Pattern of mineralization after regenerative periodontal therapy with enamel matrix proteins

A derivative (EMD) of enamel matrix proteins (EMPs) is used for periodontal regeneration because EMPs are believed to induce the formation of acellular extrinsic fiber cementum (AEFC). Other reports, however, indicate that EMPs have osteogenic potential. The aim of this study was to characterize the nature of the tissue that forms on the root surface following application of EMD. Ten human teeth affected by periodontitis and scheduled for extraction were treated with EMD. Four to six weeks later, they were extracted and processed for analysis by light microscopy and transmission electron microscopy. Immunocytochemistry with antibodies against bone sialoprotein (BSP) and osteopontin (OPN) was performed to determine the mineralization pattern. The newly formed tissues on the root were thick and contained embedded cells. Small mineralization foci were regularly seen, and large organic matrix patches were occasionally seen, but a distinct mineralization front was lacking. While labeling for BSP was always associated with small mineralization foci and large matrix patches, OPN labeling was seen inconsistently. It is concluded that tissues resembling either cellular intrinsic fiber cementum or a type of bone were observed. The mineralization pattern mostly resembled that found in bone, except for a few areas that exhibited a hitherto undescribed mineralization pattern.

The effect of enamel matrix proteins and deproteinized bovine bone mineral on heterotopic bone formation

To evaluate the osteoinductive potential of deproteinized bovine bone mineral (DBBM) and an enamel matrix derivative (EMD) in the muscle of rats. Sixteen rats were used in this study. The animals were divided in three groups. Group A: a pouch was created in one of the pectoralis profundis muscles of the thorax of the rats and DBBM particles (Bio-Oss) were placed into the pouch. Healing: 60 days. Group B: a small pouch was created on both pectoralis profundis muscles at each side of the thorax midline. In one side, a mixture of EMD (Emdogain) mixed with DBBM was placed into one of the pouches, whereas in the contralateral side of the thorax the pouch was implanted with DBBM mixed with the propylene glycol alginate (PGA--carrier for enamel matrix proteins of EMD). Healing: 60 days. Group C: the same procedure as group B, but with a healing period of 120 days. Qualitative histological analysis of the results was performed. At 60 days, the histological appearance of the DBBM particles implanted alone was similar to that of the particles implanted together with EMD or PGA at both 60 and 120 days. The DBBM particles were encapsulated into a connective tissue stroma and an inflammatory infiltrate. At 120 days, the DBBM particles implanted together with EMD or PGA exhibited the presence of resorption lacunae in some cases. Intramuscular bone formation was not encountered in any group. The implantation of DBBM particles alone, combined with EMD or its carrier (PGA) failed to exhibit extraskeletal, bone-inductive properties.

Penetration ability and microleakage of a fissure sealant applied on artificial and natural enamel fissure caries

OBJECTIVES AND METHODS: This study investigated the sealing ability of a current available unfilled fissure sealant applied over sound (n=80), artificially created (n=80) and naturally carious fissures (n=80) under different humidity conditions (90+/-2 and 45+/-2% relative humidity) and etching times (40 and 60s). All samples were submitted to 5000 thermal cycles and examined by light microscopy after sectioning. Microleakage, penetration ability, fissure type, fissure entrance angle, sealant occlusal length, caries location and caries depth were assessed. RESULTS: The significantly longer sealant occlusal length and larger entrance angle exhibited by shallow fissures, contributed to their higher microleakage and smaller amounts of unfilled areas compared to deep fissures. Sealant microleakage was significantly influenced by the condition of the enamel (sound, artificial and natural caries) and the caries location in the fissures, but not by enamel caries depth (D1 and D2), etching time, or humidity condition. Natural caries exhibited significantly higher microleakage than sound or artificially created carious fissures. CONCLUSIONS: Based on the results of this study, it can be concluded that location of caries in the fissure rather than its depth should be taken into account when applying a fissure sealant. When the borders of the fissure sealant are on carious enamel, a significantly higher microleakage must be expected. The artificial caries model was not a suitable method to assess the behavior of natural fissure caries.

