Tissue reaction to Aroeira ( Myracrodruon urundeuva ) extracts associated with microorganisms : an in vivo study

Based on aroeira’s (Myracrodruon urundeuva) antimicrobial activity and a future trend to compose intracanal medication, the aim of this study was to assess in vivo inflamatory tissue response to the extracts by edemogenic and histological analysis containing inactivated facultative and anaerobic microorganisms. For edema quantification, eighteen animals were divided into three groups (n = 3, periods: 3 and 6 hours) and 0.2 mL of 1% Evans blue per 100 g of body weight was injected into the penile vein under general anesthesia. After 30 min the animals received a subcutaneous injection in the dorsal region of aqueous or ethanolic extract of aroeira or saline (control) containing inactivated bacteria. Samples were collected, immersed in formamide for 72h, and evaluated by spectrophotometry (630 m). For histological analysis, polyethylene tubes with the extracts were implanted in the dorsal of 30 male rats. Analysis of the fibrous capsule and inflammatory infiltrate were performed after 7 and 30 days. The aqueous extract group induced less edema in both postoperative periods compared to the other groups, but the differences were not significant (p > 0.05). Tissue repair was significantly better after 30 days than after 7 days (p < 0.01). The aqueous solution showed less inflammatory response than the ethanolic solution (p < 0.05), with tendency for better results than control after 7 days. After 30 days, the response to both extracts was similar to control. The aqueous and ethanolic aroeira extracts containing inactivated microorganisms showed a trend for better results than saline, even when associated with microorganisms, and facilitated the tissue repair process.


Introduction
The role of microorganisms, their products and by-products is observed in the canal system 1,2,3,4 in teeth with pulpal necrosis and periapical disease. 5ue to the complexity of the root canal system, the mechanical action of the instruments is not able to complete eliminate the microorganisms present in isthmus, lateral canals neither deep in the dentinal tubules 6 nor those located in areas of external reabsorption, protected by apical biofilm. 7herefore, the use of an inter-appointment medication is often essential to reduce the bacterial load. 8With this purpose, many medication protocols have been used over the years, including the use of paramonoclorofenol, phenolic derivatives, aldehydes and steroids in combination with antibiotics, and all proved antimicrobial activity or biological compatibility, but rarely both. 5Calcium hydroxide has been the most significant medication used through many years and is still used as root canal dressings for both deciduous and permanent teeth.
Recently, studies using plant extracts of araçá (Psidium cattleianum) showed the antimicrobial activity, 9,10 even against S. mutans 11 and its biocompatibility as extract with inactivated microorganism 12 or when used with calcium hydroxide. 13yracrodruon urundeuva, (also known as aroeirado-sertão), is a plant that belongs to the Anacardiaceae family, and is native of South America. 14It is one of the five most important native species used for local therapeutic indications 15 demonstrating analgesic and anti-inflammatory properties. 16Recently, aroeira's extracts were reported to have potent antimicrobial activity against oral microorganisms 10 , while presenting a satisfactory tissue response. 10,17 ic ro orga n i sm s m ay pre s e nt d i f fere nt characteristics, such as structural, metabolic or pathogenic and its contact with periapical region stimulate an inflammatory response. 18The presence of bacteria, endotoxins (lipopolysaccharides), products of bacterial metabolism, even the presence of dead bacteria, induces an inflammatory response, leading to processes that may develop a periapical disease. 19herefore, the aim of this study was to evaluate in vivo, the edema inflammatory tissue reaction to aqueous or ethanolic extracts of aroeira (Myracrodruon urundeuva) associated with inactivated microorganisms.The null hypothesis was that the association of the plant extracts with the microorganism would evoke a severe inflammatory reaction.

Methodology Animals
Thirty-eight Wistar (Rattus norvegiccus) male rats, 60 days old and 250-300 g, were obtained from Araçatuba School of Dentistry Vivarium -UNESP.The animals were housed in temperature-controlled rooms and received water and food ad libitum through the pre-experimental period.The care of the animals was performed according to the Araçatuba School of Dentistry Ethical Committee on Animal Research (Process #2007-003230), which approved the project before the beginning of the experiments.

