a promising remineralizing biomaterial in initial enamel carious treatment., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

We recently reported an amelogenin-chitosan (CS-AMEL) hydrogel as a promising biomimetic material for future in situ human enamel regrowth. To further optimize the necessary conditions for clinical applicability of CS-AMEL hydrogel, herein we studied the effects of viscosity and supersaturation degree on the size and orientation of synthetic crystals by means of scanning electron microscopy (SEM) and X-ray diffraction (XRD). Raising the hydrogel viscosity by increasing chitosan concentration from 1% to 2% (w/v) improved the orientation of the crystals, while a higher supersaturation (σ(HAp) >10.06, [Ca(2+)] >5 mM) resulted in the formation of random crystals with larger sizes and irregular structures. We conclude that optimal conditions to produce organized enamel-like crystals in a CS-AMEL hydrogel are: 2% (w/v) chitosan, 2.5 mM calcium, and 1.5 mM phosphate (degree of supersaturation = 8.23) and 200 µg/ml of amelogenin.

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.)

Tooth-supporting periodontium forms a complex with multiple tissues, including cementum, periodontal ligament (PDL), and alveolar bone. In this study, we developed multiphase region-specific microscaffolds with spatiotemporal delivery of bioactive cues for integrated periodontium regeneration. Polycarprolactione-hydroxylapatite (90:10 wt%) scaffolds were fabricated using three-dimensional printing seamlessly in three phases: 100-μm microchannels in Phase A designed for cementum/dentin interface, 600-μm microchannels in Phase B designed for the PDL, and 300-μm microchannels in Phase C designed for alveolar bone. Recombinant human amelogenin, connective tissue growth factor, and bone morphogenetic protein-2 were spatially delivered and time-released in Phases A, B, and C, respectively. Upon 4-week in vitro incubation separately with dental pulp stem/progenitor cells (DPSCs), PDL stem/progenitor cells (PDLSCs), or alveolar bone stem/progenitor cells (ABSCs), distinctive tissue phenotypes were formed with collagen I-rich fibers especially by PDLSCs and mineralized tissues by DPSCs, PDLSCs, and ABSCs. DPSC-seeded multiphase scaffolds upon in vivo implantation yielded aligned PDL-like collagen fibers that inserted into bone sialoprotein-positive bone-like tissue and putative cementum matrix protein 1-positive/dentin sialophosphoprotein-positive dentin/cementum tissues. These findings illustrate a strategy for the regeneration of multiphase periodontal tissues by spatiotemporal delivery of multiple proteins. A single stem/progenitor cell population appears to differentiate into putative dentin/cementum, PDL, and alveolar bone complex by scaffold's biophysical properties and spatially released bioactive cues.

Background: In a previous study, the authors obtained a synthetic peptide (SP) for useful periodontal tissue regeneration. Periodontal ligament stem cells (PDLSCs) have multiple potentiality to contribute to tissue regeneration. The aim of this experiment is to investigate the effect of SP on human PDLSCs., Methods: Periodontal ligament cells were obtained from healthy adult human third molars and used to isolate single PDLSC-derived colonies. The mesenchymal stem cell nature of the PDLSCs was confirmed by immunohistochemical evaluation of STRO-1 expression. Proliferation and osteoblastic differentiation were investigated by culturing PDLSCs in normal or osteogenic medium with and without SP (100 ng/mL). Osteoblast differentiation was assessed by measuring alkaline phosphatase (ALP) activity, osteocalcin production, mRNA expression of osteonectin, mineralization, and calcium deposition., Results: Isolated PDLSCs were immunohistochemically positive for vimentin and STRO-1 and negative for cytokeratin. A greater number of calcified nodules were observed in osteogenic medium culture with SP than without. In the early and later stages of PDLSC culture with SP, osteonectin production and osteocalcin production were increased. SP in culture with osteogenic medium significantly enhanced proliferation of PDLSCs, as well as ALP activity, expression of osteonectin, osteocalcin production, formation of calcified nodules, and mineralization., Conclusions: SP enhances the formation of calcified nodules and osteocalcin production in the culture of PDLSCs into osteoblast-like cells and is a useful material for periodontal tissue regeneration.