CO2 laser-irradiation through topically applied fluoride increases acid resistance of demineralised human enamel in vitro

PURPOSE: To evaluate the effect of CO2 laser treatment through topically applied amine fluoride solution on demineralised enamel. MATERIALS AND METHODS: Sixty extracted human molar crowns were selected and cut longitudinally into half. One half was subjected to a 10-day pH-cycling procedure to create caries-like lesions, whereas the other was left non-demineralised. The following treatments were randomly assigned (one treatment per tooth, on respective non-demineralised and demineralised matched specimens): exposure to a 1% amine fluoride solution for 15 s without irradiation (group I), irradiation for 15 s with a continuous-wave CO2 laser (group II), or laser-treatment for 15 s through the amine fluoride solution applied immediately beforehand (group III). Fluoride uptake (n = 30) and acid resistance (n = 30) were determined after treatment. Enamel surface alterations after laser irradiation were monitored using scanning electron microscopy. RESULTS: In groups I and III, an increased fluoride uptake was detected (p < or = 0.05). Laser irradiation through topical fluoride resulted in an increased acid resistance of sound and demineralised enamel specimens in deeper layers (p < or = 0.05). In addition, less surface alterations were observed in SEM examination of specimens irradiated through the amine fluoride solution compared with counterparts treated with laser only. CONCLUSIONS: CO2 laser light application through an amine fluoride solution may be instrumental in enhancing acid resistance of sound and demineralised enamel.

Effects of various forms of calcium added to chewing gum on initial enamel carious lesions in situ

The purpose of this randomized, cross-over in situ study was to determine the effects of 4 chewing gums on artificial caries-like subsurface lesions. Two chewing gums (1 with zinc citrate and 1 without) contained dicalcium phosphate (3.9%), calcium gluconate (1.8%) and calcium lactate (0.45%), 1 chewing gum contained casein phosphopeptide-amorphous calcium phosphate nanocomplexes (0.7%), and another one contained no calcium. Fifteen subjects without current caries activity (7 male, 8 female; mean age: 27.5 +/- 2.5 years) wore removable buccal appliances in the lower jaw with 4 bovine enamel slabs with subsurface lesions. The appliances were inserted immediately before gum chewing for 20 min and then retained for an additional 20 min. This was performed 4 times per day. Every subject chewed 4 different chewing gums over 4 periods of 14 days each. During a fifth period (control) the subjects only wore the appliances without chewing gum. At completion of each period the enamel slabs were embedded, sectioned and subjected to transversal microradiography. With regard to change of mineral loss and of lesion depth no significant differences could be found between chewing gums containing calcium and calcium-free chewing gums. Moreover, the chewing gum groups and the control group did not differ significantly if adjustments were made for baseline values (p > 0.05; ANCOVA). Under the conditions of the present study it may be concluded that the use of chewing gum offers no additional remineralizing benefit to buccal tooth surfaces, even if the chewing gum contains calcium compounds.

Is right coronary artery hypoplasia and sudden death an underdiagnosed association?

Determining whether hypoplasia of a coronary artery has caused or contributed to death is often complicated by an absence of histologic evidence of myocardial ischemia in the area of the heart supplied by the affected artery and also by the lack of data for assessing coronary artery size at autopsy. A 45-year-old woman is reported who collapsed and died and who was found at autopsy to have a dominant, small-caliber, right coronary artery, with acute and chronic ischemic changes in the posterior interventricular septum supplied by the diminutive vessel. This case provides evidence that small-caliber coronary arteries may be associated with a lethal outcome. Given the difficulties that may occur in determining whether there is a causal link between small coronary artery caliber and death, it is possible that this may be an underdiagnosed cause of sudden cardiac death, rather than a coincidental finding of minimal significance.

