**Abstract**

The potential use of calcium phosphate cements in endodontic therapy is an active area of research. Hydroxyapatite is one of the most commonly used calcium phosphate materials in medicine and dentistry. Biocompatibility of hydroxyapatite is closely related to its chemical composition, similar to dental and bony tissues. Recent studies have focused on new and modified formulations of calcium-phosphate-based biomaterials with improved mechanical and maintained favorable biological properties. Recently, two non-commercial new nanomaterials based on calcium silicates and hydroxyapatite have been synthesized. One is a calcium silicate system of tricalcium and dicalcium silicates (CS), and the other one is a mixture of the calcium silicate system and hydroxyapatite (HA-CS). Both CS and HA-CS are nanostructural materials. Particle size affects cement hydration and consequently setting time and final quality of the cement. Fast setting is a clear clinical advantage while cement composition and internal nanostructure are expected to provide biological behavior in vital tissues. The problem with furcation perforation repair is still not agreed upon as no currently available materials meet all the requirements of an ideal repair material as defined in the literature. Therefore, this study aimed to compare the tissue reaction of two new repair materials for furcation perforations.

**Keywords:** calcium silicate cement, hydroxyapatite, nanotechnology, furcation defects, nanostructured materials

## **1. Introduction**

Root perforations represent communication between the root canal system of the tooth and the outer surface of the tooth root. Most often, they arise as a result of pathological processes, such as deep caries or resorption, and are revealed by a detailed clinical examination or X-ray analysis. However, most perforations occur mechanically, due to iatrogenic factors (during the opening of the access cavity, finding the entrance to the canals, mechanical processing of the root canal, or during the preparation phase for an intracanal post) [1–4]. The resulting iatrogenic perforations affect the success of endodontic therapy and the long-term prognosis of the tooth. Root perforations

leading to failure of endodontic treatment account for approximately 10% of all failures [5]. Errors during endodontic therapy can occur at any stage of the work. Predisposing factors for the occurrence of perforations are the presence of denticles, calcification, obliterated canals, internal resorptions, inability to identify canal orifices, extensive caries, inclined or rotated teeth, and presence of canal posts [1–4]. Root perforation is a serious complication that requires rapid diagnosis, and timely and adequate therapy. Root perforations often lead to an inflammatory response and destruction of periodontal tissue and alveolar bone. The proliferation of granulomatous tissue, the proliferation of the epithelium, the formation of an endoperiodontal lesion and finally the loss of teeth can occur depending on the size of the perforation, its localization, and the intensity of the chronic inflammatory reaction [1, 6, 7].

Perforations of single-rooted teeth have a better prognosis than perforations of multi-rooted teeth. Perforations in the furcation area of multi-rooted teeth are a special problem. Iatrogenic perforations in the area of the furcation occur in approximately 2–12% of endodontically treated teeth and can have serious consequences on the outcome of endodontic therapy, but also on the preservation of the tooth itself [1, 8]. This perforation is an open door for bacteria to enter, either from the root canal or from the periodontal tissue, which causes an intense inflammatory response. The consequences of the inflammatory reaction can be bone resorption, growth of the gingival epithelium, and the appearance of a fistula, which further worsens the prognosis of the tooth [1].

Factors that determine the prognosis of a tooth with a perforation include the size and localization of the defect, the time elapsed from the diagnosis to the closure of the perforation, the duration of the contamination, the physical and chemical properties of the material used for its repair, or its sealing capabilities [9, 10]. The prognosis would be relatively good if the perforation was quickly detected and closed with a biocompatible material [11, 12]. There are now various biocompatible materials on the market that are used to close perforations in order to reduce the inflammatory reaction in the surrounding tissues [13–15]. The ideal material for closing these perforations should be non-toxic, non-resorbable, radiopaque, bactericidal, or bacteriostatic, and should provide a hermetic seal and prevent microleakage [16, 17].

