Morphological analysis of tissue reaction caused by a new endodontic paste in subcutaneous tissue of rats.

AIM: To assess the biocompatibility of an experimental endodontic paste based on the ethyl acetate fraction of Pothomorphe umbellata + calcium hydroxide, using propylene glycol as vehicle, in connective tissue of rats.
MATERIALS AND METHODS: Fifteen rats had four polyethylene tubes implanted in their backs, with each one containing the experimental paste. The tube side was considered the control group. After 7, 21, and 42 days, animals were euthanized.
RESULTS: Intense inflammatory reaction was noticed after 7 days for experimental paste and it was moderate for control group. At 21 days, the inflammatory reaction was moderate for experimental paste and discrete for control group; and at 42 days, it was discrete for experimental paste and control group. Statistical analysis (Dunn's test, P < 0.01) demonstrated significant difference between the fibrous capsule area at 7 and 42 days (P > 0.01) for experimental paste.
CONCLUSIONS: Experimental endodontic paste presented satisfactory tissue reaction in the connective tissue of rats.

INTRODUCTION

The significant global growth of phytotherapy within preventive and curative programs has stimulated the assessment of the activity of different plant extracts.[1] The practice of the members of the community using plants to reestablish health, common to all people but almost forgotten for decades, has become increasingly more intense in the civilized world in recent years.[1]

There is research involving herbal medicines in several areas of the health sciences, including Dentistry. Research involving herbal medicines in Dentistry is recent and innovative, particularly in Endodontics, the science that deals with the study of dental pulp and root canal system present in the dental organ.[23]

Investigations about the biological compatibility and physicochemical properties of the essential oil and resin of Copaifera multijuga, associated with calcium hydroxide, have been conducted.[23] Studies have also been conducted with the plant Arctium lappa and ethyl acetate fraction of Pothomorphe umbellata, whose extracts showed antimicrobial activity against microorganisms found in infections present in the root canal systems.[4-7]

The Pothomorphe genus is composed of a small number of species. Several authors believe that the number of species does not exceed more than half a dozen. This family includes shrub and herbaceous-sized plants with wide round leaves, a cordate or peltate base and an acuminate apex.[8] The flowers are gathered in dense spiciform inflorescences and situated in axils of petioles dilated into sheaths. In Brazil, there are two species: Pothomorphe umbellata and Pothomorphe peltata, both known by the common names of “pariparoba” and “caapeba”,[8] which are used to treat several infirmities due to their choleretic, cholagogic, gastric, antiepileptic, antimalarial activities and for treating diabetes and hepatic diseases.[5-8] Moreover, the main component of the plant, N-benzoylmescaline, presents significant antibacterial activity against Helicobacter pylori strain.[8]

In an endeavor to find more effective medications that could be used in Endodontics, researchers in the field of Dentistry could not abstain from this new treatment perspective which uses natural medicines with antibacterial activity, at a lower cost and less toxic activity to the organism.[46]

Thus, the aim of the present study was to assess, in a preliminary manner, the tissue compatibility of the ethyl acetate fraction of P. umbellata, with the purpose of enabling its application in Endodontics in the future.

MATERIALS AND METHODS

This study was conducted with the approval of the Ethics Committee on Research with Animals of Federal University of Amazonas and in compliance with the ethical concepts for use of laboratory animals at all stages of the experiment. The leaves of P. umbellata used to prepare the medication were collected at the Medicinal Plant Collection of the University of Ribeirão Preto. The aerial parts of the plant were dried in an oven with forced air circulation and then macerated with ethanol/water (8:2) in an amber receptacle for 48 hours. The resulting tincture was concentrated in a rotary evaporator and then lyophilized. The fraction studied was obtained by liquid/liquid partition in a separatory funnel, adding 100 ml of distilled water and successively, the following solvents of increasing polarity scale: hexane, ethyl acetate and butanol, thus obtaining hexane, ethyl acetate, butanol and aqueous fractions, respectively, after evaporation and drying.

Then, column chromatography was performed to separate the above-mentioned constituents and identify the tested chemical fraction.

To obtain the polyethylene tubes (GoldLab, Ribeirão Preto, SP, Brazil), an urethra catheter with an internal diameter of 0.8 mm was used, which was sectioned at intervals of 10 mm and sealed on one of the extremities with cyanoacrylate ester-based adhesive (Super Bonder, Aachen, Germany) to prevent extravasation of the material to be tested.

The medication was dispensed first by adding 0.167 mg of the ethyl acetate fraction of P. umbellata + 1 ml of propylene glycol, with the purpose of obtaining a favorable consistency, and this mixture was called “base paste”. This was followed by mixing 23 μl of the “base paste” with 30 mg of calcium hydroxide P.A. (Pro Analysis) (Merck, Jacarépagua, RJ, Brazil).