Biological mediators and periodontal regeneration: a review of enamel matrix proteins at the cellular and molecular levels

BACKGROUND: Despite a large body of clinical and histological data demonstrating beneficial effects of enamel matrix proteins (EMPs) for regenerative periodontal therapy, it is less clear how the available biological data can explain the mechanisms underlying the supportive effects of EMPs. OBJECTIVE: To analyse all available biological data of EMPs at the cellular and molecular levels that are relevant in the context of periodontal wound healing and tissue formation. METHODS: A stringent systematic approach was applied using the key words "enamel matrix proteins" OR "enamel matrix derivative" OR "emdogain" OR "amelogenin". The literature search was performed separately for epithelial cells, gingival fibroblasts, periodontal ligament cells, cementoblasts, osteogenic/chondrogenic/bone marrow cells, wound healing, and bacteria. RESULTS: A total of 103 papers met the inclusion criteria. EMPs affect many different cell types. Overall, the available data show that EMPs have effects on: (1) cell attachment, spreading, and chemotaxis; (2) cell proliferation and survival; (3) expression of transcription factors; (4) expression of growth factors, cytokines, extracellular matrix constituents, and other macromolecules; and (5) expression of molecules involved in the regulation of bone remodelling. CONCLUSION: All together, the data analysis provides strong evidence for EMPs to support wound healing and new periodontal tissue formation.

Controlled toothbrush abrasion of softened human enamel

The aim of this in vitro study was to compare toothbrush abrasion of softened enamel after brushing with two (soft and hard) toothbrushes. One hundred and fifty-six human enamel specimens were indented with a Knoop diamond. Salivary pellicle was formed in vitro over a period of 3 h. Erosive lesions were produced by means of 1% citric acid. A force-measuring device allowed a controlled toothbrushing force of 1.5 N. The specimens were brushed either in toothpaste slurry or with toothpaste in artificial saliva for 15 s. Enamel loss was calculated from the change in indentation depth of the same indent before and after abrasion. Mean surface losses (95% CI) were recorded in ten treatment groups: (1) soft toothbrush only [28 (17-39) nm]; (2) hard toothbrush only [25 (16-34) nm]; (3) soft toothbrush in Sensodyne MultiCare slurry [46 (27-65) nm]; (4) hard toothbrush in Sensodyne MultiCare slurry [45 (24-66) nm]; (5) soft toothbrush in Colgate sensation white slurry [71 (55-87) nm]; (6) hard toothbrush in Colgate sensation white slurry [85 (60-110) nm]; (7) soft toothbrush with Sensodyne MultiCare [48 (39-57) nm]; (8) hard toothbrush with Sensodyne MultiCare [40 (29-51) nm]; (9) soft toothbrush with Colgate sensation white [51 (37-65) nm]; (10) hard toothbrush with Colgate sensation white [52 (36-68) nm]. Neither soft nor hard toothbrushes produced significantly different toothbrush abrasion of softened human enamel in this model (p > 0.05).

Tin-containing fluoride solutions as anti-erosive agents in enamel: an in vitro tin-uptake, tissue-loss, and scanning electron micrograph study

Tin-containing fluoride solutions can reduce erosive tissue loss, but the effects of the reaction between tin and enamel are still not clear. During a 10-d period, enamel specimens were cyclically demineralized (0.05 M citric acid, pH 2.3, 6 x 5 min d(-1)) and remineralized (between the demineralization cycles and overnight). In the negative-control group, no further treatment was performed. Three groups were treated (2 x 2 min d(-1)) with tin-containing fluoride solutions (400, 1,400 or 2,100 ppm Sn2+, all 1,500 ppm F-, pH 4.5). Three additional groups were treated with test solutions twice daily, but without demineralization. Tissue loss was determined profilometrically. Energy-dispersive X-ray spectroscopy was used to measure the tin content on and within three layers (10 mum each) beneath the surface. In addition, scanning electron microscopy was conducted. All test preparations significantly reduced tissue loss. Deposition of tin on surfaces was higher without erosion than with erosion, but no incorporation of tin into enamel was found without demineralization. Under erosive conditions, both highly concentrated solutions led to the incorporation of tin up to a depth of 20 mum; the less-concentrated solution led to small amounts of tin in the outer 10 mum. The efficacy of tin-containing solutions seems to depend mainly on the incorporation of tin into enamel.

Quantitative comparison of 3 enamel-stripping devices in vitro: how precisely can we strip teeth?

