Biocompatibility of new bioactive resin composite versus calcium silicate cements: an animal study

This study was conducted after being approved by the ethical committee of Central South University for animal experiments (No: 20180025). Seventy-five male rats (Rattus norvegicus albinus, Wistar) weighing approximately 180–200 g provided by Institute of Basic and Clinical Pharmacology Research Centre of Hunan Medical University were used according to the ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments, 2013) and the procedures conformed to the requirements of the ISO 10993-1 (1992) and ISO 10993-2 (1992) standards [16‐18]. Animals were kept in an aerated chamber with 12-h dark and light intervals, divided according to study period. The cages were cleaned on a daily basis, and free access to food and water was allowed. Animals were then given water only 12 h before surgery.

Three hundred sterile polyethylene tubes (10 mm in length and 1.3 mm in diameter) were used and filled with the test materials then implanted in the dorsal region underneath the skin of each rat which were divided into 4 groups: (Fig. 1a)

The materials were manipulated according to manufacturers’ instructions. A small condenser was used to insert the test materials into the tubes. The tubes were entirely filled with the tested materials and left for setting. Before surgery, the animals were anesthetized with ketamine hydrochloride and xylazine hydrochloride Intraperitoneal injection (3, 1 respectively; 0.05 mL/100 g of weight; I/P). The dorsal region of each rat was submitted to trichotomy, and antisepsis was done using a gauze moistened with 0.12% chlorhexidine. The dorsal region was then swapped with a gauze soaked with saline solution to remove any antiseptic residue. Four incisions were performed on the dorsal region of each rat (2 anteriorly and 2 posteriorly). The skin lateral to the incisions was pinched, and subcutaneous dissection was done using blunt-ended scissors.

Each animal received 4 tubes: 2 in the anterior dorsal region (Group1 to the left side and group 4 to the right) and 2 tubes in the posterior region (Group 2 to the left side and group 3 to the right). Then, the incisions were closed with Nylon 3–0 sutures. The rats were further subdivided into 3 groups according to the time period of sacrifice into: Group A after 1 week, Group B after 2 weeks and Group C after 4 weeks. (Fig. 1b) By the end of each period, 25 animals were euthanized by administering an anesthetic overdose with ketamine hydrochloride and xylazine hydrochloride Intraperitoneal injection (1: 1 respectively; 0.15 mL/100 g of weight; I/P). Biopsies of skin and subcutaneous tissues (2 × 2 cm) containing the implants were obtained with 1-cm safety margins.

The subcutaneous tissues containing the tubes were excised, fixed in 10% neutral formalin for 48 h. After that, the specimens were trimmed parallel to the tube leaving at least 2 mm of tissue on each side, cut into two equal halves, and the tubes were removed. Then, the specimens were inserted in serial ascending concentration of ethyl alcohol for dehydration, followed by clearance in xylene and embedded in paraffin at 58–62 C°. Samples were casted parallel to the long axis of the tube to show the region of interest (tube opening) then serial sections of 4 μm thickness were prepared for further staining with hematoxylin & eosin stain (H&E).

Sections from the prepared paraffin blocks were mounted on super frosted coated glass slides (Fisher Thermo Scientific, Nepean, Ontario, Canada) for immunohistochemistry. The sections were cleared, hydrated and subjected to antigen retrieval in EDTA solution (pH = 8) for 15 min at 37 °C. Subsequent incubation of the sections was performed with primary antibodies against α-smooth muscle actin (Thermo Fisher Scientific, Fremont, CA, USA; Cat. No. A1–70007) and caspase 3 (cleaved form of concentrated polyclonal antibody, CP 229A, B, C, 1:200, rabbit, Biocare medical, Pacheco, USA). The slides were rinsed in phosphate buffer saline and incubated with anti-rabbit IgG secondary antibodies (EnVision + System HRP; Dako) for 30 min at room temperature, visualized with di-aminobenzidine commercial kits (Liquid DAB+Substrate Chromogen System; Dako), and finally counterstained with Mayer’s haematoxylin. The negative control sample was prepared with replacing the primary antibody with normal mouse serum.

