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Year : 2022  |  Volume : 12  |  Issue : 1  |  Page : 67-75

Effect of two calcium-silicate sealers and a resin sealer on collagen matrix integrity of root dentin after different treatments. An in vitro and in vivo study

1 Department of Endodontics, Faculty of Dentistry, Suez Canal University, Ismailia, Egypt
2 Department of Surgery, Anesthesiology and Radiology, Faculty of Veterinary Medicine, Suez Canal University, Ismailia, Egypt

Date of Submission12-Mar-2021
Date of Acceptance14-May-2021
Date of Web Publication8-Jan-2022

Correspondence Address:
Dalia Mohamed Mukhtar Fayyad
Department of Endodontics, Faculty of Dentistry, Suez Canal University, Ismailia
Dalia Abd-Allah Mohamed
Faculty of Dentistry, Suez Canal University, Ismailia
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/sej.sej_57_21

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Introduction: This research aimed to examine and compare the effect of two calcium silicate sealers with AH plus resin sealer on collagen extraction and surface collagen degradation of dog's root dentin, using different irrigants.
Materials and Methods: In vitro part; 180 standardized root dentin cylinders obtained from dog's incisors were randomly allocated into three groups (n = 60) according to the final irrigant used; Group A: 17% Ethylenediaminetetraacetic acid, Group B: 2.5% NaOCl, Group C: 0.9% saline. Each group was classified into 4 subgroups (n = 15) according to the type of sealer used for obturation; subgroup 1: BioRoot RCS, subgroup 2: Total Fill BC, subgroup 3: AH Plus sealer, and subgroup 4: Unfilled (control). After preparation, irrigation, obturation, and aging in a storage medium; hydroxyproline (HYP) released in the medium was determined after 1 day, 1 and 3 months using spectrophotometer. In vivo part, sixty incisors of five dogs were randomly allocated as in the vitro part into three groups and four subgroups in accordance to the type of irrigation and sealer used. After 3 months of preparation, irrigation, and obturation, dogs were euthanized and teeth were extracted for the assessment of surface collagen degradation using scanning electron microscope. HYP released was analyzed using repeated-measures analysis of variance and post hoc of Bonferroni. Surface collagen degradation was analyzed using Kruskal–Wallis test and Dunn's test.
Results: BioRoot RCS and TotalFill BC showed a significant higher (P < 0.001) HYP release and surface collagen degradation than AH plus sealer and control with all irrigants.
Conclusions: Calcium silicate sealers significantly affected the collagen microstructure of dentin surface. AH Plus resin sealer has no effect on collagen dissolution or microstructure.

Keywords: Calcium silicate, collagen, dentin, hydroxyproline, scanning electron microscope

How to cite this article:
Ismail SM, Fayyad DM, Eldaharawy MH, Mohamed DA. Effect of two calcium-silicate sealers and a resin sealer on collagen matrix integrity of root dentin after different treatments. An in vitro and in vivo study. Saudi Endod J 2022;12:67-75

How to cite this URL:
Ismail SM, Fayyad DM, Eldaharawy MH, Mohamed DA. Effect of two calcium-silicate sealers and a resin sealer on collagen matrix integrity of root dentin after different treatments. An in vitro and in vivo study. Saudi Endod J [serial online] 2022 [cited 2022 Jan 25];12:67-75. Available from: https://www.saudiendodj.com/text.asp?2022/12/1/67/335247

  Introduction Top

Dentin is a biologic tissue composed of collagen matrix packed with calcium deficient, carbonate-rich apatite crystals nanometer in size.[1] Collagen Type I represents 90% of the organic matrix of dentin, while the remaining 10% is made up of noncollagenous proteins such as phosphoproteins and proteoglycans. Collagen fibers Type I constitute a three-dimensional network that makes up the matrix of dentin and responsible for dentin cohesiveness.[2] The organic matrix of dentin accounts for viscoelasticity, mechanical stability, tensile strength, and toughness of dentin.[3]