INTRODUCTION In this in-vitro study, we aimed to investigate the predictability of the expected amount of stripping using 3 common stripping devices on premolars. METHODS One hundred eighty extracted premolars were mounted and aligned in silicone. Tooth mobility was tested with Periotest (Medizintechnik Gulden, Modautal, Germany) (8.3 ± 2.8 units). The selected methods for interproximal enamel reduction were hand-pulled strips (Horico, Hapf Ringleb & Company, Berlin, Germany), oscillating segmental disks (O-drive-OD 30; KaVo Dental, Biberach, Germany), and motor-driven abrasive strips (Orthofile; SDC Switzerland, Lugano-Grancia, Switzerland). With each device, the operator intended to strip 0.1, 0.2, 0.3, or 0.4 mm on the mesial side of 15 teeth. The teeth were scanned before and after stripping with a 3-dimensional laser scanner. Superposition and measurement of stripped enamel on the most mesial point of the tooth were conducted with Viewbox software (dHal Software, Kifissia, Greece). The Wilcoxon signed rank test and the Kruskal-Wallis test were applied; statistical significance was set at alpha ≤ 0.05. RESULTS Large variations between the intended and the actual amounts of stripped enamel, and between stripping procedures, were observed. Significant differences were found at 0.1 mm of intended stripping (P ≤ 0.05) for the hand-pulled method and at 0.4 mm of intended stripping (P ≤ 0.001 to P = 0.05) for all methods. For all scenarios of enamel reduction, the actual amount of stripping was less than the predetermined and expected amount of stripping. The Kruskal-Wallis analysis showed no significant differences between the 3 methods. CONCLUSIONS There were variations in the stripped amounts of enamel, and the stripping technique did not appear to be a significant predictor of the actual amount of enamel reduction. In most cases, actual stripping was less than the intended amount of enamel reduction.

Enamel matrix derivative inhibits adipocyte differentiation of 3T3-L1 cells via activation of TGF-βRI kinase activity

Enamel matrix derivative (EMD), an extract of fetal porcine enamel, and TGF-β can both suppress adipogenic differentiation. However, there have been no studies that functionally link the role of EMD and TGF-β in vitro. Herein, we examined whether TGF-β signaling contributes to EMD-induced suppression of adipogenic differentiation. Adipogenesis was studied with 3T3-L1 preadipocytes in the presence of SB431542, an inhibitor of TGF-βRI kinase activity. SB431542 reversed the inhibitory effect of EMD on adipogenic differentiation, based on Oil Red O staining and mRNA expression of lipid regulated genes. SB431542 also reduced EMD-stimulated expression of connective tissue growth factor (CTGF), an autocrine inhibitor of adipogenic differentiation. Moreover, short interfering (si)RNAs for CTGF partially reversed the EMD-induced suppression of lipid regulated genes. We conclude that the TGF-βRI - CTGF axis is involved in the anti-adipogenic effects of EMD in vitro.

In vitro evaluation of demineralized freeze-dried bone allograft in combination with enamel matrix derivative

BACKGROUND Preclinical and clinical studies suggest that a combination of enamel matrix derivative (EMD) with demineralized freeze-dried bone allograft (DFDBA) may improve periodontal wound healing and regeneration. To date, no single study has characterized the effects of this combination on in vitro cell behavior. The aim of this study is to test the ability of EMD to adsorb to the surface of DFDBA particles and determine the effect of EMD coating on downstream cellular pathways such as adhesion, proliferation, and differentiation of primary human osteoblasts and periodontal ligament (PDL) cells. METHODS DFDBA particles were precoated with EMD or human blood and analyzed for protein adsorption patterns via scanning electron microscopy. Cell attachment and proliferation were quantified using a commercial assay. Cell differentiation was analyzed using real-time polymerase chain reaction for genes encoding Runx2, alkaline phosphatase, osteocalcin, and collagen 1α1, and mineralization was assessed using alizarinred staining. RESULTS Analysis of cell attachment revealed no significant differences among control, blood-coated, and EMD-coated DFDBA particles. EMD significantly increased cell proliferation at 3 and 5 days after seeding for both osteoblasts and PDL cells compared to control and blood-coated samples. Moreover, there were significantly higher messenger ribonucleic acid levels of osteogenic differentiation markers, including collagen 1α1, alkaline phosphatase, and osteocalcin, in osteoblasts and PDL cells cultured on EMD-coated DFDBA particles at 3, 7, and 14 days. CONCLUSION The results suggest that the addition of EMD to DFDBA particles may influence periodontal regeneration by stimulating PDL cell and osteoblast proliferation and differentiation.

