Preparation of tooth specimens
Fifty straight single-rooted teeth with relatively similar dimensions and morphology and closed apices were extracted for orthodontic or periodontal reasons collected with the patients´ informed consent. This study design was approved by the Ethics in Human Research Committee of Shiraz University of Medical Sciences (Ethics ID no. IR.SUMS.DENTAL.REC. 1398.138). Proximal view radiographs were taken to confirm the presence of a single patent canal. Teeth with root caries, cracks, curved canals, endodontic treatment, internal resorption, or calcification were excluded. Teeth were thoroughly cleaned of any soft tissue or calculus deposits and stored in isotonic saline solution at room temperature until the time of use. The crowns of all specimens were cut transversally at the coronal level of the roots with a double-faced diamond disc (Microdont, LDA, Brazil) at low speed with water coolant to ensure a uniform sample length of 14 mm (± 1 mm root length).
Specimen Preparation For The Micro-hardness Evaluation
Specimens were longitudinally sectioned in the buccolingual direction using a double-faced diamond disk at low speed, without passing through the canal space. A mallet and chisel were used to split the root. The root segments were horizontally embedded in auto polymerizing acrylic resin (Acrostone, Dent Product, Egypt), leaving their dentin surface exposed. The dentin surface of the mounted specimens was ground flat and smooth with a series of ascending grades of carbide abrasive papers (500, 800, 1,000, and 1,200 grit) (Bigo, Dent Product, Germany) under distilled water to remove any surface scratches and finally polished with a 0.1-Mm alumina suspension on a rotary felt disc (Microdont, LDA, Brazil) to obtain a smooth glassy mirror-like surface.
The samples were divided randomly into one control and four experimental groups based on the immersion solution and incubation time:
Control group: Normal saline for 15 min
G1: 2.5% NaOCl (Chloraxid, Cerkamed, Poland) for 15 min without an incubation period
G2: 2.5% NaOCl for 15 min
G3: 2.5% NaOCl for 15 min irrigated with normal saline followed by 5% STS (Merck, Darmstadt, Germany) for 10 min
G4: Normal saline for 15 min followed by 5% STS for 10 min
All groups except group 1 were incubated for 1 week in an incubator (37 °C with 100% humidity) before the micro-hardness test. The group 1 samples were tested immediately after immersion in NaOCl.
Dentin Micro-hardness Measurements
The microhardness measurements were taken either on the buccal or lingual side of each root. The sectioned root was divided equally into three-thirds representing the coronal, middle and apical thirds, and each area was tested separately. An indentation was made in the dentin surface approximately 200 µm from the canal-dentin interface for standardization. The Vickers hardness value was obtained by dividing the test force by the area of the sloping faces of the indentation. The resulting impression of the two diagonals was observed with an optical microscope and the average length of the two diagonals was measured with the built-in scaled micrometer and converted into the Vickers hardness number (VHN) with the following equation:
VHN (HV) = 1,854(F/D²).
The constant value of the equation was calculated from the specific geometry of the indenter, F is the applied load in grams and D is the diagonal of the indentation in µm (27).
Specimen Preparation For Sem Evaluation:
One specimen of each group was dehydrated, mounted and gold-sputtered, for evaluation under a scanning electron microscope (Nova NanoSEM 450, FEI, Eindhoven, Netherlands) operated at 20KV. Photographs were taken from 3 points of each sample at 1000 × magnifications (Fig. 1).
Data were analyzed using the Kruskal-Wallis test for pairwise comparisons. A p-value < 0.05 was considered significant, and all analyses were carried out using SPSS software (SPSS version 16, SPSS INC., Chicago, IL, USA).