During endodontic treatment procedures, chemical substances used for chemomechanical preparation, intracanal medications, and root fillings may change the construction of dentin, mainly collagen. Damage to dentin collagen may result in alternation of dentin toughness and interfacial failure at the filling material/dentin interface which decrease the dentin mechanical strength and in turn decreases the clinical survival of root canal treated tooth.[3],[4] So that, considerations to dentinal tissue properties and assimilation of the effects of endodontic materials specially root canal sealers on dentin are important. Different calcium silicate-based sealers were recently inserted to the market.[5],[6] The potential advantages of these sealers are related to the biocompatible and bioactive di- and tricalcium silicate constituents. Furthermore, the chemical reaction at the dentin interface beside the micromechanical tag-like structures results in effective adhesion of sealer to root dentin.[7] This adhesion strengthens the root canal-treated teeth and thus reduces fracture risk.[3]

The discharge of calcium hydroxide (Ca (OH)2) from calcium silicate sealers as a result of hydration and the interaction with phosphates from dentinal fluids leads to deposition of calcium phosphate and/or calcium carbonate on the surface material[8] and formation of hydroxyapatite.[9] Furthermore, calcium silicate sealers form mineral infiltration zone at the interfacial dentin.[10] However, despite calcium silicate material's bioactivity, others[11],[12],[13] showed that, the high alkalinity of these materials may have detrimental effects to dentin collagen matrix. They degenerate the collagenous component of the interfacial dentin and raising their permeability by the breakdown of collagen fibrils intermolecular bonds.[10] Hence, they possess a negative impact on root mechanical properties.[11]

Multiple in vitro researchers studied the effect of different calcium silicate materials and irrigants on root dentin microstructure separately. In these studies, they used different teeth types, different techniques, and variable findings were found.[10],[12],[13],[14],[15],[16],[17],[18],[19] However, the mutual effect of different irrigants with calcium silicate sealers was not clearly studied, especially in vivo. Thus, this research was carried out both in vitro and in vivo. The aim was to analyze the biochemical effects of BioRoot RCS and TotalFill BC sealers in comparison to AH Plus resin sealer on the collagen matrix integrity of dog's root dentin after treatment with different irrigating solutions.

  Materials and Methods Top

Ethical approval was granted to utilize dog's incisors teeth by the Research Ethics Committee of the Faculty of Dentistry, Suez Canal University (61/2017). Dogs selected in the present study were already requested and scheduled for euthanasia due to veterinarian reasons (e.g.,; behavioral issues, life changing circumstances, convenience, and overpopulation).

Part one: Assessment of collagen extraction (in vitro study)

Sample size calculation

A repeated measures analysis of variance (ANOVA) was proposed. The difference between different time points (T1, T2, and T3) was assessed by repeated measures ANOVA. A minimum calculated total sample size of 175 samples was sufficient to detect the effect size of 0.146, a power (1− β) of 90% with a partial eta-squared of 0.021, and at a significant level of P < 0.05. A total sample size of 180 was applied, each irrigant Type (A, B, and C) was represented by sixty samples and each sealer subgroup (sealers: 1, 2, 3, and 4) was represented by 15 samples at different time points (T1, T2, and T3). G*Power software version (https://www.psychologie.hhu.de/arbeitsgruppen/allgemeine-psychologie-und-arbeitspsychologie/gpower), was used to calculate the sample size.[20],[21]

Preparation of root segment specimens

One hundred and eighty freshly extracted dog's anterior teeth were used to prepare 180 standardized root dentin cylinders. A slow-speed, water-cooled double-faced diamond disc (Brasseler, Savannah, Georgia, USA) was used to cut the teeth horizontally at the cementoenamel junction (CEJ) to remove their crowns. Another cut was done at 10 mm from the CEJ to produce a 10 mm length root segment from the coronal third of each root. Then, root canal content was removed using a ≠ 2 peeso reamer 0.7 mm diameter (Shenzhen Feihuan Medical Instruments Co., Ltd, Shenzhen City, China) to obtain standardized internal canal diameter. A symmetrical cylindrical dentin specimen of uniform wall thickness and 5 mm external diameter was prepared using saline cooled bone biopsy hole saw (Stryker Corp., Kalamazoo, MI, USA) at low speed 100 rpm.