Plant extract preparation
The Aroeira (Myracrodruon urundeuva) plants were obtained from ecological reserves in Carolina, Maranhão state in Brazil.The leaves were collected if they appeared healthy and free of disease and pests.After collection, the plants were washed in water, dried at room temperature and in placed in a heater at 37°C until they became dry and friable, as previous protocols. 13,17,20Subsequently, they were pulverized.
The ethanolic and aqueous extracts were prepared according to the methodology described by Machado et al. 17

as follow:
Ethanolic extract: 20 g of leaf powder were mixed to 250 ml of 80% ethanol.The flask was mixed vigorously and manually for three minutes, five times a day, for 12 days.Subsequently the filtration was performed.The product was sterilized by filtration in cellulose ester membrane of 0.22 μm (Millipore ® ).
Aqueous extract: 20 g of leaf were added to 250 ml of distilled water, boiled at 100°C for 5 minutes and kept at 55°C for 1 hour at room temperature for 72 hours, agitated every 24 hours.The solution was filtered and sterile as mentioned before.

Microorganisms association
All microorganisms (Table 1) were grown in BHI and incubated under anaerobiosis conditions (90% N2 + 10CO2), at 37 o C for 24-48 hours.After this period, a suspension containing 10 9 cel/mL was prepared, and subjected to 3 successive washes in saline solution by centrifugation at 14,000 rpm for 5 minutes, to remove residues of the culture medium.Then, the precipitate was re-suspended in 3 mL of saline solution and maintained by 30 minutes in water bath at 60°C, for bacterial inactivation.

Edemogenic test -immediate reaction
For edema quantification, 18 Wistar rats were divided into 3 experimental groups, with 2 analysis periods (3h and 6h).The animals were anesthetized with xylazine (10 mg/kg) and ketamine (25 mg/kg), and received an intravenous injection of 1% Evan's blue (Evan's Blue; Difco Lab, Detroit, USA) with a concentration of 0.2 mL/100g body weight, in the penile vein.After 30 min, 0.1 mL of the inactivated bacteria-containing extracts, or saline, was injected in the dorsum, using the median line as a reference.After 3 and 6h, the animals were euthanatized by an anesthetic overdose and a tissue fragment standardized with 23mm diameter, containing a blue halo in the center, was dissected, macerated, and immersed in 4mL of formamide for 72 h at 45°C, and filtered for spectrophotometric analysis at a wave length of 630 nm. 17,20lyethylene tubes Sixty polyethylene tubes (Abbott Lab of Brazil, Sao Paulo, Brazil) with 1.0mm internal diameter x 1.6mm outer diameter x 10mm length, were obtained and the groups were accommodated inside.
In order to prevent leakage, one end of the tube was sealed with 1mm of gutta percha.Then, a sterile paper cone with a diameter compatible with the tube, was inserted, but accurately cut 0.2 mm below the tube length, avoiding direct contact with the tissue, offering conditions to retain the solutions inside the tubes.

Subcutaneous implant
For the histological analysis, 20 male Wistar albino rats (n = 10) were divided into groups according to the experimental period of analysis (7 and 30 days).The anesthetized animals were shaved in the back and the area was disinfected with a 5% iodine solution (Riodente, Rioquímica, São José do Rio Preto, Brazil).A 2 cm longitudinal incision was made in the dorsal region with a #15 blade.The tubes were implanted and the skin was closed with a 4/0 silk suture (Ethicon, Johnson & Johnson Produtos Profissionais Ltda., São José dos Campos, Brazil).Each animal received 3 implants containing the aqueous, ethanolic aroeira extracts and saline with inactivated bacteria, implanted in the right and left side of the animals' subcutaneous tissue.After the experimental time periods of 7 and 30 days, the animals were euthanatized by anesthetic overdose and the tubes with the surrounding tissue were removed and fixed in 10% formalin at pH 7.0 for 48 hours and then washed in water for 12 hours.The pieces were dehydrated, clarified, and included in paraffin, followed by longitudinally cut with 6 μm thickness, to be stained with hematoxylin and eosin for microscopic analysis (Leica, Germany).
The results obtained for inflammatory tissue response from extracts was compared to those of the control group under 100X and 400X magnification, to measure the thickness of the fibrous capsule and to count inflammatory cells, respectively.A descriptive analysis was performed for the three experimental groups.Tissue reactions at the open end of the tubes were scored according to previous studies 13,20,21 as follows: 0, few inflammatory cells or no reaction; 1, less than 25 cells and mild reaction; 2, between 25 and 125 inflammatory cells and moderate reaction; and 3, 125 or more inflammatory cells and severe reaction.Fibrous capsules were considered thin when < 150 μm and thick when > 150 μm. 13,20,21,22