In the literature, calcium silicate-based materials are most often used for such clinical situations. They are used in direct pulp capping procedures, closure of the root apex in apex surgery, therapy of root perforations, pulpotomy, and apexification [18, 19]. Biocompatibility, bioactivity, and sealing ability of calcium silicate cement have been proven in many studies due to their potential for dentin, cementum, bone, and periodontal ligament regeneration [18, 19]. The good biological properties of calcium-silicate cement are associated with calcium hydroxide, which is released during the bonding of the material and stimulates the proliferation and differentiation of various cells for tissue regeneration [20].

Mineral trioxide aggregate (MTA) was introduced in endodontics in 1990, as a new material with the property of closing the communication between the tooth and the outer surface of the root [18, 19]. *In vitro* and *in vivo* studies have shown that MTA has significant sealing ability and marginal adaptation [18, 19]. This material has a long initial setting time (3 h), as a result of its chemical composition and hygroscopic nature, and it also contains traces of heavy metals [21]. MTA contains bismuth oxide as an X-ray contrast agent, which interferes with cement hydration processes [22] and can react with dentin collagen, leading to tooth discoloration [23]. Newer generations of MTA cement have a slightly shorter initial setting time, as a result of changes in chemical composition, and contain non-toxic radiocontrast agents such as zirconium

#### *Application of New Nanostructured Materials in Furcation Defects Therapy DOI: http://dx.doi.org/10.5772/intechopen.109643*

oxide [24]. The main disadvantages of MTA are the long setting time and difficult handling of this material, despite its optimal sealing ability and other advantages [25].

Despite its biocompatibility and bioconductivity, hydroxyapatite has not found its place in endodontic therapy, mainly due to inadequate mechanical properties. In order to improve the mechanical properties and bioactivity of hydroxyapatite, it was added to calcium silicate cements [26].

Technological progress and the influence of nanotechnology in the field of biomedical research have led to the emergence of new nanostructured materials. This new field of science deals with controlling matter, energy and/or information at the atomic and molecular level and enables the synthesis of new materials that are increasingly similar to natural biomolecular structures [27].

Nanomedicine is a branch of medicine that is based on the medical application of nanotechnologies, through the application of nanomaterials, nanoelectronic biosensors, and molecular nanotechnology [27, 28]. The very size of nanomaterial particles (< 100 nm), which is similar to the size of biological molecules and structures (proteins 5 nm, organelles 100–200 nm), points to the possible application of nanomaterials in *in vivo* and *in vitro* biomedical research [29]. Nanomedicines made from nanomaterials and nanoparticles (compared to drugs made from the same materials in a classical way) have up to ten times greater interactive surface, which unequivocally indicates a possible improvement in the pharmacokinetic and pharmacodynamic properties of the drug. Chen (2012) points out that the bioactivity, biocompatibility, stability, and mechanical properties of nanomaterials are determined by their composition, structure, morphology, and crystal size, as well as the method of synthesis [30].

Nanomaterials have revolutionized medicine and are increasingly being used in this field. These materials have the ability to mimic the surface properties of natural tissues, are highly cytocompatible and biocompatible, and therefore show excellent properties for use in tissue engineering and regenerative medicine [27]. The pronounced activity of nanoparticles improves the hydration of nanostructured calcium silicate cement, improving their hardening and bonding, as well as their physical and chemical properties [31]. However, there are concerns about the biological behavior of these materials, as nanoparticles usually deposit in mitochondria, causing structural damage to cells. Commercial nanostructured calcium silicate cement with hydroxyapatite (BioAggregate, Innovative bioceramics, Vancouver, BC, Canada) shows similar toxicity in cell cultures, but lower systemic toxicity compared to commercial MTA, which was related to differences in the content of heavy metals and differences in production [32].