Grouping and randomization of samples

Root dentin cylinders were randomly and blindly classified into three experimental groups (n = 60 each) according to the irrigation protocol that was used during and after preparation as follow:

  • Group A: Samples were irrigated with 6 mL 2.5% sodium hypochlorite (NaOCl) (Clorox, Egyptian company for house hold products, Egypt) during preparation and 3 mL 17% ethylenediaminetetraacetic acid (EDTA) (Maillefer, Dentsply, Ballaigues, Switzerland) as final irrigant
  • Group B: Samples were irrigated with 6 mL 2.5% NaOCl during preparation and 3 mL 2.5% NaOCl as final irrigant
  • Group C (control): Samples were irrigated with 6 mL 0.9% saline solution during preparation and 3 mL saline as final irrigant.

Samples in each group were then subdivided into four subgroups (n = 15) according to the type of sealer that was used for obturation as follow:

  • Subgroups 1: Root dentin cylinders were filled with BioRoot RCS (Septodont, Saint Maur-des-Fosses, France)
  • Subgroups 2: Root dentin cylinders were filled with Total Fill BC (FKG Dentaire, La-Chaux-de-Fonds, Switzerland)
  • Subgroups 3: Root dentin cylinders were filled with AH Plus sealer (Dentsply De Trey, Konstanz, Germany)
  • Subgroups 4: (control): Root dentin cylinders were left unfilled.


This study was double blinded by the operator and the assessor. Dentine cylinders were randomly assigned to be filled with either BioRoot RCS, TotalFill BC, AH plus sealer, or left unfilled. Masking tape concealed the contents of the obturating materials from the operator was kept with the allocator. After mechanical preparation and irrigation, the allocator mixed the sealer and then gave it to the operator as a ready mixed paste at the time of obturation. The operator did not know the type of sealer used. Computer software, (http://www.random.org) was used to generate the random sequence.

Filling and aging of dentin cylinders

After canal preparation with #2 pesso-reamer and irrigation with the corresponding irrigant to each group, dentin cylinders were gently dried using absorbent paper points # 35 (FKG Dentaire, La-Chaux-de-Fonds, Switzerland). These specimens were obturated using gutta-percha points # 35 and taper 0.02 (FKG Dentaire, La-Chaux-de-Fonds, Switzerland) and one of the tested sealers in single cone technique. A size #35 gutta-percha point was selected based on the internal diameter (0.7) of the dentin cylinders as prepared by the type and size of peso reamer. At the D16 (maximum taper at the CEJ), the diameter of gutta-percha is 0.32 + 0.35 = 0.67 mm. This matches the size of preparation and allows a room for sealer placement that is near to the clinical situation. Excess gutta-percha was removed from the apical part of the specimen using scalpel, and the specimens were placed on a sterilized glass slab to support sealers until initial setting. Samples were then saved in an incubator at 100% humidity and at 37°C for 24 h for complete sealer setting, as according to the manufacturers, this time was enough for all tested sealers setting.

Five samples from each subgroup were assigned for each designated aging time (1 day, 1 month, and 3 months) (5 samples × 12 subgroups × 3 aging times = 180 samples). All root dentin cylinders were stored in a 400 μL of storage medium at 37°C (Sigma-Aldrich Co. USA) in a polypropylene microcentrifuge tube. The storage medium was composed of 50 mmol/L HEPES buffer, 5 mmol/L CaCl2.2H2O, 0.001 mmol/L ZnCl2, 150 mmol/L NaCl, and 3 mmol/L NaN3 to prevent bacterial growth with pH 7.2.[22]

Evaluation of collagen extraction (hydroxyproline assay)

Assessment of the amount of hydroxyproline (HYP) released in the storage medium was performed using HYP assay to determine the solubilized collagen peptides from the dentin cylinders,[12],[22],[23] as following:

The pH values of the storage media at the end of each specified aging time were measured using a pH meter (Microprocessor Laboratory pH/mV/ORP Meter, Model BT-500, BOECO-Germany). Then, 200 μL of vortexed storage medium was gathered from each tube and placed in an individually labeled ampule. The HYP content was analyzed using a spectrophotometer (Tokyo, Japan) in the transmission mode at 558 nm. The HYP released from solubilized collagen peptide fragments (Sa) was calculated using regression equation derived from absorbance values obtained from known concentrations of HCl hydrolyzed HYP [Figure 1]. The hydrolyzed HYP amount was expressed as pg/mg of dehydrated mineralized dentin. The final concentration of HYP in each sample was obtained from the following equation;
Figure 1: Hydroxyproline standard curve (Spectrophotometric absorbance as a function of collagen concentration)

Click here to view

Sa/Sv = C

Sa = the amount of HYP, calculated from the standard curve (in μg)

Sv = the volume of sample hydrolysate added to the tube (in μL)

C = Concentration of HYP in sample.

Part two: assessment of surface collagen degradation (in vivo animal study)

Sample size calculation

A repeated measures ANOVA was proposed. A minimum calculated total sample size of sixty samples was sufficient to detect the effect size of 0.25, a power (1− β) of 85% with a partial eta-squared of 0.06, and at a significant level of P < 0.05. A total sample size of 60 was applied and each irrigant type (Irrigants: A, B, and C) was represented by twenty samples. Each sealer type subgroup (1, 2, 3, and 4) was represented by five samples for surface degradation analysis. G*Power software version was used to calculate the sample size.[20],[21]

Grouping and randomization of teeth

Sixty upper and lower incisors of five mongrel dogs were used. Teeth of each dog were randomly and blindly classified (as previously mentioned) into three groups according to the irrigation protocol that was used during and after preparation using 30G needles (n = 20) as follow:

  • Group A: Samples were rinsed with 6 mL 2.5% NaOCl during preparation and 3 mL 17% EDTA as final irrigant
  • Group B: Samples were rinsed with 6 mL 2.5% NaOCl during preparation and 3 mL 2.5% NaOCl as final irrigant
  • Group C (control): Samples were irrigated with 6 mL 0.9% saline solution during preparation and 3 mL saline as final irrigant.

Samples in every group were then subdivided into four subgroups (n = 5) (3 experimental and one control) in accordance to sealer type that was applied for obturation as follow:

  • Subgroups 1: Teeth were filled with BioRoot RCS
  • Subgroups 2: Teeth were filled with Total Fill BC sealer
  • Subgroups 3: Teeth were filled with AH Plus sealer
  • Subgroups 4: (control): Teeth were left without filling.

Preoperative considerations and anesthesia of the dogs

Food was withheld before treatment by 6–8 h. Fifteen minutes before induction of general anesthesia, each dog was premedicated with intramuscular injection of chloropromazine hydrochloride (Hikma Maple; Usl Pharma; Sandoz) in a dose of 1 mg/kg. General anesthesia was accomplished by intravenous injection of thiopental sodium (EPICO, Egypt) 2.5% solution until the main reflexes were abolished.[24]

Mechanical preparation of dog's teeth

Instrumentation of root canal was performed with I Race rotary files (FKG Dentaire, La-Chaux-de-Fonds, Switzerland) and finishing up using ≠ 35 file 4%.[25] Each root canal was irrigated with its corresponding irrigation protocol using 30G needles and then flushed with sterile distilled water to remove any residual of the irrigant. Absorbent paper points # 35 were used for gentle dryness of each root canal.

Thereafter, each root canal was obturated using size 35 gutta-percha points 4% and the corresponding sealer according to its group using single cone technique. The adequacy of the root canal filling was checked by periapical radiograph. A glass ionomer, Fuji® IX GP FAST (Fuji® IX, GC Corporation, USA.), was used as a coronal seal for each tooth. The dogs were euthanized after 3 months using an intravenous barbiturate overdose of 6% pentobarbital dose (120 mg/kg).[25] The incisors were extracted, decoronated, and placed in capped test tubes with saline to avoid dehydration of samples and properly labeled.

