Highlights
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The effects of a novel continuous chelation (NaOCl/HEDP) protocol on dentin were characterized using several approaches.
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The conventional NaOCl/EDTA protocol exposed bundles of “naked” collagen fibrils on the surface.
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NaOCl/HEDP resulted in minimal alterations to the surface and the subsurface matrix.
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These findings profoundly contribute to structural failures of dentin and/or dentin-biomaterial interfacial failures.
Abstract
Objective
Proteolytic and demineralizing agents have a profound influence on the dentin ultrastructure, which plays a key role in the mechanical integrity of the tooth and integrity of dentin-biomaterial interfaces. In-depth characterization of dentin treated with a novel root canal irrigation protocol comprising sodium hypochlorite (NaOCl) and etidronate (HEDP) is lacking. This study comprehensively characterized and compared the effects of the continuous chelation (NaOCl/HEDP) and sequential chelation (NaOCl/EDTA) protocols on dentin.
Methods
Dentin blocks, dentin powder and root canals of mandibular premolars were distributed into Group 1, Saline (control); Group 2, NaOCl/EDTA; and Group 3, NaOCl/HEDP. Ultrastructural characteristics of the treated dentin were investigated using electron microscopy and light microscopy, while the surface roughness was analyzed using atomic force microscopy. Chemical compositional changes were characterized using Fourier transform infrared spectroscopy (FTIR) and energy-dispersive-X-ray spectroscopy (EDS), while collagen degradation was determined using ninhydrin assay. Data were statistically analyzed using multiple-factor one-way ANOVA and Tukey HSD tests (P = 0.05).
Results
NaOCl/HEDP resulted in partially degraded, yet mineralized collagen fibers, with minimal alteration to the subsurface matrix. Conversely, NaOCl/EDTA dissolved the hydroxyapaptite encapsulation, exposing collagen fibre bundles. There was no significant difference in the surface roughness between the two protocols (P > 0.05). NaOCl/HEDP resulted in homogenous distribution of organic and inorganic components on the treated surface.
Significance
This study highlighted that continuous chelation (NaOCl/HEDP) resulted in a frail surface collagen layer while sequential chelation (NaOCl/EDTA) exposed bare collagen fibres. These surface and sub-surface effects potentially contribute to structural failures of dentin and/or dentin-biomaterial interfacial failures.
1
Introduction
Dentin is a biological composite containing a structured arrangement of collagen fibres embedded in a calcified matrix [ ]. Unlike bone, it does not have an ability to remodel, once it is fully mineralized [ ]. This highly organized tissue is exposed to a variety of proteolytic and demineralizing chemicals during endodontic treatment procedures. Sodium hypochlorite (NaOCl), a non-specific proteolytic agent is used in a range of concentrations (0.5–6%), for its remarkable tissue solvent action, microbicidal and anti-biofilm effects [ , ]. The chlorine compound undergoes saponification reaction with the organic matrix to form fatty salts. It neutralizes amino acids and releases oxidant chlorine, which inhibits the bacterial enzymes [ ]. NaOCl causes concentration and time-dependent collagen destruction, resulting in a mineral-rich, collagen-sparse ghost mineral layer [ ]. Since NaOCl is unable to remove the inorganic tissue remnants (smear layer), it is followed by a chelating agent such as ethylenediaminetetraacetic acid (EDTA), in a concentration of 15–17% for 1−2 min [ , ].
The sequential chelation protocol (NaOCl/EDTA), which is the most commonly used irrigation protocol in root canal treatment, results in widening of the dentinal tubular opening [ ] and intertubular tunnelling due to dentin erosion [ ]. NaOCl/EDTA results in complete decalcification of the superficial 1−5 μm of intertubular dentin, and up to 20 μm in the dentinal tubular walls [ ]. The above-mentioned microscopic and compositional changes significantly decrease the flexural strength of dentin [ ], potentially contributing to vertical root fractures [ ]. The effects of NaOCl alone [ ] and NaOCl/EDTA [ ] on dentin have been comprehensively characterized by others. Using microscopic and spectroscopic investigations, it has been shown that NaOCl removes the superficial subsurface organic phase from mineralized dentin in a concentration and time-dependent manner. The resultant dentin substrate is more brittle than untreated dentin [ , ]. EDTA then removes the mineral (apatite), which is observed as erosion of the dentin [ , ].
