*2.1.1 Results*

After the application of CS and HA-CS materials in the artificial furcation defects of the teeth of Vietnamese pigs, no inflammatory reaction was recorded in the periodontal and bone tissue of any sample (grade 1). No giant cells or microorganisms were found (score 1). A small number of material particles were detected in most of the CS and HA-CS group samples (grade 2).

The results of cytohistological and stereological analyzes of bone after implantation of CS and HA-CS materials in the furcation of porcine teeth are shown in **Tables 1** and **2**; and **Figures 1**–**6**.

The results showed that the volume and numerical density of osteocytes, as well as the number of osteocytes, was higher after implantation of HA-CS material than after implantation of CS material in the area of the tooth furcation, which represented a statistically significant difference. The volume density of the mineralized extracellular matrix in the bone tissue in the HA-CS group was higher compared to


#### **Table 1.**

*Values of stereological parameters of bone in the root perforation of Vietnamese pigs teeth with two types of materials CS and HA-CS (mean value±SD).*

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


#### **Table 2.**

*Results of morphometric and stereological values of the periodontium in pig teeth with CS and HA-CS (mean value ± SD, p<0.05).*

#### **Figure 1.**

*Micrographs of histological sections of pig teeth: (A) CS, Goldner, and (B) HA-CS, H&E, applied material in the perforation area (yellow arrow), newly formed bone (black arrow), complete healing at the perforation site (green arrow), ×40.*

#### **Figure 2.**

*Micrographs of histological sections of pig teeth: (A) CS and (B) HA-CS, histochemical technique Picrosirius red, applied material in the area of the furcation (blue arrow), newly formed bone (green arrow), collagen depots (black arrow), ×40.*

the CS group. A higher percentage of newly formed bone was observed in the HA-CS group (25.66%) compared to the CS group. The mean bone marrow volume density was lower in the HA-CS group compared to the CS group, and the numerical bone

#### **Figure 3.**

*Micrographs of histological sections of pig teeth: (A) CS and (B) HA-CS, histochemical technique Picrosirius red, periodontium (green arrow), newly formed bone (black arrow), blood vessels (white arrow), ×40.*

#### **Figure 4.**

*Micrographs of histological sections of the peridontia of pig teeth: (A) CS, and (B) HA-CS, different width of peridontium (black bracket), blood vessels (red arrow), and peridontium (black arrow), Goldner, ×450.*

#### **Figure 5.**

*Micrographs of histological sections of pig teeth: (A) CS, and (B) HA-CS, H&E, periodontium (white arrow), osteoblast activity surface (black arrow), and newly formed bone (yellow arrow), ×100.*

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

**Figure 6.**

*Micrographs of histological sections of pig teeth: (A) CS, and (B) HA-CS, Goldner, remnants of applied materials (red arrow), periodontium (yellow arrow), newly formed bone (white arrow), and newly formed tendon tissue (black arrow), ×100.*

marrow density was higher in the HA-CS group compared to the CS group. These data imply that the mitogenic effect led to an increase in the number of bone marrow cells and not to an increase in its volume in the bone tissue. This was the reason to make individual measurements of volume and numerical densities, as well as the number of mesenchymal cells and fibroblasts in the bone marrow.

The results of these measurements showed that the volume and numerical density, as well as the number of the mentioned cells in the bone marrow, were higher in the HA-CS group compared to the CS group. Also, less extracellular substance was observed in the same tissue in the bone marrow. Stereological parameters of blood capillaries showed a slight increase in values in the tissue of the HA-CS group compared to the CS group, which is a consequence of increased mitosis of all types of bone tissue cells.

Newly formed calcified tissue was observed in all samples after the application of both CS and HA-CS (**Figure 1**). In all samples of the HA-CS group, the implanted material was completely separated from the adjacent tissue by newly formed, properly structured, calcified continuous tissue (grade 1) (**Figure 1B**), whose thickness was 190–210 μm. Incomplete closure of the perforation by newly formed calcified tissue (green arrow) 125–160 μm thick is present in CS material in most samples. The newly formed calcified tissue in the CS group mostly had an irregular morphology (grade 2).