Evaluation of surface collagen degradation

Each root was sectioned longitudinally into two halves using IsoMet 4000 slow-speed micro saw (Buehler, Ltd, Lake Bluff, IL, USA) under water cooling and the half with no defect was selected for scanning. The gutta-percha and sealer were removed gently from the root dentin using an ultrasonic scaler tip (Satelec, Acteon, France) and copious water cooling. The samples were fixed by glutaraldehyde 2.5% and dehydrated through ascending series of ethanol. The processed specimens were gold sputter coated in a vacuum evaporator,[26] and the collagen matrix architecture was tested by the aid of scanning electron microscope (SEM) (JSM-5500 LV; JEOL Ltd-Japan) operated at 15 kV. Five images were obtained for each root at 3, 6, 9, 11, and13 mm from the CEJ.

The SEM images were transferred to a computer and assessed by three blinded examiners for determining the degree of surface collagen degradation according to the following criteria;[27] Score 0: Nonaltered collagen fibrils, Score 1: Slight collagen fibrils degradation, Score 2: Moderate degradation of collagen matrix, and Score 3: Severe collagen degradation. Three values were assigned for each image, and the average was calculated resulting in a single value per image and five values per subgroup.

Statistical analysis

Analyzing the data for normality was through measuring the distribution of data and applying normality tests (Kolmogorov-Smirnov and Shapiro–Wilk tests). HYP parametric data were provided as mean ± standard deviation values. Repeated ANOVA test was applied to investigate the impact of sealer type, irrigant, time, and their interaction on mean HYP concentration release. When ANOVA test is significant, post hoc test of Bonferroni was applied for pair-wise comparisons. Collagen degradation scores were nonparametric data presented as median and range values. To compare between different sealer types and irrigants, Kruskal–Wallis test was applied. For pair-wise comparisons between the groups, Duncan's test was applied. The level of significance was adjusted at P ≤ 0.05. IBM SPSS Statistics for Windows version 23.0., Armonk, NY, USA: IBM Corp, was applied for statistical analysis.

  Results Top

Collagen extraction

The pH values of the storage media after 3 months aging were (10.13 ± 0.08) for BioRoot RCS, (9.8 ± 0.06) for TotalFill BC, (7.35 ± 0.07) for AH Plus, and (7 ± 0.09) for control. Both calcium silicate sealers (P < 0.001), irrigants (P < 0.001), and storage period (P < 0.001) significantly affected the HYP amount extracted from dentin [Figure 2]. Furthermore, the three factors interaction was also statistically significant (P < 0.001). Both BioRoot RCS and TotalFill BC showed the highest statistically significant HYP released values than AH Plus and control with the three irrigants and at the three aging times [Figure 2]. BioRoot RCS and TotalFill BC showed a significant increase in HYP release from day 1 to 1 month and from 1 to 3 months (P < 0.001) [Figure 3]. AH Plus and control showed no statistically significant increase in HYP release. Both 17% EDTA and 2.5% NaOCl showed the highest statistically significant HYP release than saline (P < 0.001) [Figure 4].
Figure 2: Bar chart representing mean and standard deviation values for the effect of the three sealers on released hydroxyproline concentration after irrigation with the three irrigants and at the three aging times

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Figure 3: Bar chart representing mean and standard deviation values for the effect of time on released Hydroxyproline concentration after irrigation with the three irrigants and obturation with three sealers

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Figure 4: Bar chart representing mean and standard deviation values for the effect of tested irrigants on released Hydroxyproline concentration after obturation with the tested sealers at the three aging times

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Collagen degradation

Severe-to-moderate collagen degradation zone was observed in dentin surface specimens in contact with BioRoot RCS and TotalFill BC after 3 months of aging. While, minimal collagen degradation could be noticed in dentin surfaces specimens in contact to AH plus and there was no collagen degradation in control group. Both 17% EDTA and 2.5% NaOCl showed the highest statistically significant median surface collagen degradation scores than saline (P < 0.05) [Table 1] and [Figure 5], [Figure 6], [Figure 7], [Figure 8].
Table 1: The median, range values and results of Kruskal-Wallis test for comparison between collagen degradation scores of different sealers.

