Pulpal and periapical tissue response after direct pulp capping with endosequence root repair material and low-level laser application

Full effort was done to utilize the least number of animals based on the data collected from the pilot test. The power of the test was calculated using independent t-test. The power of 0.95 was reached for the given value from it with alpha level of 0.05 and minimal sample size of 6 teeth per group for each outcome.

The study followed the standard practices for biological evaluation of dental materials. All the experiment’s steps followed the European Communities Council Directives of 24th November 1986 (86/906/EEC) recommendations. The study was approved by a Research Ethics Committee, under (Proposal No. 127-09-09). Eight Adult male New Zealand rabbits were selected from the animal housing with age of 8 ± 2 weeks with weight of 2382.5 ± 324.7 g. The animals were kept in polypropylene cages (60 × 45 × 45 cm) in singles under a temperature of 23 ± 2 °C and relative humidity of 55% and 12 h. photoperiod. The experimental procedures were approved by the ethical committee before starting the project with ethical approval no (127-09-19). Laboratory experiments and animal care strictly followed the ethical guidelines of the declaration of Helsinki of the World Medical Association regarding using animals for laboratory experiments.

Six out of eight rabbits were selected and randomly distributed into two groups, three rabbits were sacrificed after 2 weeks and considered as group 1 and the other three rabbits were sacrificed after 2 months and considered as group two. Four anterior teeth were included in each animal in each group interval (twelve teeth per each group per interval). Low-level laser was applied to the two lower anterior teeth in all tested animals. The other two animals were sacrificed without intervention to serve as a negative control of pulp tissue baseline reading to compare the changes in tested groups.

Animal fasting was done 2 h before surgery. Intra-muscular Pentobarbital injection in dose of 0.5 mg/kg followed by Ketamine hydrochloride injection at a dose of 5 mg/kg (Ketamine10%, Alfasan, Netherland) were administered [27].

The teeth surface was disinfected with 2% chlorhexidine solution (Chlor-X, Prevest DenPro, Jammu, India), using a small cotton pellet. Then teeth were isolated by rubber dam and a class V cavity with a diameter of 1–2 mm was done in the labial surface of anterior teeth with a sterile TC round bur (edednta, Liechtenstein, Switzerland) in 45° to the long axis at low speed of 20,000 rpm and under saline spray coolant. One bur was used for each cavity. Mechanical pulp exposure was performed using endodontic explorer with diameter of 0.15 mm (DG16; Hu-Friedy Co., Chicago, IL). The bleeding was controlled by gentle pressure with sterile cotton pellets [28]. A team of two operators was responsible for all surgical procedures with the aid of 3.5× magnifying dental loupes (Zumax Medical Co Ltd, Suzhou New District, China).

Diode laser of 980 nm wavelength and output power of 0.25 W (Doctor Smile, Lambada Spa, Brendola, Italy) was applied in direct contact technique with the exposure site in continuous mode to the lower teeth for 90 s with a total dose of 1.25 J/cm².

Pulp capping was done with regular-set ERRM (ERRM; Brasseler USA, Savannah, GA) using hand pluggers No.2 (Dentsply, Maillefer). After that, cavities were sealed by Glass ionomer (Fuji IXGP; America Inc., Alaip, USA).

The animals were kept on soft diet. The surgical site was examined daily to check the sealing of the filling, abscess formation, or presence of infection. In the meantime, animal health monitoring was performed by observing their normal diet consumption, sleeping hours, and weight [27].

Animals were sacrificed after 2 weeks and after two months by ketamine overdose. The anterior teeth and the surrounding bone were dissected in buccolingual direction then fixed in 10% buffered formalin with pH of 7.1 for 48 h then samples were subjected to decalcification using Shandon TBD-1 (HCl based; Thermo Electron Corporation, Basingstoke, Hampshire, UK) for 4 weeks at room temperature of 25⁰C. Demineralized specimens were washed under running tap water for 1 h. Histological processing was done after insertion of samples in enclosed perforated cassettes using Excelsior™ AS Tissue Processor (Thermo Fisher Scientific, Fair Lawn, UK). Then paraffin blocks were cut in buccolingual serial sections of 3–4 μm thickness using The Accu-Cut® SRM™ 200 Rotary Microtome (Sakura Finetek, Tokyo, Japan) and sections were stained with hematoxylin and eosin stain (H&E) and Masson Trichrome using Tissue-Tek Prisma device (Sakura Finetek, Tokyo, Japan).

