Application Of Enamel Matrix Proteins Research Articles

Re-understanding the role of enamel matrix proteins in periodontal regeneration

Enamel matrix proteins (EMP) are deposited on the surface of developing roots before dentin formation and may play a role in osteogenesis. Amelogenins (Am) are the main and active component in EMPs. Studies had showed great clinical values of EMPs in periodontal regenerative treatment and other fields. By affecting the expression of growth factors and inflammatory factors, EMPs could act on various periodontal regeneration-related cells to promote angiogenesis, anti-inflammation, bacteriostasis and tissue healing, achieving the clinical effect of periodontal tissue regeneration (newly generated cements and alveolar bones as well as functionally penetrated periodontal ligament). EMPs alone or combined with bone graft material and barrier can be used for regenerative surgical treatment in intrabony defects and degree Ⅱ furcation-involved at maxillary buccal or mandibular teeth. EMPs can also be adjunctively used to treat recession type 1 or 2 gingival recessions, forming periodontal regeneration on the exposed root surface. Through a comprehensive understanding of the principle and current clinical application of EMPs on periodontal regeneration, we can look forward to their future development. The development of recombinant human amelogenin to replace animal-derived EMPs through bioengineering technologies, the study of the clinical application of EMPs combined with other collagen biomaterials, and the specific application of EMPs in severe soft and hard periodontal tissue defects and peri-implant lesions are all important development directions of future EMPs-related researches.

Current Status of Regenerative Periodontal Treatment

Periodontal regenerative therapy aims to completely restore the tooth’s supporting tissues (e.g., periodontal ligament, root cementum, alveolar bone, and gingiva) which have been lost following periodontal disease or trauma. The histologic outcomes should translate in substantial clinical improvements evidenced by probing depth reduction, gain in clinical attachment level, and bone fill. The present review provides a brief overview of the current status of periodontal regenerative therapy. There is plenty of evidence to support the use of guided tissue regeneration (GTR) techniques and the application of enamel matrix proteins (EMD) onto the exposed root result in true periodontal regenerative. The use of various growth factors and autologous blood concentrates for periodontal regeneration also have produced positive results, but more studies are needed. In general, both GTR and EMD, alone or in combination with bone grafts or substitutes, result in larger clinical improvements compared to conventional periodontal treatment for deep intrabony and furcation Class II defects. Important factors that negatively impact on the clinical outcomes of periodontal regeneration procedures are smoking, large number of remaining sites with bleeding on probing, bad oral hygiene, and increased tooth mobility. The magnitude of improvement arising from periodontal regenerative techniques and procedures depends largely on operator skills, not only in terms of dexterity but also on the ability to choose the best approach regarding the surgical technique and the regenerative technology depending on patient- and site-specific criteria. The clinical improvements after regenerative treatment can be preserved on a long-term basis on the majority of treated sites, provided that patients do not smoke, keep high oral hygiene standards, and attend regularly SPT.

In vitro re-hardening of artificial enamel caries lesions using enamel matrix proteins or self-assembling peptides.

ABSTRACTObjectives To assess the re-hardening potential of enamel matrix derivatives (EMD) and self-assembling peptides in vitro, hypothesizing that these materials may increase the mineralization of artificial carious lesions and improve hardness profiles.Material and Methods Forty-eight enamel samples were prepared from extracted bovine lower central incisors. After embedding and polishing, nail varnish was applied, leaving a defined test area. One third of this area was covered with a flowable composite (non-demineralized control). The remaining area was demineralized in an acidic buffer solution for 18 d to simulate a carious lesion. Half the demineralized area was then covered with composite (demineralized control), while the last third was left open for three test and one control treatments: (A) Application of enamel-matrix proteins (EMD - lyophilized protein fractions dissolved in acetic acid, Straumann), (B) self-assembling peptides (SAP, Curodont), or (C) amine fluoride solution (Am-F, GABA) for 5 min each. Untreated samples (D) served as control. After treatment, samples were immersed in artificial saliva for four weeks (remineralization phase) and microhardness (Knoop) depth profiles (25-300 µm) were obtained at sections. Two-way ANOVA was calculated to determine differences between the areas (re-hardening or softening).Results Decalcification resulted in significant softening of the subsurface enamel in all groups (A-D). A significant re-hardening up to 125 µm was observed in the EMD and SAP groups.Conclusions This study showed that EMD and SAP were able to improve the hardness profiles when applied to deep demineralized artificial lesions. However, further research is needed to verify and improve this observed effect.

Histopathological evaluation of the effects of variable extraoral dry times and enamel matrix proteins (enamel matrix derivatives) application on replanted dogs' teeth.