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Figure 5: Scanning electron microscope micrographs at ×10,000 magnification represent root dentin surfaces of control Group (a); specimen treated with ethylenediaminetetraacetic acid showing intact three-dimensional network of collagen fibrils on demineralized dentin surface (Score 0). (b); specimen treated with NaOCl showing smear layer covering the mineralized dentin surface with no exposed collagen fibrils (Score 0). (c); specimen treated with saline showing uniform collagen network on the intertubular and intratubular dentin surface (Score 0)

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Figure 6: Scanning electron microscope micrographs at ×10,000 magnification represent root dentin surfaces; (a1) specimen treated with ethylenediaminetetraacetic acid + BioRoot rich communication services showing severe loss of collagen matrix (arrow), exposing the underlying mineralized dentin (Score 3). (b1) specimen treated with ethylenediaminetetraacetic acid + TotalFill showing moderate loss of surface collagen fibrillar network (Score 2). Degraded collagen fibrils (arrows). (c1) specimen treated with ethylenediaminetetraacetic acid + AH plus showing slight collagen network loss of integrity (star) (Score 1)

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Figure 7: Scanning electron microscope micrographs at ×10,000 magnification represent root dentin surfaces: specimens treated with NaOCl + BioRoot rich communication services (a2) and NaOCl + TotalFill (b2) showing moderate loss of collagen fibers from root dentin surface (Score 2). Specimen treated with NaOCl + AH plus (c2) showing mild surface collagen degradation (Score 1). Arrows (degraded collagen fibrils)

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Figure 8: Scanning electron microscope micrographs at ×10,000 magnification represent root dentin surfaces: specimens treated with saline + BioRoot rich communication services (a3), saline + TotalFill (b3), and saline + AH plus (c3) showing no loss of surface collagen fibrillar network from the root canal surface Score (0). Bacteria-like structure (arrow)

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  Discussion Top

In this study, we found a consequent increase in HYP release as the contact time of calcium silicate sealers (BioRoot RCS and TotalFill BC) with dentin increased. This indicated that the high alkalinity induced dentin collagen degradation is a time dependent. Similarly, many studies reported that prolonged contact of mineralized dentin with materials based on calcium silicate adversely affect the dentin collagen matrix integrity.[12],[13],[28] Leiendecker et al.[12] showed the same finding when they measured the HYP extracted over time from mineralized dentin that was placed in direct contact with MTA Plus and Biodentine. Furthermore, Huang et al.[13] found that the molecular conformation of collagen polypeptide chains gradually disappeared in the transmission electron microscopic images of dentin surfaces, as the contact time of ProRoot® MTA cement increased.

This was attributed to the ability of calcium silicate-based materials to release Ca (OH)2 up on set which creates highly alkaline environment, consequently induces a caustic degradation impact on exposed collagen by the breakdown of fibrillar collagen intermolecular bonds, enhancing their water absorption and swelling.[12],[28] This was confirmed by Huang et al.[13] who found small-sized tricalcium silicate-derived hydroxyl ions penetrated the intrafibrillary compartment of mineralized collagen and continuously degrade the collagen fibrils without altering the surface degradation zone thickness. Moreover, there was a time-dependent disintegration of the organic components from Ca (OH)2-treated dentin surfaces when analyzed using Attenuated total reflection-Fourier transform-infrared spectroscopy in their study. Furthermore, using the enzyme-linked immunosorbent assays, Cerebrotendinous xanthomatosis (a peptide fragment of Type I collagen) was found to be extracted from dentin exposed to Ca (OH)2 for 3 months.[13]

In addition, the high alkalinity of calcium silicates could induce a caustic denaturation and increase the permeability of the of interfacial dentin organic collagen component. However, because of its lower collagen content, this alkaline caustic effect has virtually no effect on the highly mineralized peritubular dentin.[6],[13] On the other hand, AH plus had a neutral pH 7.35 that did not affect the collagen integrity, but the open epoxide rings form covalent bonds to any exposed amino groups in collagen network.[29],[30]

Scanning electron microscopic findings of this study were in accordance with Atmeh et al.[10] who found band of structurally altered dentin immediately beneath the tricalcium silicate cement (Biodentine) when seen by SEM. Moreover, Leiendecker et al.[12] and Huang et al.[13] found collagen degradation in dentin surface directly in contact with calcium silicate materials when seen by transmission electron microscope.