Statistical analysis
Edemogenic test results were analyzed with an ANOVA and Tukey's test, and the Mann-Whitney and Kruskal-Wallis tests were used to analyze the histological data, using the SigmaPlot software (Systat V12.0).The significance level was set to 95% for all analyses (p < .05%).

Edemogenic analysis
Although there was no statistically significant difference (p > 0.05) between the two experimental time periods (3h and 6h), the inactivated bacteria-containing ethanolic aroeira extract induced more edema than the aqueous extract or saline solution (p < 0.05) (Figure 1).

Histological analysis
Histological analysis was conducted after 7 and 30 days, and a significant difference was observed for all three groups (p < 0.01) with decreased inflammation in the 30-day period (Figure 2).

Saline + Bacteria -7 days
The inflammatory response achieved a median score 3. The capsule was thick (> 150 μm) with inflammatory cells, predominated with macrophages, lymphocytes some neutrophils, and other mononucleated cells.Presence of rare fibroblasts, interspersed with few collagenous fibers, which appeared in a disorganized and complex arrangement (Figure 3A, Table 2).

Saline + Bacteria -30 days
This group presented a higher degree of tissue organization (median score 2) when compared to 7 days period, presenting a thin fibrous capsule composed of fibroblasts arranged parallel to the implant area.A macrophages conglomerate on the surface was also observed in contact with the implant area and presence of some lymphocytes and few other inflammatory cells.No sample showed capsule thickness greater than 150μm (Figure 3B; Table 2)

Aqueous Aroeira + Bacteria -7 days
Most of samples in this group presented severe inflammation (median score 3) in a thick capsule, with predominance of macrophages and numerous lymphocytes.A dense network of small caliber blood vessels within the fibrous capsule and larger adjacent tissues were observed.All samples had a capsule thickness greater than 150μm (Figure 3C; Table 2).

Aqueous Aroeira + Bacteria -30 days
The microscopic images indicate a decrease of the inflammatory infiltrate (median score 1) compared to the period of 7 days.The inflammatory cells agglomerate was replaced by a thin fibrous capsule.It was possible to identify the presence of some fibrocytes and some few fibroblasts among the collagen fibers.In general, the macrophage layer was more discrete and the amount of lymphocytes and other mononuclear cells was diminished.No capsule was thicker than 150 μm (Figure 3D; Table 2)

Ethanolic Aroeira + Bacteria -7 days
The fibrous capsule was also thick, rich in macrophages, lymphocytes, few leukocytes and other mononuclear inflammatory cells (median score 3).Some plasmocytes could also be observed.It was possible to observe predominance of fibroblasts, accompanied by rare and discrete collagen fibers presented in a disorganized tissue.In most of the samples the thickness of fibrous capsule was greater than 150 μm (Figure 3E; Table 2).

Ethanolic Aroeira + Bacteria -30 days
The fibrous capsule was clearly thinner and condensed, with a considerable reduction in the number of inflammatory cells (median score 1), evidencing macrophages along the entire surface of the implant area.The collagen fibers were more organized and the fibroblasts were with more elongated and thin nucleus (indicating a decrease of the collagen metabolism).

