Histopathological Study of Radicular Cysts Diagnosed in a Brazilian Population

Radicular cysts (RCs) are the most common cysts affecting the jawbones. They are related to extensive caries lesions, pulp necrosis and infection of the root canal (1). RCs arise to control the infection as the lowintensity chronic stimulus triggered by bacterias and their products provides conditions to the organism, confining the aggression to the periapical region. During these events of defense, different cells produce cytokines, mainly interleukin 1 (IL-1), IL-6 and tumor necrosis factor alpha (TNF-α), which are involved in this immunoinflammatory response and play a role in bone resorption and in the stimulation of mitogenesis (2). In addition, foreign body-type material and other morphological findings might be observed in RCs, either potentiating the inflammatory process (3,4) or as a result of the defensive mechanism of the organism itself. Histopathologically, RCs consist of a cavity lined Histopathological Study of Radicular Cysts Diagnosed in a Brazilian Population


INTRODUCTION
Radicular cysts (RCs) are the most common cysts affecting the jawbones.They are related to extensive caries lesions, pulp necrosis and infection of the root canal (1).RCs arise to control the infection as the lowintensity chronic stimulus triggered by bacterias and their products provides conditions to the organism, confining the aggression to the periapical region.During these events of defense, different cells produce cytokines, mainly interleukin 1 (IL-1), IL-6 and tumor necrosis factor alpha (TNF-α), which are involved in this immunoinflammatory response and play a role in bone resorption and in the stimulation of mitogenesis (2).In addition, foreign body-type material and other morphological findings might be observed in RCs, either potentiating the inflammatory process (3,4) or as a result of the defensive mechanism of the organism itself.
Histopathologically, RCs consist of a cavity lined

Histopathological Study of Radicular Cysts Diagnosed in a Brazilian Population
by stratified squamous epithelium exhibiting spongiosis and/or exocytosis and are delimited by a fibrous wall containing a predominantly chronic inflammatory infiltrate (1).However, little has been described about the histopathological features and variations of RCs diagnosed in Brazilian individuals specifically.Thus, the aim of the present study was to describe the histopathological features and possible variations of RCs in a Brazilian population.

MATERIAL AND METHODS
After approval from the Ethics Committee of the Federal University of Bahia, 73 cases diagnosed as RCs were included in the study sample.This accounted for 4.9% out of 1,480 biopsies performed at the Laboratory of Surgical Pathology of the Dental School of the Federal University of Bahia, between 2001 and 2008.
The formalin-fixed, paraffin-embedded material L.C.S. Santos et al. was cut into 4-μm thick sections for morphological analysis.After staining with hematoxylin-eosin, the slides were revised by one experienced pathologist (J.N.S) who recorded the representative histopathological features of each lesion.The criterion used to name the lesions of radicular cyst was a well-delimited cavity lined by stratified squamous epithelium defined by Nair (1).
The inflammatory infiltrate was classified as discrete, moderate or intense, and the epithelium was classified as atrophic (2 to 10 cell layers) or hyperplastic (>10 cell layers) (5).Furthermore, other morphological features as apoptosis, mucosal cells, odontogenic epithelial remnants, eosinophilic bodies of Rushton, foamy macrophages, cholesterol crystals, and others were also included.Data regarding the gross pathology as size and cystic aspect were also obtained.Differences between groups were evaluated using Fisher's exact and Kruskal-Wallis tests.All statistical calculations were performed using the GraphPad Prism 4.0 program (GraphPad Inc., San Diego, CA, USA).A p-value <0.05 was considered to be statistically significant.

RESULTS
The histopathological findings are summarized in Table 1.Most lesions (69%) presented as a cystic formation by the gross pathology.The diameter of the cysts ranged from 0.5 to 7.6 cm, with a mean size of 1.94 cm (Fig. 1A,B).No information regarding the latter aspect was available in one case.
Statistical analysis was performed to determine a possible influence of these histological findings on the occurrence of RC.Comparison of the presence of exogenous material and epithelial thickness showed that among the 16 cases presenting exogenous material, 10 exhibited an atrophic epithelium and 6 a hyperplastic epithelium, with no significant difference between groups (Fisher's test, p=0.2448).No significant association between exogenous material and the degree of inflammation was observed (Kruskal-Wallis test, p=0.6514).A significant positive association was observed between the presence of Rushton bodies and apoptotic cells, with Rushton bodies being detected in 5 of the 21 cases presenting apoptotic cells (Fisher's test, p=0.0014).However, no significant association was observed between the presence of apoptotic cells and epithelial thickness (Fisher's test, p=0.2695).Analysis of the concomitant presence of foamy macrophages and cholesterol crystals in the same cyst revealed no significant association between these structures, with only one specimen containing  foamy macrophages out of 10 cases, in addition to the cholesterol crystals (Fisher's test, p=0.9507).
The inflammatory infiltrate observed in the sample was mainly chronic.This infiltrate was intense in 36 cases (49.3%), discrete in 22 (30.1%) and moderate in 15 (20.5%).Acute inflammation characterized by the presence of neutrophils was observed in 7 cases, and the infiltrate was mixed in 2 cases.