HA-CS performed significantly better than CS in terms of continuity and thickness of newly formed calcified tissue (p = 0.008).

Immunostaining for collagen (Picrosirius red) is a method by which, based on the detection of collagen, the quality of newly formed bone is determined. The results showed that the newly formed bone under the HA-CS material was of better quality and denser (**Figure 2**) and that there were more depots of extracellular collagen in the form of strips compared to the CS group (black arrow).

The newly formed bone was more regular and denser in contact with the periodontium in the HA-CS group than in the CS group (**Figures** 2 and **3**). A thin layer of periodontium (green arrow) is observed in most samples under the applied material (**Figure 3**). The activity of osteoblasts leading to new bone formation (black arrow) and the presence of newly formed blood vessels (white arrows) are also observed. Blood vessels are more numerous and larger in the HA-CS than in the CS group.

The results of morphometric and stereological measurements of the periodontium tissue are shown in **Table 2**; and **Figures 4**–**6**.

The thickness of the newly formed periodontium in the area of the furcation in the HA-CS group was 26.88% higher than in the CS group. The surface of the periodontium in the area of the furcation was 16.34% higher in the HA-CS group compared to the CS group. The volume density of the periodontium was 27.75% higher in HA-CS compared to CS, which was a statistically significant difference.

Histological and morphometric and stereological data show that the newly formed periodontal tissue was better and more regular in HA-CS material (**Figure 4**, black bracket) (**Table 2**). The connective tissue of the periodontium under the applied HA-CS material was more regular and dense (**Table 2**, **Figure 4**, black arrow), and the blood vessels were narrower and more numerous (red arrow). The volume density of the periodontium was statistically significantly higher (p = 0.021673) in the HA-CS group compared to the periodontium of the CS group. The blood vessels of the periodontium in the CS group were wider, dilated, with remnants of clustered erythrocytes.

Histological analysis shows that the newly formed periodontium in the HA-CS group is thicker compared to the CS group. Mesenchymal cells with osteoblastic differentiation were observed at the periphery of the newly calcified tissue. These cells are more numerous and fill the entire space between the periodontium and newly formed bone in the HA-CS material, while they are found in smaller numbers in the CS group (**Figure 5**).

The periodontium is histologically more regular, thicker, and more reactive to staining in the HA-CS group compared to CS. Newly formed bone (white arrow) is observed below the periodontium and the beginnings of tendon tissue formation (black arrow) are observed below it (**Figure 6**). Newly formed bone is histologically more regular and thicker, as well as tendon tissue which is thicker in teeth with HA-CS compared to CS.

#### **2.2 Implantation of CS and HA-CS in root canals of rabbit teeth**

Experimental research was carried out at the Institute of Surgery of the Faculty of Veterinary Medicine, University of Belgrade. Permission for experimental work with animals was obtained from the Ethics Committee of the Faculty of Dentistry, University of Belgrade, Serbia (Protocol No. 36/21/2013), conducted according to international standards ISO10993-2 (Requirements for animal welfare) and ISO 7405 [41].

Four rabbits from the genus Orictolagus cuniculus, aged 12 months and with an average weight of 4 kg, were included in the research.

Animals were kept in standard, individual cages, given ad libitum access to water, and standard rabbit chow. Daily monitoring of the animals was performed while the experiment lasted. Before the surgery, the animals were put under general anesthesia with Xylazine (2% Xylazine, Czech Republic) 35 mg/kg body weight and Ketamidor (100% Ketamidor 100 mg/ml, Richter Pharma AG, Austria) 5 mg/kg body weight.