Enamel matrix proteins (EMP) have been shown to promote regeneration of periodontal ligament and root cementum, and sometimes to enhance the differentiation of bone-forming cells in vitro and new bone growth in vivo. However, the inconsistent and unpredictable effects of EMP that have been reported for bone regeneration may be due to the highly variable composition of this heterogeneous material, which is comprised mainly of amelogenin and amelogenin-derived peptides. The present study has therefore examined the effects of naturally occurring low-molecular-weight (LMW) and high-molecular-weight (HMW) fractions of Emdogain(®) (EMD; Institut Straumann, Basel, Switzerland), a commercially available form of EMP, on osteogenic differentiation of bone precursor cells in vitro. In addition, the effects of chemically synthesized specific components of LMW and HMW-namely, the tyrosine-rich amelogenin peptide (TRAP), a specific amelogenin isoform derived by proteolytic clipping, and a leucine-rich amelogenin peptide (LRAP), an isoform derived by alternative splicing-on bone-forming cell activity were also investigated. Our findings demonstrate that while TRAP suppressed the formation of bone-like mineralized nodules, LRAP upregulated osteogenic differentiation. Furthermore, synthetically produced TRAP and its unique C-terminal 12 amino acid sequence (TCT) also suppressed bone-forming cells, whereas LRAP and its unique C-terminal 23 amino acid sequence (LCT) markedly enhanced terminal differentiation of bone-forming cells. These findings suggest that the differential effects of amelogenin-derived peptide sequences present in EMP could be of potential clinical value, with the novel bioactive TCT peptide as a useful tool for limiting pathological bone cell growth and the unique LCT sequence having therapeutic benefits in the treatment of periodontal and orthopedic diseases.

Background: Enamel matrix derivative (EMD), the active component of Emdogain®, is a viable option in the treatment of periodontal disease owing to its ability to regenerate lost tissue. It is believed to mimic odontogenesis, though the details of its functioning remain the focus of current research., Objective: The aim of this article is to review all relevant literature reporting on the composition/characterization of EMD as well as the effects of EMD, and its components amelogenin and ameloblastin, on the behavior of various cell types in vitro. In this way, insight into the underlying mechanism of regeneration will be garnered and utilized to propose a model for the molecular arrangement and functioning of EMD., Methods: A review of in vitro studies of EMD, or components of EMD, was performed using key words "enamel matrix proteins" OR "EMD" OR "Emdogain" OR "amelogenin" OR "ameloblastin" OR "sheath proteins" AND "cells." Results of this analysis, together with current knowledge on the molecular composition of EMD and the structure and regulation of its components, are then used to present a model of EMD functioning., Results: Characterization of the molecular composition of EMD confirmed that amelogenin proteins, including their enzymatically cleaved and alternatively spliced fragments, dominate the protein complex (>90%). A small presence of ameloblastin has also been reported. Analysis of the effects of EMD indicated that gene expression, protein production, proliferation, and differentiation of various cell types are affected and often enhanced by EMD, particularly for periodontal ligament and osteoblastic cell types. EMD also stimulated angiogenesis. In contrast, EMD had a cytostatic effect on epithelial cells. Full-length amelogenin elicited similar effects to EMD, though to a lesser extent. Both the leucine-rich amelogenin peptide and the ameloblastin peptides demonstrated osteogenic effects. A model for molecular structure and functioning of EMD involving nanosphere formation, aggregation, and dissolution is presented., Conclusions: EMD elicits a regenerative response in periodontal tissues that is only partly replicated by amelogenin or ameloblastin components. A synergistic effect among the various proteins and with the cells, as well as a temporal effect, may prove important aspects of the EMD response in vivo.