Two new experimental nanostructured cements were recently developed at the Institute of Nuclear Sciences "Vinča" according to the recipe of V. Jokanović and associates. The goal was to synthesize materials with good biological properties, short bonding time, and without heavy metals and bismuth oxide. The first cement (CS) was based on dicalcium and tricalcium silicate, and the second (HA-CS) was a mixture of hydroxyapatite with CS, in a ratio of 2:1. CS was synthesized using a hydrothermal sol-gel methodology and self-propagating combustion waves [33]. Hydroxyapatite was synthesized by the hydrothermal method. Both materials contain barium sulfate as an X-ray contrast agent. The setting time of CS and HA-CS is 10 minutes and 15 minutes. *In vitro* testing of CS and HA-CS showed no genotoxic effects on human cells [34]. Their biocompatibility was confirmed in studies where these materials were applied in subcutaneous tissue in rats or as direct pulp capping material in rabbits [35, 36].

Examination of the cytotoxicity of these materials on cell culture of fibroblasts and MRC-5 cells showed better results in comparison with MTA [37].

Calcium silicate hydrate gel (CSH) and calcium hydroxide, the main soluble fraction of cement that dissolves into Ca2+ and OH− ions, are formed by the hydration of calcium silicate materials. The reaction of released calcium with phosphates from tissue fluids represents the physicochemical basis of the bioactivity of calcium silicate materials [38]. It was established that the formation of an apatite layer on the surface of the material is not only a consequence of the release of calcium, but also the formation of Si-OH groups on the surface of the cement, which acts as centers of apatite nucleation and precipitation. Consequently, the synergistic action of calcium silicate hydrate gel as an apatite nucleator and calcium from dissolved calcium hydroxide as a precipitation accelerator is responsible for the rapid precipitation of apatite. Additionally, the hydrophilic substrate facilitates the bond with the apatite layer due to the presence of OH- groups on the surface of the cement [39]. The formation of apatite on the surface of calcium silicate actually goes through several stages. Amorphous calcium phosphate first forms on the surface of the material and then transforms into apatite, which later matures into type B carbonate apatite, which represents the biological phase of hydroxyapatite of bone, cementum, and dentin.

Calcium silicate cements are superior to most of the endodontic materials known to date in terms of their biocompatibility, bioactivity, and sealing properties. The fact that they possess a strong inductive potential for the regeneration of damaged tissues has contributed to the fact that these cements are now considered the materials of choice for numerous clinical indications.

The improved physical properties of calcium silicate cements obtained by the application of nanotechnology in terms of lower degree of solubility, higher structural stability of the material, and shorter bonding time, as well as lower cytotoxicity and better biocompatibility of these materials [40, 41], are sufficient reasons for examining their application *in vivo* conditions. The chemical nature of the material and the method of synthesis should ensure satisfactory biological behavior of these materials in living tissues. Quality marginal sealing, that is, adequate marginal adaptation of the material, should prevent the flow of tissue fluids and consequent bacterial microleakage, which is a significant factor for the long-term success of endodontic treatment.

The aim of this study was to evaluate the inflammatory reactions of the periradicular tissue and the formation of calcified tissue after implantation of CS and HA-CS in the furcation defects of the teeth of Vietnamese pigs and in the root canals of the teeth of rabbits.

## **2. Experimental procedure**

#### **2.1 Application of CS and HA-CS in furcation defects of teeth of Vietnamese pigs**

Experimental research was carried out at the Institute of Surgery of the Faculty of Veterinary Medicine, University of Belgrade, and at the Institute for Biological Research "Siniša Stanković," University of Belgrade.

Permission for experimental work with animals was obtained from the Ethics Committee of the Faculty of Veterinary Medicine and from the Ethics Committee of the Faculty of Dentistry (16/29), conducted according to international standards ISO10993-2 (Requirements for animal welfare) and ISO 7405.

Three Vietnamese pigs (*Sus scrofa* verus), both sexes, aged 24 months and with an average weight of 25 kg were the animal model in this experimental research. The protocol of the European Good Laboratory Practice (86/609/EEC), which implies the

#### *Application of New Nanostructured Materials in Furcation Defects Therapy DOI: http://dx.doi.org/10.5772/intechopen.109643*

implementation of the main principles of asepsis and antisepsis, the realization of the experiment in the minimum necessary time without physical and mental pain of the animals (International Organization for Standardization, 1997), was respected during the work.