After obtaining the paste, the polyethylene tubes were filled with the aid of a sterile Lentulo bur (Dentsply/Maillefer, Ballaiges, Switzerland), compatible with the internal diameter of the tube, and later inserted into the dorsum of the rats.

Fifteen young adult male rats (Rattus novergicus, Albinus, Holtzman) with body weight ranging from 200 to 250 g were used. The animals were anesthetized with 10% ketamine hydrochloride (Calier Laboratories S.A., Barcelona, Spain) at a concentration of 0.2 ml per 100 g body weight of the animal, administered intraperitoneally.

The incisions to implant the tubes were performed on the dorsum of the animal, after trichotomy, being two incisions close to the scapular region and two close to the pelvic region. A scalpel blade n° 15 was used to make the incision that measured approximately 5 mm, compatible with the width of the tube. The tubes were inserted and the edges of the wounds were sutured.

Once the implants were performed, while the animals were still anesthetized, they all received 0.2 ml intramuscular dose of veterinary pentabiotic (Wyeth Laboratory, New York, NY, USA).

After 7, 21, and 42 days, the rats were euthanized by anesthetic overdose. After trichotomy and antisepsis, the implant areas were dissected and the tubes were removed for histological analysis.

The analysis of the results was made based on the tissue response caused by the tested paste by comparing it with the lateral portion of the polyethylene tube (control group). By means of the histopathological analysis, the following events were assessed: inflammatory infiltrate (polymorphonuclear cells and mononuclear cells), cellularity, vascularization, macrophage activity (mononuclear phagocytes or macrophages and giant inflammatory cells) and the fibrous capsule area. Histopathological analysis was performed under a light microscope (Nikon, Tokyo, Japan) at 120× magnification, based on the tissue responses stimulated by the tested materials and the control group. Morphological analysis was performed by assessing and quantifying the above-mentioned events within these areas situated in the fibrous capsule.

The total area of the fibrous capsule was measured using the Image Tool software [The University of Texas Health Science Center in San Antonio (UTHSCSA), San Antonio, TX, USA] and the values were submitted to statistical analysis (Dunn's multiple comparison test, P < 0.01).

RESULTS

In the period of 7 days, the fibrous capsule presented an area of 1.367 mm2. The histopathological analysis showed high population of fibroblasts and vascularization with minimal collagenization. Minimal necrotic residues at the interface of the space previously occupied by the polyethylene tube were observed. The inflammatory infiltrate was intense, with predominance of mononuclear cells. Moderate macrophage activity was observed, mainly carried out by mononuclear phagocytes and giant inflammatory cells on the dispersed material residues, both inside the capsular cone and at the surface interface with the polyethylene tube and sporadically, in the adjacencies. The general rate of inflammation was intense.

The lateral side of the polyethylene tube showed no fibrous capsule formation, presenting high populations of fibroblasts and vascularization with minimal collagenization, as well as minimal necrotic residues at the interface of the space previously occupied by the polyethylene tube. The inflammatory infiltrate was moderate, with predominance of mononuclear cells with discrete macrophage activity, mainly carried out by mononuclear phagocytes and giant inflammatory cells with general rate of inflammation considered moderate [Figures 1a and b].

Figure 1

Histological images. 7 days: (a) Intense inflammatory reaction on the tubular opening containing the experimental paste; (b) moderate inflammatory reaction on the lateral side of the polyethylene tube. 21 days: (c) Moderate inflammatory reaction on the tubular opening containing the experimental paste; (d) discrete inflammatory reaction on the lateral side of the polyethylene tube. 42 days: (e) Discrete inflammatory reaction on the tubular opening containing the experimental paste; (f) discrete inflammatory reaction on the lateral side of the polyethylene tube (Hematoxylin–Eosin, ×120)

In the period of 21 days, the fibrous capsule presented an area of 0.720 mm2. The analysis showed moderate populations of fibroblasts and vascularization with minimal collagenization. The inflammatory infiltrate was moderate with predominance of mononuclear cells and moderate macrophage activity, mainly carried out by mononuclear phagocytes and giant inflammatory cells on the dispersed material residues, both at the surface of the interface conjunctive tissue/polyethylene tube. The general rate of inflammation was moderate.

The lateral side of the tube showed no fibrous capsule formation; mild inflammatory infiltrate with discrete populations of fibroblasts and vascularization with discrete collagenization were observed. Macrophage activity mainly carried out by mononuclear phagocytes and giant inflammatory cells was also observed. The general rate of inflammation was discrete [Figures 1c and d].