The extra-alveolar dry period and storage medium in which the tooth was kept prior to replantation remain the critical factors affecting the survival and regeneration of the damaged periodontium. When the replantation is delayed, replacement root resorption is the most common complication following replantation of an avulsed tooth. The aim of this histological study was to evaluate the periodontal healing of replanted dogs' teeth after 20 min (short) and 60 min (long) extraoral dry time with and without the application of enamel matrix proteins. Eighty mature premolar roots (40 teeth) maxillary and mandibular premolars were extracted, the root canals were accessed, instrumented, and filled using a lateral condensation technique, and the access cavity was restored with amalgam. Each root was randomly assigned to one of experimental groups: Groups I and II: Roots were replanted after an extraoral dry time of 20 min. In group II, Emdogain(®) (Biora, Malmo, Sweden) was applied directly to the external root surface with complete coverage. Groups III and IV: Roots were replanted after an extraoral dry time of 60 min. In group IV, Emdogain(®) was applied to the whole external root surface before replantation. Roots that replanted within a total extraoral dry time of 10 min were used as negative controls, while those replanted after 90 min of extraoral dry time were assigned as positive controls. After 4 months, the dogs were euthanized, and the maxillary and mandibular processes were processed for histology and microscopically evaluated. Statistical analysis showed no significant differences (P = 0.1075) among the experimental groups. The results of this study show that 20 min of extraoral dry time is as detrimental to the PDL cells as 60 or 90 min of extraoral dry time, with avulsed dogs' teeth, even when replanted with an inductive material such as EMD. This study provides strong evidence in relation to the threshold of the extraoral dry time of avulsed teeth, suggesting that the extraoral dry time threshold of PDL cell viability is significantly less than that which current guidelines promote.

Tracking endogenous amelogenin and ameloblastin in vivo.

Research on enamel matrix proteins (EMPs) is centered on understanding their role in enamel biomineralization and their bioactivity for tissue engineering. While therapeutic application of EMPs has been widely documented, their expression and biological function in non-enamel tissues is unclear. Our first aim was to screen for amelogenin (AMELX) and ameloblastin (AMBN) gene expression in mandibular bones and soft tissues isolated from adult mice (15 weeks old). Using RT-PCR, we showed mRNA expression of AMELX and AMBN in mandibular alveolar and basal bones and, at low levels, in several soft tissues; eyes and ovaries were RNA-positive for AMELX and eyes, tongues and testicles for AMBN. Moreover, in mandibular tissues AMELX and AMBN mRNA levels varied according to two parameters: 1) ontogenic stage (decreasing with age), and 2) tissue-type (e.g. higher level in dental epithelial cells and alveolar bone when compared to basal bone and dental mesenchymal cells in 1 week old mice). In situ hybridization and immunohistodetection were performed in mandibular tissues using AMELX KO mice as controls. We identified AMELX-producing (RNA-positive) cells lining the adjacent alveolar bone and AMBN and AMELX proteins in the microenvironment surrounding EMPs-producing cells. Western blotting of proteins extracted by non-dissociative means revealed that AMELX and AMBN are not exclusive to mineralized matrix; they are present to some degree in a solubilized state in mandibular bone and presumably have some capacity to diffuse. Our data support the notion that AMELX and AMBN may function as growth factor-like molecules solubilized in the aqueous microenvironment. In jaws, they might play some role in bone physiology through autocrine/paracrine pathways, particularly during development and stress-induced remodeling.

Successful Management of Aggressive Periodontitis By Regenerative Therapy: A 3‐Year Follow‐Up Case Report

This case report presents a new treatment for localized aggressive periodontitis using surgery (papilla preservation technique [PPT]) combined with enamel matrix proteins and bioactive glass. Eight intrabony defects in a 19-year-old woman with localized aggressive periodontitis were treated by PPT and the application of enamel matrix proteins or enamel matrix proteins in combination with bioactive glass. Probing depth, gingival recession, and clinical attachment level (CAL) were evaluated at baseline, 6 and 12 months, and 2 and 3 years after treatment. The primary outcome variable was CAL. After 3 years, the sites treated with enamel matrix proteins demonstrated a mean CAL change from 8.3 +/- 3.2 mm to 4.0 +/- 3.6 mm (P < 0.39), and the sites treated with enamel matrix proteins combined with bioactive glass showed a mean CAL change from 8.6 +/- 2.4 mm to 3.7 +/- 0.8 mm (P < 0.001). In the present case of aggressive periodontitis, application of enamel matrix proteins with or without the addition of bioactive glass resulted in the successful treatment of intrabony defects.