A novel root canal irrigation protocol, continuous chelation, was introduced to counteract the problems associated with the use of NaOCl/EDTA [ , ]. Here, NaOCl is mixed with the salt of a weak chelator, 1 hydroxyethylidene-1, 1-bisphosphonate or etidronate (HEDP), since the tetrasodium HEDP salt is highly compatible with NaOCl [ ]. This non-nitrogenous bisphosphonate has been used extensively in the fields of food disinfection, as well as in cleansers such as soaps and dishwasher tablets [ ]. A substantial body of work [ , , , ] has demonstrated the effects of this regimen on dentin surface effects, smear layer and microbiota. NaOCl/HEDP prevents smear layer formation without significant demineralization of the dentin substrate [ , ], removes biofilms [ ], and improves the adhesion of root canal fillings [ , ], significantly better than NaOCl/EDTA.
Biomechanical studies of teeth highlight the key role played by the collagen microstructure, mineral content and water in the mechanical integrity of dentin. Specifically, collagen and water contribute to the fracture toughness of dentin [ , ]. The main component of the dentin organic matrix is Type 1 collagen, a strong three-dimensional fibrous polymer [ ], which is responsible for its structural integrity [ ]. The inorganic component is intrafibrillar and interfibrillar carbonated hydroxyapatite [ ]. Thus, any procedure that undermines the dentin matrix and alters its fibrillary arrangement, results in a significant reduction in the flexural strength, potentially resulting in dentin fracture [ , ]. Therefore, ultrastructural characteristics of the dentin play a key role in the mechanical integrity of the tooth and the integrity of dentin-biomaterial interfaces [ ]. Taken together, studies on the characterization of dentin should comprehensively analyze both the organic and inorganic contents. Compositional and surface changes have been reported following treatment of dentin with different irrigation protocols [ , , ]. However, the ideal root canal irrigation protocol remains a matter of conjecture. To make clinical recommendations that will strategically minimize chemically-induced damage to the dentin, detailed investigations of irrigation protocols are necessary. Yet, the dentin composition and ultrastructural effects of NaOCl/HEDP on dentin have never been characterized in detail. Therefore, the aim of this study was to investigate the effects of the continuous chelation protocol (NaOCl/HEDP) on the ultrastructural, matrix characteristics and the chemical composition of dentin, and compare it with sequential chelation protocol (NaOCl/EDTA) and saline-treated dentin. The null hypothesis was that there was no significant difference between NaOCl/HEDP, NaOCl/EDTA and saline on dentin characteristics and composition.
2
Materials and methods
2.1
Chemicals and reagents
All chemicals and reagents were of reagent grade and purchased from Sigma Aldrich (MO, USA) unless otherwise indicated. Normal saline (0.9% NaCl), NaOCl (6% and 3%) and EDTA (12.5% and 17%) were prepared with deionized water solvent. A pH of 7.5 was maintained for EDTA. A commercially available 18% HEDP powder (Dual Rinse HEDP, Medcem, Vienna, Austria) was freshly mixed with 6% NaOCl before use.
2.2
Teeth selection and specimen preparation
Single-rooted human mandibular premolars (n = 24) were collected from a pool of intact, non-carious teeth extracted for orthodontic reasons, based on a protocol approved by the Institutional Review Board and Ethics Committee of the University (UW 17-467). The teeth were stored in 0.9% saline with 0.5% thymol (1:1 proportion) at 4 °C until use. The storage period did not exceed 1 month.
2.3
Experimental procedures
The sample allocation to the different characterization techniques has been shown in Fig. 1 .
2.3.1
Preparation of dentin blocks
The premolars were decoronated using a water-cooled diamond saw, and 48 dentin blocks (4 mm × 3 mm × 1 mm) were prepared from the mid-coronal dentin. Each tooth yielded 2 dentin blocks of 1 mm height each. A smear layer was developed in 36 blocks using a previously described method [ ]. Briefly, silicon carbide abrasive papers were used on the dentin blocks, in increasing order of 400, 800, 1200 and 4000 grit, under continuous water irrigation using a two-speed grinder-polisher (Ecomet 5 Polisher, Buehler). Pilot studies confirmed the formation of a uniform smear layer. These 36 blocks were pooled and then randomly divided into 3 groups (n = 12): Group 1 (Control): Normal saline; Group 2 (NaOCl/EDTA): 3% NaOCl/17% EDTA; and Group 3 (NaOCl/HEDP): 3% NaOCl/18% HEDP. The surfaces of the dentin block were covered with nail varnish with the exception of only one flat surface, which was exposed to the protocols mentioned above for 15 min. The specimens were then thoroughly rinsed in sterile water for 2 min.