Histological analysis was done blindly by two evaluators under light microscope (Eclipse E1000; Nikon, Tokyo, Japan). Images were captured using Nikon DS-Fi2 digital camera at different magnification powers. H&E slides were scanned using IScan Goreo (Ventana Medical System, Roche, Tucson, AZ) [29]. Histological assessment was done for, inflammatory response, odontoblasts cell status, cell density, predentin thickness analysis, presence of vacuoles, and condition of the apical part and periapical area. [28, 30] as follows. The inflammatory response was given grades 1–4, in which grade 1 refers to no or sparse inflammatory cells, grade 2 refers to mild or less than 10 inflammatory cells, grade 3 refers to severe inflammation with abscess or dense infiltration of inflammatory cells that includes more than one third of the pulp space, and grade 4 refers to necrotic pulp [31, 32]. Odontoblast cells status was also given grades 1–4, in which grade 1 refers to palisade odontoblasts, grade 2 refers to the presence of odontoblast cells, and odontoblast-like cells, grade 3 refers to the presence of odontoblast-like cells only, and grade 4 refers to no cells [31, 33]. Regarding predentin thickness analysis, the captured images of predentin at 10× magnification for each group were used for measurements. Predentin thickness was measured in pixels by using ImageJ v1.53e software [31, 34]. All values were calculated from three sites of predentin for each tooth in all groups and we calculated the means and standard deviation. Cell density was evaluated by checking the cells (fibroblasts and undifferentiated mesenchymal cells) density at the central zone of the pulp [35]. The cell counting was performed and analyzed by ImageJ v1.53e software under 40× magnification power.

After 2 weeks of pulp capping with ERRM material the microscopic images showed mild inflammation and organized palisaded odontoblasts mixed with odontoblast-like cells in teeth treated with laser. But in non-lased teeth, there was severe inflammation detected in the pulp and the disorganized pre-odontoblasts cells found next to dentine wall. The pulp central space of lased teeth exhibited low cellularity and more dilated congested blood vessels while the non-lased teeth showed high cellularity and newly formed small size blood vessels (Fig. 1). The predentin thickness was thicker in the lased group compared to non-lased teeth with a statistically significant difference (p < 0.05) (Table 1). Vacuoles were observed near the pulp horn in the coronal part only and were associated with missing odontoblasts. Vacuoles were observed in the control and tested groups. No areas of necrosis or micro-abscess were observed.

Table 1 shows histological assessment for inflammatory response; odontoblasts cell status; cell density; predentin thickness analysis for group 1 In which the animals were sacrificed after 2 weeks of pulp capping procedures. The predentin thickness was statistically significant (p < 0.05) thicker in lased group compared to non-lased teeth. There was no statistically significant difference in the other variables between lased and non-lased groups.

After two months, the inflammatory cells were sparse in both lased and non-lased teeth and the odontoblasts are organized and palisaded. Odontoblast-like cells were more noticeable in the non-lased group. The central pulp zone of lased teeth showed more collagenous fibrous tissue components compared to non-lased teeth but both groups exhibited multiple dilated congested blood vessels (Fig. 2). In Addition, the predentin was observed in the labial side of the teeth. The thickness was variable in the pulpal side, and it was thicker in lased too but no statistically significant difference was found (Table 2).

Table 2 shows histological assessment for inflammatory response; odontoblasts cell status; cell density; predentin thickness analysis for group 2 in which the animals were sacrificed after two months of pulp capping procedures. There was no statistically significant difference in the other variables between lased and non-lased groups.

The histological morphology of the periapical area in group one (2 weeks) with or without laser therapy showed dilated blood vessels either in complete or incomplete apex closure. Although the blood vessels were numerous in periapical tissue, there is no significant infiltration of lymphocytes in the area. In the complete closure, the preapical area shows thick fibrous connective tissue (Fig. 3B, D). In teeth with complete apex closure, the cementum is formed and attached to the mature and well-organized periodontal ligament (Fig. 3B, D), while open apex teeth exhibited irregular PDL attachment (Fig. 3A, C).

The same was found in group two (Fig. 4), with more numerous maturations of PDL fibers observed with complete development of root apex of the root (Fig. 4A, B). The periapical area of lased teeth showed more fibrosis than non-lased teeth (Fig. 4C, F). Bone in all groups showed normal pattern of growth and viability. In teeth from the non-lased group, there is mild inflammation and scattered blood vessels in the periapical area (Fig. 4D). Closed apex teeth in this group shows mature PDL and scattered inflammatory cells in the periapical area with congested blood vessels (Fig. 4E).