The working field was disinfected with 5% tincture of iodine. Round diamond bur was used for preparing the class I cavities on the upper and lower central incisors. Access cavities were formed and coronary pulp tissue was removed with sterile, round, and carbide burs. Root canals were instrumented with K files #40 (VDV Gmbh, Germany) after extirpation of the radicular pulp and irrigated with 5 ml saline between each instrument. A new set of endodontic instruments was used for each animal. Then, the canals were dried with paper points and filled with freshly mixed material. Experimental and nanostructured cements, CS and HA-CS, were mixed

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

with distilled water in a ratio of 2:1 [33]. The control material, mineral trioxide aggregate (White MTA, Angelus® Solu oes odontologicas, Londrina, Brazil) was mixed according to the manufacturer's instructions, in a powder-to-liquid ratio of 3:1. MTA was applied in the right maxillary incisors of all animals. In both mandibular incisors and left maxillary incisors of two animals were applied CS, HA-CS was applied in the left maxillary incisors and both mandibular incisors of remaining two animals. The materials were introduced with a lentulo spiral and condensed with a hand compactor into the root canals. The cavities were then closed with voice ionomer cement (GC FUJI VIII, GC Corporation, Tokyo, Japan). Postoperatively, the animals received subcutaneously an analgesic (Butorphanol, 10 mg/ml, Richter Pharma AG Austria), 0.1 mg/kg body weight, every 8 h for the next three days and an antibiotic (Baitril®, 25 mg/ml, KVP Pharma und Veterinar Produkte GmbH), 10 mg/kg body weight, daily for the next five days. After 28 days, animals were sacrificed by intravenous injection of 10 ml of pentobarbital solution (pentobarbital sodium salt 100 mg ml-1, Sigma-Aldrich Chemie GmbH, Steinheim, Germany).

The treated teeth together with the bone tissue of the upper or lower jaw were cut with a diamond disc in the form of block sections and fixed in 10% formalin after removing the soft tissues and separating the upper and lower jaw. 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 (eight per sample) of 5 μm thickness were cut from paraffin blocks and stained with hematoxylin eosin (HE) according to standard procedure. Glass histological slides were analyzed with an optical microscope (Olympus 5 microscope) using the morphometric software package "Cell-B" (Olympus), at magnifications of 40x, 100x, and 200x, by an experienced pathologist who did not know the types of examined materials. Histological parameters were analyzed qualitatively (presence of inflammation, general tissue condition, continuity, and morphology of calcified tissue), semi-quantitatively (presence of giant cells, particles of material, and microorganisms), and quantitatively (intensity of inflammatory reaction, thickness of calcified tissue). Histomorphometric analysis was performed according to the cellularity and thickness of the calcified tissue. Parameters were scored using a scoring system from 1 to 4 according to the modified criteria of Accorinte et al. [42].

#### *2.2.1 Results*

Half of the samples showed no inflammatory reaction after CS material implantation (grade 1) (**Figure 7**). In two samples, inflammatory reactions were moderate (grade 3). In one sample, inflammatory reaction was pronounced (grade 4), with deep tissue infiltration of inflammatory cells and abscess formation (grade 3). Particles of implanted material were detected in all samples, but in different numbers (grades 2–4). Giant cells were detected in half of the samples—(grade 1), and giant cells were detected in the other half in small numbers (grade 2). Microorganisms were not detected in any sample.

The tissue was unchanged after implantation of the HA-CS material (grade 1) in most samples (**Figure 8**). Mild inflammatory reaction (grade 2) was detected in two samples (score 2). Material particles were detected in most samples (grade 2). Microorganisms and giant cells were not detected (grade 1).

#### **Figure 7.**

*CS. Photomicrograph of calcified tissue with proliferation of the connective tissue, moderate cellularity with slight macrophage infiltration. Discontinuous newly formed calcified tissue (black arrow) with a clear border between old and new osteoid (black line). Irregular structure of newly formed calcified tissue (HE, 200×).*

#### **Figure 8.**

*HA-CS. Continuous calcified tissue with lamellar structure (HE, 40×). Calcified tissue with regular mineralization. Viable osteocyte is presented in newly formed bone (black arrows).*

Different inflammatory reaction was observed after MTA implantation (grades 2–3), with inflammatory cells near the implanted material (grade 2) (**Figure 9**). Material particles were detected in all samples (grade 2). Few giant cells were found in most samples (grade 2). Microorganisms were not detected (grade 1).