Background: Amelogenin is a major component of developing extracellular enamel matrix proteins and plays a crucial role during the formation of tooth enamel. In addition, amelogenins are suggested to exert biologic functions as signaling molecules through cell-surface receptors. The purpose of this study is to examine the effect of recombinant human full-length amelogenin (rh174) on the mineralization of human cementoblasts (HCEMs) and human periodontal ligament cells (HPDLs)., Methods: HCEMs, namely, a cell line immortalized by transfection of human telomerase reverse transcription gene, and HPDLs isolated from human first premolars were cultured and treated with 0 to 1,000 ng/mL rh174. The messenger ribonucleic acid (mRNA) levels of alkaline phosphatase (ALP), osteocalcin (OCN), and bone sialoprotein (BSP) were examined by real-time polymerase chain reaction analysis. The protein levels of OCN and BSP were examined by Western blot analysis. ALP activity and calcium deposition of cell cultures were also determined. Mineralization of cells was evaluated by red dye staining., Results: The treatment of HCEMs with rh174 upregulated the ALP, OCN, and BSP mRNA levels. In addition, the protein levels of OCN and BSP, ALP activity, and calcium deposition were enhanced, resulting in enhanced mineralization. Conversely, there were no significant effects of rh174 on the mineralization of HPDLs., Conclusion: The present study shows that rh174 enhances mineralization accompanied by upregulation of mineralization markers in HCEMs, whereas it has no effect on that in HPDLs, suggesting different effects of amelogenin on PDL and cementum.

Background: Among the various sutureless techniques, fibrin glue has proved to be effective in the treatment of peptic ulcer perforation as an alternative to classical suture repair. Albeit rare, a potential disadvantage of fibrin glue use is viral transmission or anaphylaxis. The aim of this study is to introduce a new technique for the closure of duodenal perforation using a novel recombinant enamel protein called amelogenin., Methods: In this case-control experimental study, 32 adult male Wistar Albino rats weighing 250-300 g were randomly divided into four groups, each containing 8 rats. Duodenal perforation of 0.2 cm were performed in the postpyloric region in all rats. Each group received primary repair, primary repair with omentoplasty, fibrin glue, and amelogenin, respectively. All animals were killed on the postoperative day five and the bursting pressure measurements, hydroxyproline levels and histopathologic values of the wound site were evaluated., Results: Bursting pressure levels of the fibrin glue and amelogenin groups were significantly lower than the primary repair and primary repair with omentoplasty groups (P < 0.05) However, no significant difference existed between the fibrin glue and amelogenin groups in this respect (P > 0.05). There was also no statistically significant difference among all groups regarding tissue hydroxyproline levels and histopathologic values (P > 0.05)., Conclusion: Application of amelogenin as an alternative sutureless repair technique did not improve wound healing in this animal model of duodenal perforation.

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.

Amelogenins are the major constituent of developing extracellular enamel matrix proteins and are understood to have an exclusively epithelial origin. Recent studies have demonstrated that amelogenins can be detected in other tissues, including bone marrow mesenchymal stem cells (MSCs), but the role of amelogenins in MSCs remains unclear. The purpose of this study was to examine the effect of recombinant human full-length amelogenin (rh174) on the osteogenic differentiation of cultured human MSCs. MSCs isolated from human bone marrow were cultured in osteoblastic differentiation medium with 0, 10 or 100 ng/ml rh174. The mRNA levels of bone markers were examined by real-time PCR analysis. Alkaline phosphatase (ALP) activity and calcium concentration were determined. Mineralization was evaluated by alizarin red staining. The mRNA levels of ALP, type I collagen, osteopontin and bone sialoprotein in the MSCs treated with rh174 became significantly higher than those in non-treated controls. Treatment of MSCs with rh174 also enhanced ALP activity and calcium concentration, resulting in enhanced mineralization, as denoted by high intensity of alizarin red staining. In conclusion, the present study showed that rh174 enhances the mineralization accompanied by the upregulation of bone markers in human bone marrow MSCs during osteogenic differentiation, suggesting a certain role of amelogenin in the modulation of osteogenic differentiation of MSCs., (Copyright © 2012 S. Karger AG, Basel.)

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.)

Objectives: To determine whether all or only certain proteins in an enamel matrix derivative (EMD) are angiogenic., Materials and Methods: The angiogenic effect was analysed using an in vivo angiogenesis assay. Silicon tubes were filled with or without potential and known angiogenic-modulating factors: (i) an EMD parent, (ii) nine pools of EMD proteins, (iii) fibroblast growth factor/vascular endothelial growth factor and (iv) amelogenin. Silicon tubes were implanted subcutaneously in mice. Dextran-fluorescein isothiocyanate (FITC) was injected via the tail vein, mice were euthanized and tubes were retrieved. Neovascularization was determined by measuring the amount of dextran-FITC within the tubes., Results: The greatest angiogenic potential of the EMD parent was at a weight of 125 ng, resulting in a 4.3-fold increase compared with the negative control. Five pools of EMD proteins showed a stronger angiogenic activity than the EMD parent. Pool 5 showed the greatest angiogenic activity, when compared with the negative control (8.1-fold increase) and with 125 ng of the EMD parent (4.2-fold increase). Amelogenin demonstrated a significantly higher angiogenic activity than the negative control (increase up to 4.0-fold) and the EMD parent (increase up to 1.6-fold)., Conclusions: EMD parent, recombinant porcine amelogenin and certain pools of EMD proteins induced significant angiogenesis compared with the controls using a standardized in vivo assay., (© 2010 John Wiley & Sons A/S.)