Discussion
The effect of natural compounds, such as plant extracts, on the oral biofilm and their impact on microbial ecolog y has been widely stud ied. 9,10,11,12,20,23,24,25,26The null hypothesis was rejected, once the extracts induced a mild inflammatory reaction after 30 days, similar to the control group.The biocompatibility of the aroeira extracts containing inactivated microorganisms were chosen, because the previously reported antimicrobial effect. 10he microbiota used in this study was composed of commonly isolated microorganisms from refractory and/or acute endodontic infections.It has been shown that associations of facultative anaerobes and anaerobes are capable of inducing inflammation. 2,27he methodology of inflammatory tissue response assessment with plant extract containing inactivated bacteria was recently reported by Machado et al. 20 Some anaerobic species lost their viability within the connective tissue of mice, even before the first trial period. 27For this reason, we opted to use inactivated microorganisms to avoid obtaining false positive results.Since the objective was to evaluate the influence of products and toxic by-products of microorganisms, standardization was required.
The P. gingivalis, P. endodontalis and P. intermedia were previously found in periapical disease 28 and they were selected due to presence of the lipopolysaccharide (LPS), which produces histolytic enzymes and the antigens induce the inflammatory response, 29 so as the F. nucleatum, capable of inducing a cutaneous inflammation in rats. 30The P. micros is also found in pulp necrosis and periapical lesion 28,31 and the E. faecalis is present in more than 80% of endodontic disease, considered a very resistant microorganism. 32,33he endotoxin from alive or dead bacteria triggers the release of inflammatory mediators such as TNF, interleukin-1, interleukin-6, interferon-alpha and prostaglandins, 34,35 even in rats, since several researches have reported that virulence factors in dead bacteria are capable of inducing inflammation in animals. 36,37ccording to Lima et al. 38 the aroeira has antimicrobial activity, anti-inflammatory and cicatrizant potential, which are related to chemical components present in different parts of the plant, such as tannins, flavonoids and triterpenes.Due to these properties, the aroeira presents an ample use within the medicine popular, which allows their choice in clinical studies.
Even though inactivated bacteria associated to ethanolic aroeira extract induced more edema than the other associations, after 30 days of subcutaneous implantation, the tissue response was similar to the aqueous extract.
The subcutaneous implantation method can result in an inflammatory process due to surgical trauma. 39herefore, edemogenic analysis is a more reliable way to test biocompatibility at the early time points.The edema was similar at the 3 and 6h time points for the three experimental groups.This corroborates the study by Machado et al. 17 who surveyed the same extracts without the addition of microorganisms, leading us to conclude that the presence of inactivated microorganisms does not affect edema.Both the aqueous and ethanolic aroeira extract-treated groups showed a slight decrease of edema over time; however, the ethanolic extract-induced edema was greater than that induced by the aqueous extract or saline.This could be due to the initial irritating effect of ethanol, and its metabolism in the body within 6h.
Histological analysis showed tissue repair as a function of time, as expected for all groups.This suggests that the inflammatory effect of inactivated microorganisms is greater at the early time points, because virulence factors and components, present in the membrane and/or the bacteria capsules, and were decreased by the body's inflammatory response over time.Further, other studies have demonstrated surgery trauma-induced inflammatory responses detected by histological analysis, 13,20,21 but the degree of inflammation was lower than that observed with microorganisms.
The results from this study confirmed that the tested extracts did not interfere negatively in the biological response, allowing normal tissue repair to occur.It is possible to speculate that the extracts inactivated bacterial toxins, once the aqueous extract had a beneficial effect at 7 days and both extracts had the tendency to be superior to saline after 30 days.This suggests that a component of the extracts can inhibit toxic compounds present in inactivated microorganisms.

Conclusion
Within the limitations of this study, the aqueous and ethanolic leaf extracts of Myracrodruon urundeuva containing inactivated microorganisms showed favorable tissue response, similarly to that obtained from the saline group.The slightly lower inflammation degree after 30 days for both extracts indicates a possible interference against the bacterial components, with a trend for better results than saline, suggesting future applications of these extracts in the dentistry field.

Figure 1 .
Figure 1.Graphical representation of the average edema value in both experimental time periods of 3 and 6 hours.

Figure 2 .Figure 3 .
Figure 2. Graphical representation of the position occupied by the average scores in the two experimental periods, for the three groups.

Table 1 .
Bacterial species used for the preparation of the suspension.

Table 2 .
Percentage of samples in each group categorized according to the inflammatory score and the rating thickness of fibrous capsule.