There were no statistically significant differences in the intensity of inflammatory reactions between the tested materials. There were statistically significant differences between HA-CS and CS regarding the extent of inflammation (p = 0.004).

Newly formed calcified tissue was mostly irregular in morphology in samples with CS (score 2), deposited in a thickness greater than 250 μm (scores 1–2) in most samples, but discontinuous with foci of fibrovascular tissue (scores 2–3) (**Figures 7**A, B).

The implanted material was completely separated from the adjacent tissue by newly formed, regularly structured, and calcified continuous tissue in most samples with HA-CS (score 1). The thickness of the newly formed tissue varied between 150 and 250 μm (scores 1–2). Mesenchymal cells with osteoblastic differentiation were observed at the periphery of the newly calcified tissue (**Figures 8**A, B).

New calcified tissue was deposited in small amounts, up to 150 μm thick, in all samples with MTA (score 3). The newly formed calcified tissue was irregularly structured and discontinuous with foci of fibrovascular proliferation (scores 2–3) (**Figures 9**A, B).

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

**Figure 9.**

*MTA. Foci of fibrovascular proliferation (black arrow) in partially discontinuous newly formed calcified tissue (HE, 40×). Regular structure of calcified tissue with many osteocytes. Material particles (blue arrow) (HE, 200×).*

HA-CS performed significantly better than MTA and CS in terms of continuity of newly formed calcified tissue (p = 0.03 and p = 0.010, respectively). There were significant differences in calcified tissue thickness between CS and MTA (p = 0.004) and between HA-CS and MTA (p = 0.012).

#### **3. Discussion**

The complex interaction between the material and the host tissue is best demonstrated by *in vivo* tests. These tests, in addition to biocompatibility, enable the evaluation of the biofunctionality of the material. In these two experimental studies, the effects of the materials were evaluated after their implantation in artificially created perforations in the furcation area of the teeth of Vietnamese pigs and application in the root canals of rabbit teeth.

In a rabbit study, the CS and HA-CS materials induced an inflammatory reaction of the periradicular tissue that was similar in intensity to the control material (MTA). Inflammatory reactions were rated as mild to moderate in most samples, indicating a good tolerance of the host tissue to the applied materials. These findings are consistent with the results of other authors who examined the biocompatibility of materials with similar chemical composition [42].

In another study on Vietnamese pigs, no inflammatory reaction was noted in any sample after implantation of CS and HA-CS in the furcation area of the tooth. The results of de Silva and the authors show that the inflammatory reaction is most intense in the first seven days after the application of the tested material and that the intensity of the inflammation decreases over time [43].

Inflammatory reactions after the application of calcium silicate cements are the result of the release of calcium hydroxide during the setting of the material. High pH causes local tissue necrosis with the development of local inflammatory reactions [44]. As a consequence of the alkaline pH, silicate cement induces the expression of proinflammatory cytokines (IL-6 and IL-8) [45]. It is known that tissue necrosis is the initiator of the mineralization process [46]. Some studies show that repair processes could start even without necrosis or acute inflammation [47]. As the material sets as a function of time, the amount of calcium hydroxide released from calcium silicate cements decreases [47]. By binding the material, favorable conditions are created for the beginning of the reparation process.

Although no statistically significant differences were found between CS, HA-CS, and MTA in the rabbit tissue regarding the inflammatory response, the tissue condition in the samples with HA-CS was rated as the best. This finding may be a consequence of the composition of such material. HA-CS consists mainly of hydroxyapatite with a slightly lower pH value than CS and MTA [35]. Lower pH values are thought to promote alkaline phosphatase activity but cause a smaller zone of surface necrosis compared to highly alkaline materials such as calcium silicate cements [48].

The low number of giant cells in samples with CS and MTA, or their absence in samples with HA-CS in both experiments, indicates a low activity of tissue histocytes and a good tissue tolerance to the implanted materials.

Newly formed calcified tissue was observed in all samples of the investigated materials in both experiments. It confirms that the examined nanostructured cements have an inductive potential. Previous studies also confirm the formation of mineralized tissue after the implantation of materials with the similar chemical composition [49].

