Effect of an enamel matrix derivative (Emdogain) on the microhardness and chemical composition of human root dentin: an in vitro study

The advantage of using an Enamel matrix derivative EMD Emdogain as an intracanal medication could be a manner to strength the tooth structure, improving the physical and chemical properties of dentin. We tested, in vitro, the effect of Emdogain on the surface microhardness and chemical composition of root dentin. Ten human teeth were used to produce dentin specimens originated from the canal walls (n = 30) that remained in contact to Emdogain gel for 90 days. Baseline and 90-days after Emdogain treatment measurements were performed using Fourier Transform Infrared Spectroscopy (ATR/FTIR), Scanning Electron Microscopy/Energy Dispersive Spectroscopy (SEM/EDS) and Knoop indenters. The use of EMD (Emdogain) for 90 days in contact with human root canal dentin specimens did not alter the microhardness and morphology of dentin. The elemental structure of dentin was altered because there was a reduction in carbonate content.

medication 22 . Although EMD Emdogain has been investigated as a potential substance for use in endodontics, producing good results in relation to its clinical indications, the information on its influence in dentinal properties has not been addressed.
When necrosis and apical periodontitis occur in an immature tooth, the tooth structure may become weaken, because of the incomplete apposition of dentin in the root canal walls and incomplete root development. Regenerative procedures to induce pulp revascularization is one of the options to treat these type of cases-and the use of EMD as an intracanal substance could be a manner to strength the tooth structure, improving the physical and chemical properties of dentin. Therefore, this in vitro investigation aimed to test if the EMD-gel (Emdogain) when used for 90 days had influence on mechanical or the surface chemical composition of dentin. The null hypotheses tested were: (1) EMD (Emdogain) would not alter the root dentin Knoop microhardness or morphology; and (2) EMD (Emdogain) would not affect the calcium and phosphorus content of root dentin or the ratios of mineral/matrix, carbonate/mineral, amide I/amide III and amide I/CH2.

Material and methods
Enamel matrix derivative EMD (Emdogain) was obtained from the manufacturer (Institut Straumann AG, Basel Switzerland). For this experiment approximately 6 gel-syringes of the product were needed. Ten human teeth, extracted for therapeutic reasons, were used to produce dentin specimens. Informed consent was obtained from all participants in this study. This project was approved by the local Research Ethics Committee (approval number: CAAE 15975419.1.0000.5084).
Inclusion criteria for teeth comprised complete root formation, no radiographic signals of calcification/mineralization (nor diffuse neither localized), no evidence of internal resorption, and absence of previous endodontic initiated therapy or root canal obturation.
Specimens' preparation. Ten teeth were cleaned and maintained in distilled water for a maximum of 6 months after extraction. Firstly, the crown was separated from the roots using a diamond disc attached to a cutting machine (Isomet 1000 Precision Saw Buehler). The tooth crown was discarded. Secondly, one crosssectional slice was acquired from each tooth root (from the coronal region of the root). The cross-sectional slice was, then, split (longitudinally) to produce 3 squared specimens-where the internal surface of each specimen (dentin from the canal walls) was analysed. This means that, 10 teeth originated 3 dentin specimens (n = 30). Specimens measured 3 mm diameter × 3 mm height (Fig. 1).
For SEM/EDS analysis and microhardness test, the specimens were built-in synthetic plastic polymer (polyvinyl chloride tubes) using acrylic resin (TDV, Pomerode, SC, Brasil) for fixation of the disk base. The disk dentin surfaces were polished using silicon carbide sandpaper in decreasing grains (#400, #600 and #1200). The internal surface of each dentin (the surface to be analyzed) specimen was polished with felt cloths soaked in diamond paste attached to a slow-speed handpiece (Diamond, FGM, Joinville, SC, Brazil). Then, specimens were washed in an ultrasonic vat with distilled water for 30 min. After taking the baseline measures, the specimens were stored in an oven at 37 °C, under humidity (dentin specimens remained in well plates covered with 2 × 2 gauze moistened with distilled water).
Enamel matrix derivative (EMD) (Emdogain) treatment. EMD (Emdogain) was injected directly from the manufacturer syringes into petri dishes. The dentin specimens were then set down over the EMD, having one of the disk surfaces (the surface to be analyzed) immersed into the substance. Specimens remained for 90 days into an incubator at 37 °C and 100% humidity. After EMD treatment, the specimens were again washed in an ultrasonic vat with distilled water for 30 min, and new measurements for the tests (FTIR, SEM/EDS and MH) were obtained. www.nature.com/scientificreports/ Chemical composition with fourier transform infrared spectroscopy (FTIR). Chemical composition of the samples (n = 10) was determined using attenuated total reflectance/Fourier transform infrared spectroscopy (ATR/FTIR; Vertex 70, Bruker, Ettlingen, Germany). Evaluations were made before and 90-days after EMD (Emdogain) treatment. The dentin surfaces (without being included in acrylic blocks) were positioned against the diamond crystal of the ATR/FTIR unit. Spectra were recorded in the range from 400 to 4000 cm −1 at 4 cm −1 of resolution. Each specimen was scanned 32 times in each FTIR measurement, and the final spectrum acquired was the average of all these scans. Spectra were recorded and analyzed by OPUS 6.5 software (Bruker, Ettlingen, Germany). After baseline correction and normalization, the area under each band was integrated by using the appropriate tools from the software. Each spectrum was normalized according to the phosphate band (1190-702 cm −1 ). FTIR spectra were analyzed by calculating the following parameters: (1) mineral/matrix ratio M:M (the ratio of the integrated areas of phosphate v1, v3 stretching mode at 1,035 cm −1 to the collagen amide I at 1.655 cm −1 ); (2) carbonate/mineral ratio C:M (the ratio of the integrated areas of carbonate v2 at 872 cm −1 to the phosphate v1, v3 at 1,035 cm −1) ; (3) amide I/amide III ratio (the ratio of the integrated areas of amide I at 1655 cm −1 to the amide III at 1235 cm −1 ); (4) amide I/CH 2 ratio (the band ratio of the integrated areas of amide I at 1,655 cm −1 to the CH 2 scissoring at 1450 cm −1 ) 23,24 .
Surface morphology and element analysis with scanning electron microscopy/energy dispersive spectroscopy (SEM/EDS). Images and spectra of the dentinal surfaces were obtained before and 90-days after the treatment with EMD (Emdogain) on a tabletop Scanning Electron Microscope (SEM) (TM3030, Hitachi, Tokyo, Japan). Prior to the analysis, specimens were ultrasonically cleaned and fixed in a metallic stub using a double-sided carbon tape. Samples were evaluated at 2500 × magnification in backscattered electron mode, having as reference point the central region of the sample. Subsequently, the EDS spectra were collected from the same dentinal surfaces to identify calcium and phosphorus elements, similarly to SEM images.