Background and Objective: 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., Material and Methods: 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., Results: 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)., Conclusion: 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., ((c) 2010 John Wiley & Sons A/S.)

Background: Porcine enamel matrix derivative (EMD) has a clinical use in facilitating periodontal healing by enhancing the regeneration of alveolar bone, cementum, and periodontal ligament. The mechanism of clinical use has not been elucidated, but in vitro studies suggest that EMD may enhance healing, in part, by stimulating angiogenesis. This study analyzes the effect of EMD on the production of blood vessels in the chorioallantoic membrane (CAM) of the developing chicken egg., Methods: Various amounts of EMD ranging from 15 to 125 ng/5 microl were pipetted onto a 3-mm diameter x 2-mm thick bioabsorbable hemostatic gelatin and placed onto the surface of the CAM. Recombinant human amelogenin and a purified 5-kDa protein fraction derived from EMD were also tested at various amounts ranging from 15 to 62 ng/5 microl. A mixture of fibroblast growth factor and vascular endothelial growth factor served as the positive control. The negative control was 0.9% saline. A histologic examination of the interface of the gelatin and CAM was performed, evaluating for new blood-vessel formation on an ordinal scale of 0 to 3. Non-parametric statistical analyses were applied to compare test groups with the negative controls., Results: CAM treated with EMD displayed moderate vascularity as indicated by a maximal score of 3.0 +/- 0.05 (mean +/- SEM). This compared favorably to the degree of vascularity stimulated by the mixture of fibroblast growth factor and vascular endothelial growth factor, which had a score of 3.0 +/- 0.05. Interestingly, the stimulation of angiogenesis by EMD was significant only at the lowest amounts tested. At the higher amounts, vascularity was reduced and not significantly different from the negative control. Vascularity was also increased by recombinant human amelogenin as indicated by a maximal score of 2.9 +/- 0.14. By contrast, there was only mild vascularity in sections treated with the negative control as indicated by a score of 1.7 +/- 0.4. The vascularity of the 5-kDa EMD-protein fraction was not different from the negative-control group (2.5 +/- 0.5 versus 1.7 +/- 0.4, respectively)., Conclusions: EMD stimulates angiogenesis in the CAM model. As a heterogeneous mixture of extracellular matrix components, EMD may have multiple biologic functions, but it is likely that at least part of the explanation for its observed positive clinical effects may be the stimulation of angiogenesis. The fact that vascularity was also increased by recombinant human amelogenin raises the possibility that this 28.9-kDa protein may be the source of the angiogenic activity because it is the predominant protein of the EMD mixture. These results, taken together with results from previously reported in vitro studies, suggest that EMD may increase angiogenesis directly and/or indirectly at the wound-healing site.

Background and Objective: 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., Material and Methods: 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., Results: 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)., Conclusion: We suggest that diseased gingival crevicular fluid containing various proteases leads to degradation of EMD and decreased proliferation of periodontal ligament fibroblasts.

Previous studies have assumed that amelogenin is responsible for the therapeutic effect of the enamel matrix derivative (EMD) in periodontal tissue healing and regeneration. However, it is difficult to confirm this hypothesis because both the EMD and the amelogenins are complex mixtures of multiple proteins. Further adding to the difficulties is the fact that periodontal tissue regeneration involves various types of cells and a sequence of associated cellular events including the attachment, migration and proliferation of various cells. In this study, we investigated the potential effect of a 25-kDa recombinant porcine amelogenin (rPAm) on primarily cultured periodontal ligament fibroblasts (PDLF), gingival fibroblasts (GF) and gingival epithelial cells (GEC). The cells were treated with 25-kDa recombinant porcine amelogenin at a concentration of 10 microg/mL. We found that rPAm significantly promoted the proliferation and migration of PDLF, but not their adhesion. Similarly, the proliferation and adhesion of GF were significantly enhanced by treatment with rPAm, while migration was greatly inhibited. Interestingly, this recombinant protein inhibited the growth rate, cell adhesion and migration of GEC. These data suggest that rPAm may play an essential role in periodontal regeneration through the activation of periodontal fibroblasts and inhibition of the cellular behaviors of gingival epithelial cells., (Copyright 2010 Elsevier Inc. All rights reserved.)