2.3.2
Surface characterization
Atomic force microscopy (AFM) was used to characterize the topography of the dentin surface [ ]. Three random zones were marked on the dentin blocks (n = 4) using an indelible marker. The arithmetic roughness average (R a ) of the marked areas was determined with a scan size of 100 μm and scan rate of 40 μm/s using an AFM (Dimension Edge with ScanAsyst, Bruker), maintaining a resonating frequency of 300 kHz and spring constant of 42 N/m. A Bruker’s OTESPA probe with tip height 7–15 μm and radius 7 nm was used. Image analyses were performed using the NanoScope Analysis version 1.5 software [ ].
2.3.3
Chemical compositional analysis
The dentin blocks used above, were analyzed using Fourier transform infrared spectroscopy (FTIR) and energy dispersive X-ray spectroscopy (EDS) to study the compositional changes. Type 1 collagen and hydroxyapatite served as controls for the FTIR analysis. Spectra were collected in triplicates for each specimen from the marked areas (Section 2.3.2 ) in a FTIR spectrometer (Vertex 70 FTIR spectrometer, Bruker, Germany) between 500 and 4000 cm −1 using 32 scans at 4 cm −1 resolution. The atomic percentage of different elements were measured in triplicates, using Energy Dispersive X-ray Spectroscopy (EDS) (Model 550i, iXRF systems, Austin, TX, USA) equipped with Iridium Ultra 2016, version 2.1E software (iXRF systems). The atomic percentage of C, N, O, P and Ca were measured [ ].
2.3.4
Preparation of root canals
Root canals of the decoronated premolars (n = 24) were prepared using a single-use reciprocating file size 40, 0.06 taper (AF Blue R3, Shanghai Fanta Dental Materials Company, Shanghai, China) using the experimental groups as outlined above (n = 8). All root canals were then thoroughly rinsed with 2 ml of saline and dried with paper points. The specimens were carefully split using a slow-speed diamond impregnated saw (Isomet 5000, Buehler) under copious water cooling. Since through-and-through sectioning with a saw will create a smear layer, a standardized procedure was followed wherein an absorbent paper point was placed inside the canal, and the saw was used to section through the dentin until the white paper point was visible through the thin layer of dentin. Then, a chisel and mallet were used to carefully section the specimens. The inner wall of the cross-sectioned dentin was investigated using the different techniques, as described below.
2.3.5
Ultrastructural characterization
The effects of the different chemical treatment protocols on the dentin matrix were characterized using scanning electron microscopy (SEM), transmission electron microscopy (TEM) and light microscopy. SEM analysis was used to study the surface matrix at the intertubular and peritubular dentinal regions. Dentin block (n = 4) and root dentin specimens (n = 4) were fixed with 1% osmium tetroxide for 4 h, rinsed with distilled water and then dehydrated with ascending series of ethanol. Specimens were processed with critical point drying (Bal-tec CPD 030, Bal-tec, Balzers, Liechtenstein) and sputter-coated with carbon for 30 min (Bal-tec SCD 005). The dentin sections were then viewed under a scanning electron microscope (Hitachi S4800, Tokyo, Japan) and 10 digital images were obtained from randomly chosen areas.
TEM analysis was used to characterize the orientation, length and banding pattern of the collagen fibers [ ]. Dentin block (n = 4) and root dentin specimens (n = 4) were fixed using Karnovsky’s working solution for 4 h and decalcified in buffered EDTA solution (12.5%; pH 7.5), with the decalcifying process monitored with microcomputed tomographic scans (Skyscan 1172, Bruker microCT, Antwerp, Belgium). After post-fixation with 1% osmium tetroxide for 4 h, specimens were rinsed with cacodylate buffer and dehydrated with ethanol for 2 h. The final 100% ethanol concentrations were succeeded with 3 times change of propylene oxide and then replaced incrementally (3:1, 1:1 and 1:3) with resin (TAAB 812, TAAB Laboratories, UK). After embedding in resin molds, ultrathin sections (85 nm) were obtained using a diamond knife (Ultra Jumbo, DiATOME, Electron Microscopy Sciences; 45° angle, 3 mm edge size), stained with uranyl acetate and observed under a transmission electron microscope (Phillips CM100, Philips, Eindhoven, The Netherlands) [ , ]. Ten digital images were obtained from randomly selected areas.