Within 42 days, the fibrous capsule presented an area of 0.569 mm2, with discrete populations of fibroblasts and vascularization, showing minimal necrotic residues at the interface of the space previously occupied by the polyethylene tube. Inflammatory infiltrate considered discrete with predominance of mononuclear cells and discrete macrophage activity mainly carried out by mononuclear phagocytes and giant inflammatory cells was also observed. The general rate of inflammation was discrete.

The lateral side of the tube showed no fibrous capsule formation, discrete population of fibroblasts and vascularization with discrete collagenization. Inflammatory infiltrate was discrete with predominance of mononuclear cells and discrete macrophage activity. The general rate of inflammation was discrete [Figure 1e and f].

The mean values for the fibrous capsule area (mm2) at each experimental period are shown in Table 1. The lateral side of the polyethylene tube was not considered because it did not present a significant fibrous capsule area.

Table 1

Mean values and standard deviation of the fibrous capsule area (mm2) in the different periods of analysis

Dunn's multiple comparison test showed statistically significant difference (P < 0.01) between the fibrous capsule area at 7 and 42 days [Table 2]. Figure 2 shows the involution of the fibrous capsule area at the different experimental periods.

Table 2

Dunn's test for the fibrous capsule area comparison at the different periods of analysis

Figure 2

Involution of the fibrous capsule area in the different periods of analysis

DISCUSSION

Many studies involving herbal medicines have shown that some plant extracts have an effective antibacterial action against several microorganisms found in dental infections, which may be introduced in Dentistry.[4]

The selected ethyl acetate fraction from Pothomorphe extracts was found to be effective against strains of Enterococcus faecalis, a bacterium resistant to calcium hydroxide and other antibiotics, often found in cases of failure of conventional endodontic therapy and persistent periapical lesions. This fact indicated the need to assess the biological compatibility of this fraction.[9-11]

To conduct this study, the methodology used was implanting polyethylene tubes into the subcutaneous tissue of rats, which has desirable qualities to assess the biological compatibility of different materials, enabling the assessment of histopathological events and not favoring tissue irritation, making the delimitation of the studied area possible and causing mild inflammatory reaction.[12]

Propylene glycol was used in the experiment because it is a commonly used vehicle in intracanal medications. Propylene glycol is classified as a viscous and water soluble substance, with a high molecular weight hindering the rapid dispersion of medications associated with it, keeping the medications in the desired area for a longer period of time as well as being innocuous to the human body.[13]

Calcium hydroxide was added to the fraction to increase the antibacterial spectrum of the new medication, as well as to neutralize the pH of the fraction studied.[914-16]

The different periods of analyses (7, 21 and 42 days) allowed an efficient assessment of the stages of the reaction process in the inflammatory sequence, development of the fibrous capsule and enabled the interposition of the organic response. These experimental times were strategically used to allow the follow-up of the reaction development in the subcutaneous tissue of the rat within the stages of repair reaction.[1317]

Extracts of P. umbellata are also used to treat several diseases, especially diabetes and hepatic diseases, due to its choleretic, cholagogic, gastric, antiepileptic and antimalarial properties and antibacterial activity.[518]

Among the already known active principles of the crude extract of the plant, 4-nerolidylcatechol has demonstrated innumerable important activities such as inhibiting metalloproteins present in skin cancer, and satisfactory skin penetration, and no toxic and mutagenic properties when administered in the oral cavity of rats, protecting tissue against ultraviolet rays, and effectiveness against the larvae of the Aedes aegypti mosquito, thus suggesting it as an important active principle that must be thoroughly studied.[19-21]

With regard to the histopathological analysis, a tissue reaction considered intense at 7 days, moderate at 21 days and discrete in the period of 42 days was observed. The reduction in the number of fibroblasts and blood vessels characterizes the decrease of the inflammatory reaction process and, consequently, tissue repair.[17] The activity carried out by mononuclear phagocytes and giant inflammatory cells ranged from moderate to discrete during the different periods of analysis, which is acceptable from the biological standpoint.[1317]

The ethyl acetate fraction of P. umbellata associated with propylene glycol and calcium hydroxide showed to be an irritant material to the subcutaneous conjunctive tissue of rats at the initial period of analysis, but it did not maintain a foreign body reaction, thus allowing progressive collagenization of the fibrous capsule near to the tubular opening during the course of the experimental period.[1317] Considering the sum of the histopathological events assessed by a comparative morphological analysis, the association of the extract of P. umbellata with propylene glycol and calcium hydroxide showed an acceptable irritant potential during the tested periods, proving its biological compatibility.

It is important to point out that the results obtained in this study should not be extrapolated for clinical use in humans, since it is a level 2 test research, called secondary test, conducted in the subcutaneous tissue of rats, being an aid to evaluate the irritant potential of materials. Before the ethyl acetate fraction of P. umbellata is used in dental practice, further studies regarding its properties must be conducted.