Knoop microhardness (KMH).
Baseline microhardness readings were obtained from the specimen surface before the treatment with EMD (Emdogain). Dentin microhardness was measured with a Knoop indenter at 40 × magnification (Shimadzu HMV-2000; Shimadzu Corporation, Kyoto, Japan), with a load of 10 g for 15 s. The average length of the two diagonals produced by the indenter was used to calculate the KMH value. Four indentations were made in each specimen at 20 µm far away from the root canal lumen. The representative microhardness value for each sample was the average result of the four indentations. Ninety days after EMD (Emdogain) treatment, the specimens were washed in an ultrasonic vat with distilled water for 30 min, and new microhardness measurements were obtained identically as described above.
Statistical analysis. Data

Results
Chemical composition (FTIR). The mean and standard deviation values for chemical parameters and ratios obtained by FTIR are shown in Table 1. After EMD (Emdogain) treatment, the samples showed a significant decrease in the values of carbonate (p = 0.001) and amide III (p = 0.002). The C:M ratios decreased in the samples after EMD (Emdogain) treatment (p < 0.001), while the amide I/amide III increased. Figure 2A shows the FTIR spectra of EMD (Emdogain). The EMD (Emdogain) spectrum presented three more evident bands observed at ∼ 575, 1,637 and 3,340 cm -1 wavenumbers. The peak at 3340 cm −1 is attributed to O-H vibrations in adsorbed water or as hydroxyl group, while the peak at 1637 cm −1 is dominated by the C=O stretch vibrations of the peptide linkages present in amide I 19,20 . The peak at 575 cm -1 is associated with phosphate v4 (PO4 3− ) 18,21 . Figure 2B shows the dentin spectra before and after EMD treatment. The peak at 1035 cm −1 is attributed to phosphate v1, v3 and the peak at 872 cm −1 to carbonate v2. The peak at 1655 cm −1 is associated with amide I, while the peak at 1,235 cm −1 is associated with amide III. The peak at 1450 cm −1 is attributed to the CH 2 scissoring 17,18 .

Surface morphology and element analysis (SEM/EDS).
Representative SEM/EDS images of specimens before (baseline) and 90-days after treatment with EMD (Emdogain) are also illustrated in Fig. 2. The SEM images showed that the EMD (Emdogain) treatment did not change the surface morphology. After 90 days, the EDS analysis showed a slight decrease in the intensity of the calcium (Ca) and phosphorus (P) peaks of the root dentin.