To confirm if the affected collagen fibres were exposed or encapsulated well by minerals, light microscopic analysis was performed. The classic organic fibers’ staining method, Goldner’s trichrome (GT) staining, stains the demineralized collagen fibers to red or pink in color [ ]. Disorientation of collagen fibres was studied by analyzing Picrosirius red stained sections under polarized light microscopy (PLM). This is based on the property of birefringence, an optical property of anisotropic compounds that have a certain refractive index. The Picrosirius red stain binds parallel to the collagen fibers and any change in color equivalence indicates an alteration of the morphological orientation of the fibers [ ].
Root canal dentin (n = 4) was fixed with Karnovsky’s solution for 4 h followed by complete decalcification in buffered EDTA solution and tap water rinsing for 2 h. Following dehydration with ethanol and xylene, the processed samples were embedded in paraffin wax and 6 μm sections were obtained using stainless steel blades in a microtome (Leica RM 2155, Leica microsystems). The sections were dewaxed using xylene and descending order of ethanol (100%, 95% and 70% ethanol). One set of 6 μm sections was stained with GT solutions [ , ]. Briefly, the sections were first stained with Weigert’s iron hematoxylin solutions, followed by washing in distilled water, and then stained with Ponceau 2R-Acid fuchsin. This was then washed with acetic acid and decolorized by placing on Phosphomolybdic acid-Orange G, rinsed in acetic acid and then stained with Light Green SF, which was followed by rinsing with acetic acid. Each alternate section was stained with the Picrosirius red stain for 60 min, followed by rinsing twice with 0.5% acetic acid solution [ ]. The stained sections were mounted with glass covers using SP15-500 permount (Fischer Scientific, Pittsburgh, PA, USA), and observed under a Nikon Eclipse LV 100 PDL polarizing microscope equipped with a digital camera (DS-Ri1 camera, Izasa, Barcelona, Spain). Polarizing filters with dark background were used to observe the Picrosirus red-stained specimens.
2.3.6
Preparation of dentin powder and collagen degradation assay
The ninhydrin assay was used to measure the amino acids released from the dentin powder [ ], and thereby correlate to the quantity of collagen fibers affected by the treatment protocols [ ]. The ninhydrin compound, 2,2-dihydroxyindane-1,3-dione, reacts with amine functional groups of the α-amino acids to form Ruhemann’s purple, 2-(1,3-dioxoindan-2-yl) iminoindane-1,3-dione). Since all amino acids (except proline and hydroxyproline) contain a free amino group, the results of this analysis can be correlated to the quantity of collagen degradation by the chemical sequences, and thereby support and validate the observations of the TEM and FTIR analyses.
Twelve dentin blocks were used for obtaining dentin powder for the collagen degradation assays. Following dehydration in ethanol, dentin blocks were stored overnight at −20 °C. Fine dentin powder was obtained by titrating these blocks in a stainless-steel shaking flask, containing a stainless-steel grinding ball using a dismembrator (Mikro-Dismembrator U, Braun Biotech International, Germany) at 1500 rpm for 20 min. The dentin powder was divided into 3 groups as described in Section 2.3.1
Freshly prepared dentin powder (150 mg) was taken on a 55 mm filter paper and exposed to the irrigating sequences. The treated dentin powder was suspended in 10 ml of deionized water. 500 μL of the supernatant was taken in 2 ml Eppendorf tubes. Next, 250 μL of 2% ninhydrin reagent was added and the tubes were placed in boiling water for 10 min. Following dilution with 95% ethanol [ ], the specimens were centrifuged and the dentin powder was separated as pellets. The supernatant liquid (200 μL) was placed in a 96-well plate and OD 570 readings were obtained in triplicates on three different occasions using a SpectraMax M2 spectrometer.
2.4
Data analysis
The data from the surface characterization ( i.e., R a values from AFM analysis), chemical compositional analysis (C, N, O, P and Ca from the EDS analysis) and collagen degradation (free amide ratios calculated from the ninhydrin assay) were analyzed using SPSS statistics version 25 (IBM, NY, USA). Normality of data distribution was first verified with the Shapiro-Wilk test, following which, multiple factor one-way ANOVA analysis was performed. The means of different groups were compared using the Tukey’s HSD method, with 95% level of confidence. The significance level was set at P = 0.05.
3
Results
3.1
Surface characterization
The R a values were calculated before and after treatment with the experimental protocols. While there was no significant change in the R a values in the control group, the surface roughness increased by 2-fold in the NaOCl/EDTA (0.08 R a ) and NaOCl/HEDP (0.07 R a ) groups ( Table 1 ). However, there was no significant difference in the R a values between NaOCl/EDTA and NaOCl/HEDP (P = 0.9), both of which were significantly greater than the control (P < 0.001).