Histological examination was performed using Leica Microscope (Leica Microsystems GmbH, Wetzlar, Germany) adapted with a DFC420 camera. The histopathological grading was evaluated according to the following criteria: necrosis, inflammatory cell infiltrate (polymorph nuclear and mononuclear cells), congestion, macrophage activity (macrophages and multinuclear giant cells which independently scored), vascularization (blood vessels) and fibrosis (fibroblasts). The previous analysis points were scored as (0) absent, (1) discrete, (2) moderate, and (3) severe. The thickness of the fibrous capsule was measured with ImageJ analysis software (NIH, MD, USA) [19‐21]. The scoring system was performed by an experienced blinded pathologist, and scores were organized on specific sheets. Quantitative scoring of α-SMA and caspase 3 immunostaining were done according to determination of threshold basis using ImageJ software and expressed as the percent of positive area /mm².

Semi quantitative scoring of the histopathological findings of control, ACTIVA, iRoot BP and MTA-HP was illustrated in Table 2. The statistical analysis was conducted on 60 rats because 15 rats were excluded due to either removal of tube or mortality. On the first week, control group showed thin inflammatory wall, with mild fibrin clot associated with minimal tissue necrosis and few macrophages and neutrophils cells infiltration. Marked tissue organization and angiogenesis around the tube were apparently noticed. Calcification or multinuclear giant cells infiltration were completely absent within the examined animals of this group (Fig. 2a and a1). ACTIVA group revealed slight to moderate inflammatory reaction on the tubular orifice. There was slight fibrinous inflammatory response with a pronounced granulation tissue formation and a mild inflammatory cells infiltration consisted of lymphocytes, macrophages and few neutrophils (Fig. 2b and b1). The subcutaneous tissue reaction to iRoot BP group demonstrated thick inflammatory capsule associated with extensive tissue necrosis, black brownish-leaked deposits of the test material, focal calcification as well as marked fibrosis and angiogenesis especially towards the adjacent connective tissues. The cellular inflammatory response in this group predominately showed macrophages and multinuclear giant cells infiltration (Fig. 2c and c1). MTA- HP group showed thin layer of necrotic tissue admixed with tiny fibrin clotted areas, along with the presence of particulates of brownish pigments especially at the tubular opening. Mild to moderate inflammatory reaction, consisted of mononuclear cells infiltration mainly lymphocytes, macrophages and few neutrophils without any apparent multinuclear giant cells. The fibrosis degree was scanty with immature granulation tissue formation (Fig. 2d and d1).

On the period of 2 weeks, control group showed an early remodeling of the fibrous connective tissue capsule, mild inflammatory cells infiltration and slight angiogenesis (Fig. 2e and e1). ACTIVA group demonstrated marked decrease in both inflammation and tissue necrosis with regression of inflammatory cells number. The capsule was lined with fibroblast layer, encircled by medial layer of collagen and elastic connective tissues that normally oriented in parallel pattern. The outer layer showed fibrous connective tissue associated with minimal both angiogenesis and inflammatory cells infiltration consisted of macrophages and lymphocytes (Fig. 2f and f1). iRoot BP group showed a noticeable chronic inflammatory reaction at the tube orifice associated with intense fibrosis, calcification and multinuclear giant cells infiltration. Active angiogenesis was markedly increased, not only limited to the tube opening but also extended deeper into the adjacent tissues (Fig. 2g and g1). MTA- HP group revealed similar reaction to the previous group but with a lesser degree regarding the level of calcification, fibrosis and multinuclear giant cells infiltration (Fig. 2h and h1).

After 4 weeks, control group demonstrated thin mature fibrous capsule with sparse inflammatory reaction and with normal continuity to neighboring fibrous and adipose connective tissues (Fig. 3a and a1). ACTIVA group showed progressive healing with abrogation of coagulative necrosis and inflammatory cells aggregation. A well-formed fibrous capsule was consisted mainly of multiple parallel collagen fibers. In addition, marked decrease of the inflammatory reaction was noticed by regression of lymphocytes and macrophages infiltration (Fig. 3b and b1).

iRoot BP group demonstrated moderate inflammation, slight angiogenesis and mineralization alongside the tube orifice. In addition, marked thickening of the fibrous capsule was still noticeable (Fig. 3c and c1). MTA- HP group showed restoration of the normal tissue. However, limited organization of the fibrous capsule and small tiny particulates of mineral deposition were noticed (Fig. 3d and d1).