DISCUSSION
According to previous studies, RCs accounts for 6% to 57.69% of all periapical lesions (6,7).This variation might be attributed to differences in the definition of this type of cyst.
The mean diameter of the cysts studied here was 1.94 cm, a value similar to those reported by previous studies, although exists variation (1).Histologically, the lesions fulfilled the criteria adopted for radicular cysts (1).They consisted of a cystic wall lined by stratified nonkeratinized squamous epithelium, sometimes arranged in often interconnecting rings, which exhibited spongiosis and exocytosis and was hyperplastic or atrophic.Chronic inflammatory infiltrate, multinucleated giant cells and neutrophils were also observed in the cysts.These features are comparable to those described previously (1,5).
Ciliated epithelia is uncommon in radicular cyst (7,8).In this study, pseudostratified ciliated epithelium was exclusively observed in RCs located in the anterior region of the maxilla.Similar findings also were identified by Takeda et al. (8) and Nair et al. (9).This might be attributed to a possible metaplasia of stratified squamous epithelium as a consequence of the presence of remnant respiratory tissues during embryogenesis (8,9).Another explanation might be attributed to the inflammatory stimulation on the stratified squamous epithelium that differentiates by metaplasia into ciliated columnar epithelium (1).This event is influenced by the presence of fluids inside the cavity.However, the biological significance of this phenomenon remains unknown.
In the present study, 2 out of 3 cases exhibiting a ciliated epithelium also presented mucosal cells (8).These cells were only detected in 7 cases of this series.The frequency of mucosal cells (86%) and ciliated cells (100%) was higher in the maxilla.The occurrence of mucosal cells in odontogenic cysts is probably also due to a metaplastic phenomenon (8,10).Slabbert et al. (10) demonstrated the presence of vacuolized cells in almost 10% of their series.In contrast, in the present study was observed vacuolized cells in almost 20% of the samples.These cells were associated with mucosal cells scattered or in clusters throughout the layers of the stratified pavement epithelium, except for the basal layer (10).
Apoptosis is a mechanism of genetically programmed cell death which plays an important role in the control of cell populations (11).This study showed apoptotic cells in almost 30% of the cases.In contrast, Loyola et al. (10) detected these cells in all of their cases exhibiting mainly atrophic epithelium, by using bcl-2 immunostaining.However, Suzuki et al. (11), using p53, Bax, caspase-3, Fas and Fas-L, concluded that both proliferative activity and apoptosis are more frequent in RCs exhibiting a thick epithelium.This finding is in agreement with the present results as apoptotic cells were seen in most lesions containing a hyperplastic epithelium.However, this association was not significant.
Rushton bodies or hyaline bodies exhibiting a wide variety of shapes, including linear, round, lamellar or amorphous structures, were found in the epithelium of RCs (13,14).The presence of these structures depends on the sectioning plane of the material, with their incidence ranging from 2.6% to 10.3%.In the present study, Rushton bodies were detected in few RCS.They presented a lamellar shape without calcifications.The origin of hyaline bodies has not been well established.However, it is known that hyaline bodies result from the entrapment of blood vessels by the epithelium and consequent vessel thrombosis.Another hypothesis is that cell debris or cholesterol crystals present in the cyst might stimulate epithelial cells to secrete amorphous substances that later undergo to calcification, originating Rushton bodies (13).Despite these hypotheses, Pesce and Ferloni ( 14) and Loro et al. (15) suggested that these structures are formed by apoptosis of epithelial cells accompanied by intracellular dystrophic calcification.It is important to comment that Loyola et al. (11), studying apoptosis in RCs, did not observe the presence of hyaline bodies.Further studies are necessary to better understand the presence of these structures in RCs.
In the present study, odontogenic epithelial rests were detected in some cases.Although the role of epithelial rests of Malassez is unclear, it has been suggested that they are associated with tissue remodeling and to exert reparative functions such as the prevention of ankylosis, maintenance of the periodontal space and prevention of root surface resorption (16,17).In addition, we observed epithelial plaques resembling squamous odontogenic tumor (18).The odontogenic epithelium seems to have a potential for proliferation and differentiation into squamous cells, and also participates in tooth formation.However, it is still unknown which event is involved in the formation of this squamous odontogenic tumor-like epithelial tissue.Thus, it could be recommended that cystic lesions containing squamous odontogenic tumor-like epithelial plaques to be followed up for a longer period of time as signs and symptoms of the tumor are only detectable in advanced stages of the disease.