CS and HA-CS belong to the group of bioactive materials that have the ability to release biologically active ions. The main soluble component of these cements is calcium hydroxide. It is released during the binding of the material. Considering that calcium silicate materials are characterized by a long bonding time, calcium hydroxide is released over several weeks [38]. Calcium hydroxide dissolves calcium and hydroxyl ions in contact with tissue and tissue fluids. The continuous release of calcium ions from the material is considered to be crucial for the induction of calcified tissue formation. In addition to its role in chemotaxis, calcium regulates cell proliferation, differentiation, and mineralization [39].

Calcium-releasing materials have been confirmed to induce the proliferation of periodontal fibroblasts, the growth and differentiation of pulp cells, osteoblasts, osteoblast-like cells, and cementoblasts [20].

The release of hydroxyl ions from implanted material is associated with tissue mineralization processes. An increase in alkaline phosphatase (ALP) occurs as a result of high pH, leading to the expression of growth factors and the formation of calcified tissue.

The tested materials also have Si ions in their composition, which influence the bioactivity of the material [50], and the proliferation and differentiation of cells similar to osteoblasts. High concentrations of Si ions (> 30 ppm) can inhibit osteoclast growth and resorption processes, but can also increase the level of ALP that participates in the mineralization of newly calcified tissue [51].

The result of the application of both nanostructured materials is a thicker layer of calcified tissue compared to MTA. Materials synthesized by the sol-gel method, such as CS and HA-CS in these studies, have improved bioactivity compared to the same materials obtained by other methods [52]. The topography of the surface of materials is related to their chemical composition and structure, and affects the activity of cells, especially their adhesion and vitality [47]. These results can be explained by the nanostructure of CS and HA-CS particles, which is similar to bone structure.

The newly formed calcified tissue, associated with HA-CS in both experiments, was continuous and without foci of vascularized fibroblast proliferation, which was not the case with MTA and CS. Unlike CS and MTA, HA-CS contains phosphate ions that could be related to this histological finding. Studies show that similar or better quality of calcified tissues are described after the application of calcium silicate cements containing phosphate ions, compared to pure calcium silicate cement [49]. The authors attributed these findings to the greater amount of phosphate ions available for hydroxyapatite formation. The present studies confirm that materials with hydroxyapatite have a greater potential for tissue mineralization than MTA [53].

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

There were no observed microorganisms in any sample of the implanted materials. The presence of microbes usually correlates with inadequate crown restorations and subsequent microleakage. GIC is material with good sealing properties and it may be the reason for the obtained result in this study. However, it must be emphasized that with this type of histochemical staining, microorganisms are difficult to detect and can be removed during tissue preparation for histological analysis.

#### **4. Conclusion**

The application of CS and HA-CS into the furcation defects of the teeth of Vietnamese pigs showed a complete absence of tissue inflammatory reaction, while this response was minimal after the application of these materials into the root canals of rabbit teeth, similar to the control MTA. CS and HA-CS were more effective than MTA in inducing the formation of calcified tissue after implantation in the root canals of rabbit teeth. The best organized newly formed calcified tissue was observed after the application of HA-CS in root canals of rabbits and root perforation of Vietnamese pigs. The present results serve as a solid basis for further biological studies of CS and HA-CS.

## **Conflict of interest**

The authors have stated explicitly that there is no conflict of interest in connection with this chapter.

#### **Author details**

Marijana Popović Bajić1 \*, Violeta Petrović1 , Vanja Opačić Galić1 , Smiljana Paraš2 , Vukoman Jokanović3 and Slavoljub Živković1

1 Department for Restorative Dentistry and Endodontics, School of Dental Medicine, University of Belgrade, Belgrade, Serbia

2 Faculty of Science and Mathematics, Department of Zoology, University of Banja Luka, Banja Luka, Republic of Srpska, Bosnia and Herzegovina

3 "Vinča" Institute of Nuclear Sciences, University of Belgrade, Belgrade, Serbia

\*Address all correspondence to: dr.marijanapopovic@gmail.com

© 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
