INTRODUCTION

Periapical lesions (PL) are one of the most common pathological conditions that affect peri-radicular tissues in the alveolar bone ^1,2 and are mainly classified as radicular cysts or dental granulomas ^1,3-5. The microbial invasion and subsequent infection of the root canal system play a decisive role in the initiation, progression ^1,4, and establishment of peri-radicular conditions⁴ since bacteria and their by-products act as antigens that elicit a non-specific inflammatory response as well as specific immunological reactions in the peri-radicular tissues ².

The bacterial species present in the apical region may have a significant role in the pathogenesis of apical periodontitis ⁶. Gram-negative bacteria predominate in the root canal system of teeth with pulp necrosis and PL ⁶. Some bacterial virulence factors include the structural components and products of bacterial metabolism ⁶. It has been established that the levels of endotoxins in root canal infections are directly related to the severity of peri-radicular bone destruction ⁷. Lipopolysaccharides (LPS) that form part of the bacterial cell wall and act like endotoxins are especially important in endodontic infections because of their biological effects, which lead to a complex interplay with host factors ⁶, like chemical mediators of inflammation, including the cytokines IL - 1a, IL -1b, TNF-α and prostaglandins related to the pathogenesis of periapical lesions ⁵, resulting in clinical symptomatology, inflammatory reaction, and resorption of mineralized tissues⁶. Also, teichoic acid (TA) and lipoteichoic acid (LTA) are present in gram-positive bacteria and share their pathogenic properties with LPS, resulting in well-known injuries to the dental pulp and periapical tissues ⁶.

Most PL (>90%) can be classified as dental granulomas, radicular cysts, or abscesses ³, but the precise nature of such lesions can only be determined histologically; for this reason, the true prevalence of each pathological condition is unclear ⁵. PL should be treated initially by a non-surgical approach ¹. The purpose of the non-surgical root canal treatment is to shape and clean the root canal system ^{(1, 4)} to eliminate the necrotic tissue and infective bacteria and their antigens¹, and finally seal the root canal system three-dimensionally to prevent reinfection ⁴. PL usually heals as a response to meticulous non-surgical endodontic treatment ⁴. The primary root canal treatment yields predictable results with a survival rate of 95% after a 4-year follow-up ⁴. To assess the healing potential of a PL, a period of 6 to 12 months after root canal treatment should be considered, while complete healing of the PL lesion might take up to four years. However, treatment failure is possible due to different microbial and non-microbial factors ⁴, leading to persistent intra- or extra-radicular infection, and a surgical procedure should be considered ¹.

Nowadays, histopathological evaluation is the gold standard for diagnosing PL, but CBCT, MRI, and echography show promising results in differentiating granulomas and cysts ⁴. In addition, CBCT offers relatively high-resolution and isotropic images. Potential applications in endodontics include diagnosis and evaluation of most aspects of endodontic treatment, such as determination of the configuration and length of the root canal, presence of accessory canals, and PL evaluation ⁸.

The main objective of this study was to evaluate the amount and species of microorganisms isolated from necrotic pulps, establish a correlation between these and the size of periapical lesions; and to determine how the amount and species of microorganisms decreased after the non-surgical root canal treatment.

METHODS

This study was evaluated and authorized by the Research Ethics Committee of the Faculty of Stomatology, UASLP, with the code CEI-FE-019-016.

Twenty-seven systemically healthy patients (18-60 years old) with a clinical diagnosis of dental pulp necrosis and chronic PL associate (primary endodontic infection) were selected for the study.

RESULTS

DISCUSSION

The present study contributes to the knowledge about the amount and species of microorganisms isolated and identified from necrotic pulps, and determines a correlation between the initial size of a PL and the number of bacteria. It also corroborates that the root canal shaping, cleaning, and use of intracanal medication decreased the amount and species of bacteria identified, but do not eliminate them.

According to the obtained data, the bacteria most commonly identified from the root canal system were Gram-positive facultative anaerobes, corroborating previously reported literature ⁶. It has been established that the microorganisms in the biofilm are exposed to very different environmental conditions from those in planktonic form, and many species can change their metabolism depending on the surrounding physiological and physicochemical conditions ¹⁰. Peri-radicular dental biofilm is characterized by microorganisms adhered to the cementum, to the dentin, or both, in the apical portion of the root, surrounded by an external polysaccharide matrix (biofilm) that limits the access of defense molecules (antibodies and complement) and phagocytic cells (macrophages and neutrophils) ⁶. The microorganisms forming biofilms are more resistant to antimicrobials (up to 1000 times less susceptibility to specific antimicrobials) and the host immune defenses than their planktonic counterparts ¹⁰.