Periodontal regeneration in intrabony defects after application of enamel matrix proteins with guided tissue regeneration: An experimental study in dogs

The aim of this study was to evaluate the effects of enamel matrix proteins (EMP) at the early stage of wound healing in the periodontal tissues by a combination treatment with guided tissue regeneration (GTR). Intrabony defects were produced surgically at the distal aspects of both mandibles in six beagle dogs. At 12 weeks following the surgery, the defects were exposed using a full thickness mucoperiosteal flap procedure. Subsequently, the defects were treated by the following treatments: a control group treated with GTR alone, and an experimental group treated with a combination of GTR and EMP. After one, two, four and eight weeks of the treatment, the animals were sacrificed, and sections of the tissue were stained and evaluated microscopically. After one and two weeks, the proliferating cell nuclear antigen (PCNA)-positive cell ratios of the experimental group were significantly greater than that of the control group. After 2 and 4 weeks, new bone and new cementum formation in the experimental group were significantly greater than those in the control group (P < 0.05). However, after 8 weeks, no statistical difference was found between the two groups in new bone or cementum formation. The study results suggest that a maturation of periodontal ligament cells might contribute, during the early stage of periodontal healing, to stimulate a proliferation of periodontal ligament cells.

Healing of periodontal defects treated with enamel matrix proteins and root surface conditioning—an experimental study in dogs

Application of enamel matrix proteins has been introduced as an alternative method for periodontal regenerative therapy. It is claimed that this approach provides periodontal regeneration by a biological approach, i.e. creating a matrix on the root surfaces that promotes cementum, periodontal ligament (PDL) and alveolar bone regeneration, thus mimicking the events occurring during tooth development. Although there have been numerous in vitro and in vivo studies demonstrating periodontal regeneration, acellular cementum formation and clinical outcomes via enamel matrix proteins usage, their effects on the healing pattern of soft and hard periodontal tissues are not well-established and compared with root conditioning alone. In the present study, the effects of Emdogain ® (Biora, Malmö, Sweden), an enamel matrix derivative mainly composed of enamel matrix proteins (test), on periodontal wound healing were evaluated and compared with root surface conditioning (performed with 36% orthophosphoric acid) alone (control) histopathologically and histomorphometrically by means of the soft and hard tissue profile of periodontium. An experimental periodontitis model performed at premolar teeth of four dogs were used in the study and the healing pattern of periodontal tissues was evaluated at days 7, 14, 21, 28 (one dog at each day), respectively. At day 7, soft tissue attachment evaluated by means of connective tissue and/or epithelial attachment to the root surfaces revealed higher connective tissue attachment rate in the test group and the amount of new connective tissue proliferation in the test group was significantly greater than the control group ( p<0.01). New bone formation by osteoconduction initiated at day 14 in the test and control group. At day 21, the orientation of supra-alveolar and PDL fibers established, and new cementum formation observed in both groups. At day 28, although regenerated cementum was cellular in all of the roots in the control samples, an acellular type of cementum (1.32±0.83 mm in length and 3.16±0.23 μm in width) was also noted in six roots of test samples with an inconsistent distribution on the root surfaces. The amount of new cementum was significantly higher in the test group than the control group samples ( p<0.01). The width of the cellular cementum in the control group was more than the cellular cementum in the test group, but the difference was not statistically significant ( p>0.05). A firm attachment of acellular cementum to the root dentin with functional organization of its collagen fibers was noted, and, the accumulation and organization of cellular cementum in the control group was more irregular than the cellular cementum formed in the test group. The amount of new bone was 2.41±0.75 mm in the test and 1.09±0.46 mm in the control group at day 28. The rate of bone maturation (the number of osteons) was found higher in the test group (10.75±0.85) than the control group (5.50±0.86). Under the limitations of the study, our results reveal that when compared with root surface conditioning, enamel matrix proteins have more capacity for stimulating periodontal regeneration via their positive effects on root surfaces, i.e. inhibition of gingival epithelium down growth and stimulation of connective tissue proliferation and attachment to the root surfaces during wound healing. An acellular type of cementum regeneration and new alveolar bone formation by an accelerated osteoconductive mechanism are also achieved with application of enamel matrix proteins.

Movement of periodontally affected teeth after guided tissue regeneration (GTR)--an experimental pilot study in animals.