The animals were housed in the experimental animal facility at the Faculty of Veterinary Medicine, University of Belgrade, during the experiment. Each experimental animal was housed in an individual cage in a controlled environment with a controlled diet and daily professional care. The animals were provided with appropriate care, nutrition, hygienic conditions, and their health condition was checked 3 times a day.

The animals were deprived of food for 6 hours before the operation, and water for 3 hours before the operation, in order to rule out complications during the experiment. Premedication with atropine in a dose of 0.03–0.04 mg/kg by intramuscular injection was performed in all three animals, and after 15 minutes, the animals were put under general anesthesia by administering xylazine (2% Xylazin, Cp Pharma, Bergdorf, Germany) in a dose of 1.5–2 mg/kg and ketamine (Laboratorio Sanderso S.A., Santiago, Chile) at a dose of 20–25 mg/kg intramuscularly. The average duration of anesthesia was about 100 minutes.

The surgical procedure was performed under aseptic conditions and in a way that ensures minimal trauma. Each tooth was individually cleaned, dried, and disinfected (30% hydrogen and 70% ethanol). Access cavities were formed on the lower premolars with a round diamond bur. Coronary pulp tissue was removed with a sterile, carbide, round bur. Accidental perforation of the floor of the cavum dentis in the area of the furcation of the tooth was made with the same bur, 2 mm in diameter. The cavities were washed with 5 ml of physiological solution, and hemostasis was performed with sterile cotton balls, gently and without pressure. Freshly mixed experimental, nanostructured materials, CS, and HA-CS mixed with distilled water in a ratio of 2:1 were placed on the perforation [33]. All cavities were closed with glass ionomer cement (GC FUJI VIII, GC Corporation, Tokyo, Japan) as a definitive filling.

For the next 3 days, the animals were housed in the inpatient unit of the Institute of Surgery, under constant medical supervision with analgesic therapy (Butorphanol 0.1 mg/kg/tm/i.m. for 6 h). Appropriate care, nutrition, and hygienic conditions were provided to the animals during the observation period, and their health conditions were checked three times a day.

Experimental animals were sacrificed after 30 days, by intravenous injection of 10 ml of pentobarbiturate solution (100 mg/kg).

The mandible and maxilla were separated from the rest of the skull after the removal of the soft tissues, and the tissue was fixed in 10% formalin for 48 hours. The tissue for histological analysis was taken in the form of block sections, where each block consisted of an experimental tooth with the surrounding bone. The samples were decalcified in a decalcification solution: 8% HCl from 37% (v/v) concentrate and 10% formic acid (HCOOH) from 89% (v/v) concentrate (pH = 5) at 37°C. The success of complete decalcification was evaluated subjectively and experientially. The tissue was fixed in a circular tissue processor (Leica TP 1020, Germany) after decalcification, and then molded in paraffin blocks.

Serial tissue sections of 4 μm thickness (8 from each sample) were cut from paraffin molds in the mesiodistal direction. The sections were caught on a glass slide and placed at a temperature of 56–68°C (melting point of paraffin) for one hour, to fix the samples to the glass slide and dry them. The preparations were then stained with hematoxylin-eosin (HE) and Goldner trichrome staining. H&E was used for stereological measurements, and Goldner trichrome was used for bone mineralization detection.

Stereological measurements were performed to observe the difference in the representation of bone tissue and matrix cells in the CS and HA-CS groups. Microscopic preparations were analyzed by optical microscopy using an LM Leica microscope at magnifications ×10, ×40, ×100, ×200, and ×450. Pathohistological parameters were analyzed qualitatively, semiquantitatively, and quantitatively. Histomorphometric analysis was performed according to the cellularity and thickness of the newly formed tissue. Parameters were scored using a scoring system from 1 to 4, according to the modified criteria of Accorinte et al. [42].