Gene array of primary human osteoblasts exposed to enamel matrix derivative in combination with a natural bone mineral

OBJECTIVES The application of an enamel matrix derivative (EMD) for regenerative periodontal surgery has been shown to promote formation of new cementum, periodontal ligament, and alveolar bone. In intrabony defects with a complicated anatomy, the combination of EMD with various bone grafting materials has resulted in additional clinical improvements, but the initial cellular response of osteoblasts coming in contact with these particles have not yet been fully elucidated. The objective of the present study was to evaluate the in vitro effects of EMD combined with a natural bone mineral (NBM) on a wide variety of genes, cytokines, and transcription factors and extracellular matrix proteins on primary human osteoblasts. MATERIAL AND METHODS Primary human osteoblasts were seeded on NBM particles pre-coated with versus without EMD and analyzed for gene differences using a human osteogenesis gene super-array (Applied Biosystems). Osteoblast-related genes include those transcribed during bone mineralization, ossification, bone metabolism, cell growth and differentiation, as well as gene products representing extracellular matrix molecules, transcription factors, and cell adhesion molecules. RESULTS EMD promoted gene expression of various osteoblast differentiation markers including a number of collagen types and isoforms, SMAD intracellular proteins, osteopontin, cadherin, alkaline phosphatase, and bone sialoprotein. EMD also upregulated a variety of growth factors including bone morphogenetic proteins, vascular endothelial growth factors, insulin-like growth factor, transforming growth factor, and their associated receptor proteins. CONCLUSION The results from the present study demonstrate that EMD is capable of activating a wide variety of genes, growth factors, and cytokines when pre-coated onto NBM particles. CLINICAL RELEVANCE The described in vitro effects of EMD on human primary osteoblasts provide further biologic support for the clinical application of a combination of EMD with NBM particles in periodontal and oral regenerative surgery.

Influence of enamel matrix derivative on cells at different maturation stages of differentiation

Enamel matrix derivative (EMD), a porcine extract harvested from developing porcine teeth, has been shown to promote formation of new cementum, periodontal ligament and alveolar bone. Despite its widespread use, an incredibly large variability among in vitro studies has been observed. The aim of the present study was to determine the influence of EMD on cells at different maturation stages of osteoblast differentiation by testing 6 cell types to determine if cell phenotype plays a role in cell behaviour following treatment with EMD. Six cell types including MC3T3-E1 pre-osteoblasts, rat calvarial osteoblasts, human periodontal ligament (PDL) cells, ROS cells, MG63 cells and human alveolar osteoblasts were cultured in the presence or absence of EMD and proliferation rates were quantified by an MTS assay. Gene expression of collagen1(COL1), alkaline phosphate(ALP) and osteocalcin(OC) were investigated by real-time PCR. While EMD significantly increased cell proliferation of all cell types, its effect on osteoblast differentiation was more variable. EMD significantly up-regulated gene expression of COL1, ALP and OC in cells early in their differentiation process when compared to osteoblasts at later stages of maturation. Furthermore, the effect of cell passaging of primary human PDL cells (passage 2 to 15) was tested in response to treatment with EMD. EMD significantly increased cell proliferation and differentiation of cells at passages 2-5 however had completely lost their ability to respond to EMD by passages 10+. The results from the present study suggest that cell stimulation with EMD has a more pronounced effect on cells earlier in their differentiation process and may partially explain why treatment with EMD primarily favors regeneration of periodontal defects (where the periodontal ligament contains a higher number of undifferentiated progenitor cells) over regeneration of pure alveolar bone defects containing no periodontal ligament and a more limited number of osteoprogenitor cells.