The purpose of the present experimental animal study was to examine the relevance of regenerative periodontal therapy (membrane procedure, application of enamel matrix proteins) to subsequent orthodontic tooth movement. After extraction of the third premolars in four foxhounds, 64 three-wall bony defects were artificially generated at the first and second premolars and colonized by periodontopathogenic microorganisms. After 6 months, regenerative periodontal therapy was performed: scaling, conditioning of the root surfaces with citric acid, combination of enamel matrix protein (Emdogain, Biora, Malmö, Sweden) and resorbable Vicryl membrane (Ethicon, Norderstedt, Germany). The defects at the first premolars served as the control group. In the test group eight second premolars were intruded 1 month after surgical intervention; in addition eight premolars were translatorily distalized, i.e. moved both into the defect (pressure side) and away from the defect (tension side). Qualitative histologic analysis was performed on non-decalcified microsection samples using the polychrome sequential labeling and histomorphometry. The histologic findings revealed extensive periodontal regeneration in the control group, at the intruded root segments, and on the tension sides. Cemento- and osseoneogenesis yielded mean values of 70-80%, whereas values for epithelial down-growth were low. The bony regeneration on the pressure side was markedly reduced; however, nearly 70% new formation of root cementum with Sharpey's fibers was also recorded. Periodontal regenerative procedures appear to enhance conditions for the movement of teeth with attachment loss; clinical testing is promising.

Enamel matrix proteins in the treatment of deep intrabony defects.

The present study evaluated the healing of enamel matrix derivative (EMD) proteins in the treatment of periodontal lesions with deep intrabony defects. Ten deep intrabony defects in 7 periodontal patients were treated and followed for 1 year. The sites had a probing depth (PD) > or = 8 mm; clinical attachment level (CAL) > or = 9 mm, and intrabony component depth > or = 5 mm. All subjects received therapy prior to surgery and had a plaque score (PI) < or = 10%. Full thickness flaps were elevated buccally and lingually, granulation tissue was removed from the defects, and the root surfaces were planed. A 24% EDTA gel was applied followed by the enamel matrix protein preparation. The flaps were closed with interrupted sutures. The patients rinsed with a chlorhexidine solution twice a day for 6 weeks. They were recalled every 2 weeks for 6 months for professional tooth cleaning and then every 4 weeks for an additional 6 months. The experimental sites were re-examined 6 and 12 months after regenerative surgery. At the 1-year examination, the mean CAL gain was 6.5 mm, the mean PD was 3.2 mm, and mean radiographic bone fill was 4.7 mm. The application of enamel matrix proteins in combination with open flap curettage and root planing resulted in a gain of CAL and bone fill in deep intrabony defects.

Enamel Matrix Proteins and Guided Tissue Regeneration With Titanium-Reinforced Expanded PolytetrafluoroethyleneMembranes in the Treatment of Infrabony Defects: A Comparative Controlled Clinical Trial.

Several studies have documented the clinical efficacy of guided tissue regeneration (GTR) with non-resorbable expanded polytetrafluoroethylene (ePTFE) membranes and enamel matrix proteins (EMP) in the treatment of infrabony defects. The objective of this controlled clinical study was to compare the clinical outcomes of 3 surgical modalities in the treatment of deep interproximal infrabony defects. Ninety (90) defects in 90 healthy subjects affected by chronic periodontitis were assigned to 1 of 3 treatment groups by blocking to prognostic variables. The test group was treated with the application of EMP and the simplified papilla preservation (SPP) technique; the second group was treated with titanium-reinforced ePFTE membranes and the SPP technique; and the third group was treated with the SPP technique used as access flap control procedure. No differences were observed in terms of baseline oral hygiene and defect characteristics among the 3 groups, indicating that the blocking approach was effective. A stringent infection control program was adopted for 1 year. The 1-year results indicated that: 1) all treatment modalities resulted in clinically significant improvements in clinical attachment levels (CAL) and reduction in probing depth (PD); 2) a statistically significant treatment effect was demonstrated comparing the EMP test, the membrane control, and the flap control groups in terms of CAL gains; 3) both the EMP test and the membrane control groups showed significant CAL gains compared to the flap control group; 4) a statistically significantly greater amount of CAL gain was demonstrated in GTR-treated compared to EMP-treated patients; 5) deeper residual probing depths but smaller increases in gingival recession were demonstrated following EMP therapy; and 6) smoking habits reduced the clinical outcomes of both regenerative procedures. The use of a regenerative procedure is indicated in the treatment of deep vertical bony defects since both the regenerative techniques (GTR and EMD) in the present study resulted in clinically and statistically significant improvements in clinical parameters compared to the access flap procedure. The use of EMP can be helpful in esthetically-sensitive sites and in reducing patient morbidity. J Periodontol 2002;73:3-12.