Chronic wounds come in all shapes and sizes and may be associated with co-morbidities that have a direct impact upon healing and the quality of life of patients, their families, carers, and even co-workers. Cosmetic appearance is important to both genders but especially so to female patients and unsightly wounds that are difficult to hide cause extreme problems that may have psychological implications. In addition to the effect that they have on patients, these wounds are associated with considerable resource requirements, which impact heavily on the already burdened NHS. This article presents three case studies of patients with chronic wounds that have shown a positive response, in terms of healing and impact upon the patient's quality of life, to an advanced wound care product - Xelma(R).

Background: Fibroblast senescence may delay healing of chronic wounds., Objectives: 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., Methods: 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., Results: 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)., Conclusions: 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.

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(+).

Background: Enamel matrix derivative (EMD) was shown to enhance soft tissue healing and regeneration of the periodontium; however, the mechanisms of this action are unknown. It is assumed that amelogenin, the most abundant protein in EMD, is the protein primarily responsible for the effects of EMD. The purpose of this study was to fractionate EMD and associate its specific cellular effects with different molecular weight fractions following size-exclusion chromatography., Methods: Freshly dissolved EMD was fractionated by gel filtration, and forty-five 7-ml fractions were collected, desalted, lyophilized, and resuspended. These fractions were analyzed for their effects on the differentiation of osteoprogenitor cells (C2C12) and the proliferation and differentiation of human microvascular endothelial cells (HMVECs). Alkaline phosphatase activity (C2C12) was measured as a marker for osteogenic differentiation before and after preincubation of the fractions with the bone morphogenetic protein (BMP) decoy receptor, noggin. Angiogenesis (HMVEC) was evaluated as a marker for endothelial cell differentiation. Enzymographic assays used polyacrylamide gels copolymerized with denatured type I collagen to determine gelatinolytic activities in each fraction., Results: EMD fractionated into three major protein peaks following size exclusion chromatography with cross-linked dextran particle matrix. Peak I was associated with the column void volume, whereas peak III eluted near the salt volume. Peak II eluted between these two peaks. Proliferation and angiogenic activities were associated with peaks II and III for the microvascular cells. The differentiation of osteoprogenitor cells, indicated by alkaline phosphatase activity, was induced by EMD components present in peak I and the leading edge of peak II. The additional observation that this differentiation was inhibited by prior treatment of the fractions with noggin suggested the activity was induced by BMP rather than amelogenin or other unknown proteins. Gelatinolytic activities were detected in the early fractions of peaks I and II of gel-fractionated EMD., Conclusions: The cellular activities stimulated by EMD are not associated with a single molecular weight species. The fact that noggin abolishes C2C12 alkaline phosphatase activity suggests that effects on osteoprogenitor cell differentiation are the result of a BMP-like protein(s), whereas effects on proliferation and angiogenesis are associated with lower molecular weight species present in peaks II and III. Finally, unheated EMD displays gelatinolytic activities that are also detectable following size-exclusion separation of its constituents. The masses of these activities were consistent with those reported for latent and active matrix metalloproteinase-20.

The management of hard-to-heal or chronic wounds places a high economic burden on healthcare services. This problem is exacerbated by the increasing age of the general population, an increasing diabetes population and a high prevalence of such wounds in the elderly, patients with diabetes and those with venous insufficiency. Standard treatments for such wounds, such as compression therapy in venous leg ulcers, debridement and wound care for diabetic foot ulcers, can still leave a significant population with non-healing wounds, resulting in extended hospital stays and reduced quality of life. The use of amelogenin (Xelma, Mölnlycke Health Care) for the treatment of a variety of chronic wounds has been assessed in both case studies and larger clinical trials with encouraging findings. This article examines the findings of studies relating to amelogenin in the treatment of hard-to-heal wounds.