The purpose of root canal treatment is to debride and disinfect the root canal system and to eradicate intracanal bacteria or at least reduce them to a level below that necessary to heal and prevent periapical diseases or allow their resolution ¹¹. However, in some cases when the apical seal fails (apical filtration), the pathology persists ¹², due to residual microorganisms ⁴, or extra-radicular microorganisms ⁶, both with access to peri-radicular tissues maintaining the pathology⁴. In primarily infected root canals, microorganisms were able to access and colonize the pulpal tissue and impair its function ⁶. The most common pathologic factors in the alveolar bone derived from necrotic dental pulp are PL ¹³. Their microbial profile consists of 10-30 species per canal ⁶. According to the data obtained in our study, at least 21 different species of microorganisms were identified previously to the root canal disinfection. The species number identified post-disinfection and post-medication decreased to 11 species.

It has been reported that microorganisms like Fusobacterium, Porphyromonas, Prevotella, Parvimonas, Tannerella, Treponema, Dialister, Filifactor, Actinomyces, Olsenella, and Pseudoramibacter predominated in the root canal system; also, some facultative or microaerophilic streptococci are commonly found in primary infections ⁶. According to our data, the main microorganisms identified were Actinomyces naeslundii, Enterococcus faecalis, Aerococcus viridans, Streptococcus sanguis, and Fusobacterium nucleatum, corroborating previous reported literature. Cardoso et al. ⁷ revealed a positive correlation between root canal volume, determined by CBCT analysis, and CFU count found in primary endodontic infections with apical periodontitis ⁷. It also showed that the presence of selected bacteria species, such as L. buccalis, P. intermedia, C. gracilis, C. gingivalis, and C. sputigena, as well as their interaction in the form of complexes, was positively correlated with the presence of clinical features. Cardoso et al. also revealed that larger root canals hold higher levels of culturable bacteria. Thus, the interaction of different virulent bacteria species in complexes plays an important role in the development of clinical features⁷. This data is corroborated by the results reported in this study, in which there is a relation between the number of bacteria and the presence of a PL. In a study performed in Taiwan by Li-Wan Lee ¹⁴, it was found that the main species of bacteria identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry, were Porphyromonas endodontalis, Bacteroides fragilis, Dialister invisus, Fusobacterium nucleatum and Treponema denticola¹⁴, the differences of main species reported could be due to the difference of population evaluated and the laboratory techniques to isolate, culture and identify the microorganisms. One of the main reported specie is E. faecalis, a facultative gram-positive bacterium, capable of surviving in an environment with scarce availability of nutrients and minimal commensality with other bacteria. It presents different virulence and resistance mechanisms, which hinder its eradication from root canals ⁶.

Literature has reported that at a 6-month follow-up after the root canal treatment, only half of the cases exhibit signs of healing and that after a 12-month interval, 88% of these lesions exhibit signs of recovery. In contrast, complete healing of the peri-apical lesion might take up to four years ⁴. Our study corroborates these findings since the samples evaluated by CBCT 6 months after the root canal treatment showed a size reduction of the PL. The control and resolution of the associated infection and healing of PL depend on different factors, including the amount and species of bacteria related to the infection process and the capability of the immune system to control the remaining bacteria. Interactions of bacteria species and their grouping into complexes make endodontic infections even far more complex for the immune system response, which can lead to different clinical symptoms ⁷.

It has been reported that lesions ≤ 10 mm had an 80% of success rate while the larger ones showed a success rate of 53% ¹², then the largest periapical lesions are associated with the worst prognosis ¹². In our study, it was possible to prove a directly proportional relationship between the lesion size and the amount and number of bacterial species. Also, it has been established that the pathologic nature of the PL plays an essential role in the clinical evolution of the periapical disease; a true periapical cyst is less likely to heal after non-surgical root canal treatment and might require peri-radicular surgery ⁴.

A definitive diagnosis of peri-radicular cyst is reached only through histopathologic evaluation ^{(4, 13)} by serial cross-sectioning of the lesion specimen ⁴. But nowadays, the CBCT represents a non-invasive method for differentiating periapical cysts and granulomas ⁴. Also, represents an ideal method to evaluate the healing of a PL after root canal treatment or surgical endodontic treatment, consistent with the data reported in this study. Nevertheless, according to the American Association of Endodontists (AAE), CBCT should only be used when the required imaging question cannot be answered adequately by lower-dose conventional radiography or alternate imaging modalities ⁴.

The comprehension and understanding of the microbial characteristics in the root canal system play an essential role in the treatment and resolution of periapical diseases. This study determined that the amount and species of microorganisms isolated from necrotic pulps, established a correlation between the amount/species of microorganisms and the size of periapical lesions, and showed that the decrease of microorganisms through the non-surgical root canal treatment contributes to the healing of PL, corroborating the importance of an adequate disinfection protocol. It also established that the CBCT could be used as an objective method to evaluate the evolution of a PL after root canal treatment. However, further studies are needed to confirm the data reported.