Application of Enamel Matrix Proteins in the Treatment of Root Coverage in Dogs

Application of Enamel Matrix Proteins in the Treatment of Root Coverage in Dogs

Periodontal tissue alterations following Emdogain® treatment of periodontal sites with angular bone defects.

The aim of the present study was to assess the predictability of probing attachment gain and probing pocket depth reduction following Emdogain treatment at sites with deep angular bone defects. 108 consecutively-treated periodontal patients (mean age 55.8 years) were included. Each subject exhibited at least 1 deep interproximal intrabony defect that could be identified as an experimental site based on the inclusion criteria: (i) probing pocket depth > or = 5 mm, (ii) probing attachment loss > or = 6 mm, (iii) radiographic evidence of an interproximal bone defect with a > or = 3 mm intrabony component. A total of 145 defects met the criteria for inclusion. All subjects received non-surgical periodontal therapy. This included subgingival instrumentation in all parts of the dentition. At least 6 months after the completion of this treatment, a baseline examination was performed to characterise the experimental site. Reconstructive therapy was subsequently performed. Full-thickness periodontal flaps were elevated, and the root surface scaled and planed. No bone recontouring was performed. A gel containing 24% EDTA was applied on the exposed root and was kept in place for 2 min. A preparation of enamel matrix proteins was applied to the root surface and adjacent defect space. The flaps were replaced and closed with sutures. The experimental sites were re-examined 12 months after reconstructive surgery. The re-examination demonstrated that a treatment including the application of enamel matrix proteins at periodontal sites with angular defects resulted in a mean probing attachment level gain of 4.6 mm and a probing pocket depth reduction of 5.2 mm. 87% of all sites treated exhibited a probing attachment gain of > 2 mm. One site suffered probing attachment loss. The radiographic assessments revealed that the bone defect had been reduced in depth by 2.9 mm on average. The reduction in defect size corresponded to an average bone fill of 69% of the original defect. In 43% of the defects, the bone fill amounted to > or = 80%. The overall probing pocket depth reduction, probing attachment level gain, and soft tissue recession, that results following Emdogain therapy, is similar to the corresponding outcome variables following GTR.

GTR treatment of degree III furcation defects following application of enamel matrix proteins. An experimental study in dogs.

The aim of the present study was to evaluate the effect of enamel matrix proteins (EMD) on periodontal wound healing in degree III furcation defects in dogs. The experiment was performed in 5 foxhound dogs. 2 months prior to the start of the experiment, the 2nd and 4th lower premolars were extracted. Degree III furcation defects were created in the 3rd mandibular premolars (3P3). The furcation defects were subsequently exposed to reconstructive surgery. Buccal and lingual full thickness flaps were elevated in the lower premolar regions. The exposed root surfaces of the experimental teeth were planed. A notch was placed in the roots at the base of the defect. In one side of the mandible (Test group), phosphoric acid gel was applied over the root surfaces for 15 s. The acid was removed by flushing the root surfaces with sterile saline. Subsequently, a gel of EMD was applied to cover all instrumented root surfaces. Following gel application, a resorbable barrier membrane was adjusted to cover the buccal and lingual entrances of the furcation defect. The flaps were repositioned to cover the barrier and sutured. The contralateral premolar (Control group) received the same treatment, but acid etching was not performed and EMD was not applied prior to barrier installation. 4 months after reconstructive surgery, the animals were sacrificed and biopsies from the 3P3 regions harvested. The biopsies were placed in a fixative, demineralized in EDTA, dehydrated and embedded in paraffin. 3 mesiodistal sections, representing the central portion of the furcation site, were selected for histological analysis of the defect. The furcation defects of both the Test and Control groups were clinically closed and were found to harbor bone and periodontal ligament tissue which appeared to be in structural continuity with a newly formed root cementum. The relative amounts of mineralized bone, bone marrow and periodontal ligament tissue that had formed were similar in the Test and the Control group. In the Test group, however, the cementum that had formed in the apical portion of the furcation defect was different from the corresponding tissue in the coronal portion, and also different from the cementum observed in the Control group. In the apical portion of the test defect a thin (12 microm) acellular cementum had been laid down, while in the coronal portion a thick (32 microm) cellular cementum, similar to the cementum found in the Control group, could be observed. The current observation, hence, seems to confirm that EMD when applied onto an instrumented and acid etched dentine surface may create an environment conducive for the formation of acellular cementum.

Periodontal regeneration in a buccal dehiscence model in monkeys after application of enamel matrix proteins.

Periodontal regeneration in a buccal dehiscence model in monkeys after application of enamel matrix proteins.