Discussion
The enamel matrix derivative (EMD) (Emdogain) has been indicated for promoting regeneration of dental tissues 9,10 . Emdogain is a composed mainly of amelogenin and amelin, proteins that play an important role in dentinogenesis and promote an increase in the level of mineralization markers in odontoblasts 6 . This class of proteins is known to induce the growth and proliferation of cells of the periodontal ligament, which has propylene glycol alginate (PGA) as a vehicle with an important antibacterial action [27][28][29] .
The literature has shown that EMD induced formation of mineralized tissue on root canal walls, by using available minerals from the dentinal tissue; this way, contributing to the root development and supporting the periapical healing 6,9,10 . In 2012, the efficacy of EMD was compared with a triple antibiotic paste (TAP) as an intracanal drug for regeneration of immature teeth in rats with pulp necrosis. EMD and TAP were able to reduce periapical lesion size loss and increase root length and thickness. EMD promoted narrower canals compared to TAP, a positive finding that could strengthen the tooth. Another experiment proved that EMD, when used in pulpotomy therapy, induced the formation of substantial amount of dentin-like tissue. The beginning of hard tissue formation was radiographically observed 2 weeks post-operative and it was located only in the affected analyzed by FTIR before and 90-days after EMD (Emdogain) treatment. *Indicates differences in root dentin chemical components (in rows) obtained by paired t-test (p < 0.05). **Indicates differences in root dentin ratios (in rows) obtained by paired t-test (p < 0.05).  www.nature.com/scientificreports/ pulp region. In comparison, the authors showed that dentin-like tissue was also formed in teeth treated with Dycal, but in limited amount, and at the expense of the whole width of the pulp chamber floor, narrowing of the root canals entrance. The total amount of dentin formed in the teeth treated with EMD was significantly higher than in the samples treated with Dycal 21 .

Before EDM treatment (baseline) After EDM treatment p-value
The findings of this present study added information to the body of knowledge on the benefits of using EMD (Emdogain) for 90 days in contact with root canal dentin. We showed that EMD (Emdogain gel) did not alter either microhardness of human dentin or its morphology, accepting the 1st null hypothesis.
The FTIR analysis is a reliable way to generate evidence about the presence of functional groups present in the structure of a sample, which can be used to identify a compound or to investigate its chemical composition 25 . Since EMD (Emdogain) is a mixture of hydrophobic enamel matrix proteins [derived from 6-month-old porcine tooth buds] containing amelogenin, enamelin, tuftelin, amelin, and ameloblastin, in a propylene glycol alginate (PGA) 30 its proteins guide tissue regeneration and induce remineralization of enamel and dentin 31,32 .
This current study observed a reduction in dentin carbonate values after EMD (Emdogain) treatment, which also impacted the C:M ratio. This ratio indicates the extent of carbonate incorporation in the hydroxyapatite lattice 33 . As carbonate is responsible for the acidic solubility of dental hard tissues, the reduction in carbonate content is related to the increased resistance to demineralization 34 , which occurred after EMD (Emdogain) treatment. These findings rejected the 2nd null hypothesis.
Amelogenins are responsible for regulating the mineralization process and for organizing the apatite crystals into juxtaposed prisms (Moradian-Oldak, 2001). The amelogenin protein molecule is divided into three amino acid domains, which are: central domain, C-terminus (COOH) and N-terminus (NH2) 35 . Both terminus types play key roles in proteolytic processes 36 and can interact with chemical components of the dental tissue, altering them quantitatively or changing their molecular conformation. Filamentary structure in amelogenin may induce ionic interactions, through acidic residues present in the C-terminal domain, for example 36 . This can result in modifications to the amide bands of the FTIR spectrum. In our study, these alterations found in the organic portion of dentin are represented by changes in amide III values. It is one of the amides present in the collagen structure; however, it is a very unstable and complex band depending on the details of the force field, the nature of the side chains and hydrogen bonding 24,37 . The reduction in amide III values may mean a disorganization in the secondary structure of the collagen fiber-forming protein unit 38,39 . Amide I/amide III ratio has also been altered and this represents a change in organization of collagen within the samples after Emdogain treatment 40,41 . Since collagen is the most abundant protein in dentin, its proteolysis has a significant impact on the structural integrity of this tissue, which can become mechanically and functionally compromised 23,24 . However, the chemical modifications did not repercuss in significative changes in dentin microhardness and surface morphology.
A limitation of this present study includes the assumption that the same results would be obtained in a clinical study-a challenge for any in vitro experiment. Nevertheless, we showed that EMD (Emdogain) is a potential substance to use intracanal-not interfering in dentin microhardness and contributing to increase resistance to demineralization. One of the strengthens of this study is the initial understanding of what occurs in the macro and microstructure of EMD treated dentin [the first time that it is being studied in the endodontic literature].
The findings of this current study may prompt further studies such as: tooth esthetic/color analysis when EMD is used into the root canal, analysis of other physicochemical-biological dentin properties, etc.

Conclusion
The use of EMD (Emdogain) for 90 days in contact with human root canal dentin specimens did not alter the microhardness and morphology of dentin. The elemental structure of dentin was altered because there was a reduction in carbonate content.

Data availability
The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.