In the present study, saline was used in the control group for irrigation to analyze the effect of the tested sealers individually on dentin, while NaOCl and combination of NaOCl and EDTA irrigations were used in the other two groups to simulate the most commonly used clinical irrigation protocols. The increase in HYP release and the high collagen degradation scores in specimens finally irrigated with EDTA or NaOCl than with saline might be inferred to the breakdown of NaOCl into chloramines and protein-derived radical intermediates. These broken-down products have adverse effect on crosslinks of Type I collagen, thus irrigation with NaOCl results in structurally compromised collagen in root dentin.[14],[31] In addition, NaOCl dissolves organic matter and induces the degradation of dentinal collagen through the breakdown of carbon atom bonds and disorganization of protein structure, resulting in dentin degeneration and alternation of dentin mechanical properties.[15],[32],[33]

Moreover, NaOCl has small size (molecular weight, 74.4 Da) enables it to permeate the mineralized collage intrafibrillar milieu and change the 3-dimensional conformation of tropocollagen.[16] However, further penetration of NaOCl into dentin and collagen degradation is limited by the insoluble surface hydroxyapatite crystals that reprecipitate on dentin surface and act as a barrier to deeper NaOCl penetration.[17] On the other hand, the higher release of collagen by EDTA as a final irrigant might be explained by that pretreatment with NaOCl irrigation during canal preparation enhances the EDTA final rinse to reach deeper into dentin. EDTA as an inorganic chelator results in forming stable complexes with the smear layer calcium content and removes them.[34] EDTA has a powerful demineralizing effect, induces dentinal tubule enlargement, dentin softening, and increases the denaturation of collagen fibers. It has the ability to cause excessive dentinal erosion.[35]

The finding of the current study was in adherence with Carrilho et al.[22] and Gandolfi et al.[18] who showed that the demineralization by EDTA resulted in conformational rearrangement of the collagen network and solubilization of collagen with decrease in the modulus of elasticity (E). Similarly, Ramírez–Bommer et al.[17] found that NaOCl/EDTA treatment resulted in a more degraded dentinal collagen surface in comparison to the NaOCl treated surface. Moreover, Moreira et al.[19] reported that, 5.25% NaOCl, whether correlated or not with 17% EDTA, induces dentin collagen alternations, while 17% EDTA indicated structural loss regions. Dentin loss appeared as a decrease in intertubular dentin and a consequent enlargement of the dentin canaliculi caused by the EDTA's demineralizing property. Furthermore, Calt et al.[36] stated that EDTA followed by NaOCl has the capacity to dissolve the peritubular and intertubular dentin.

While calcium silicate-based materials create a highly alkaline environment, which causes dissolution of exposed collagen, mineralized dentin is an excellent buffer. In the apatite crystallites, phosphate and carbonate ions work together to prevent progressive penetration of the hydroxyl ions into the bulk of the mineralized dentin, so the degradation of the collagenous component of mineralized dentin was stated as a self-limiting surface phenomenon.[13],[16]

Interestingly, it was reported that only 1 μm surface collagen degradation could reduce the flexural strength of the bulk mineralized dentin extensively.[13] Furthermore, it is known that the bond strength of the filling material influences the fracture resistance of the tooth.[3],[26],[29] Hence, the limitations of our study are that, the depth of collagen degradation and the change in dentin apatite/collagen ratio in response to, calcium silicate sealers contact were not measured. Furthermore, the effect of the resulted collagen degradation on dentin bonding with the tested sealers and tooth fracture resistance was not in the scope of our research. Accordingly, further studies are required to cover these limitations and to correlate between the long-term use of calcium silicate sealers and the increased risk of root fracture, especially in the presence of the cushion effect of the periodontal ligaments against the intraoral forces. Till sufficient research is conducted, it is recommended to take care clinically, when placing calcium silicate sealers in roots with thin dentinal walls as in cases with immature roots or root resorptive defects.

  Conclusions Top

Within the limitations of the present study, it can be concluded that calcium silicate sealers and root canal irrigants dissolve collagen matrix and adversely affect the collagen microstructure of dentin surface in contact with them.

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Conflicts of interest

There are no conflicts of interest.

  References Top

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]

  [Table 1]


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