In agreement with other studies, lymphocytes and plasma cells were found associated with the fibrous capsule in the present investigation (6).The intensity of the inflammatory infiltrate varied, but intense inflammation was predominant.In RCs, the degree of inflammation might be associated with the proliferative activity of their epithelial cells (11).
Russell bodies also were present in several cases.These bodies are formed by the accumulation of immunoglobulins in endoplasmic reticulum cisterns found in chronically inflamed tissue (19).It is essential to recognize these structures, as described in a recent report on periapical lesion rich in Russell bodies and plasma cells, mimicking a malignant neoplasm (19).
Macrophages are involved in the innate response of the periapical lesion, phagocytosing bacteria and preventing the dissemination of infection, as well as in the acquired response, presenting antigens to lymphocytes through the MHC II system, synthesizing cytokines and producing metalloproteinases (20).These defense cells may also phagocytose cholesterol crystals and are called foamy macrophages (3,4,21).However, phagocytosis may not occur in the presence of some crystals exhibiting a large diameter, an event that induces the release of cytokines such as IL-1, impairing the repair process after endodontic treatment (3,6,(20)(21)(22)(23).Thus, the presence of cholesterol crystals is an important factor for lesion persistence (3,4,(20)(21)(22).These crystals may originate from the crystallization of lipids ingested from the vascular system itself, or may result from the production of local metabolites in the granulation tissue of the cystic wall and from the phagocytosis of the cell membranes of dead cells (21).
Extensive clusters of foamy macrophages are rare in endodontic lesions and, when present in large numbers, are suggestive of a proliferative process or a localized bone reaction.Periapical lesions exhibiting an abundance of these cells seem to be more aggressive and are associated with a larger number or recurrences even after correct conventional endodontic treatment, with these lesions often being treated by paraendodontic surgery (24).Foamy macrophages were observed in few cases of the present series, sometimes side by side with cholesterol crystals.However, no significant correlation was found between the presence of foamy macrophages and cholesterol crystals in RCs.This finding might be attributed to the fact that foamy macrophages already phagocytosed small lipid accumulations before the formation of larger crystals.
Exogenous material compatible with endodontic filling material was also observed close to the mineralization foci and foreign body-type reactions (25).The latter was also observed in the present study.Foreign body reactions occur concomitantly with intense inflammatory infiltrates containing macrophages and multinucleated giant cells (23).In addition, an intense inflammatory infiltrate was observed in 50% of cysts containing exogenous material, but comparison between the presence of this material and the different intensities of inflammatory infiltration showed no significant association.Analysis of the presence of exogenous material and epithelial thickness showed that among the cystic lesions containing exogenous material, most were characterized by the presence of an atrophic epithelium, whereas 37.5% presented a hyperplastic epithelium, with no significant difference between groups.Possibly, conventional endodontic treatment reduced the immunoinflammatory reactions in these cysts and, consequently, the power of cytokines and other substances involved in epithelial proliferation.
Finally, the present results revealed that RCs may show distinct histopathological findings in the same lesion, and this provides an important insight into the knowledge of the morphological variations of RCs.Based on the present study, it may be concluded that the clinical and histopathological findings observed in Brazilian patients were comparable with those described for other populations.

Figure 1 .
Figure 1.Radicular cyst.A = Gross pathology showing a circumscribed and encapsulated lesion, measuring 2.5x2.2x2.0 cm.The cut surface was cystic and filled with hemorrhagic content.Small protuberances can also be seen in the inner side of the cyst wall.B = Panoramic radiograph showing a large radiolucency circumscribed on the periapex of the mandibular right first molar.

Figure 2 .
Figure 2. Radicular Cyst.A = Cystic wall covered by stratified pavement epithelium arranged in interconnecting rings and exhibiting chronic inflammation; B = Cystic lining with hyperplasia and exocytosis of leukocytes; C = Cystic wall covered by epithelium showing atrophy and strong exocytosis of leukocytes, and chronic inflammation; D = Mucosal and vacuolized cells beside squamous cells; E = Apoptotic cells exhibiting eosinophilic cytoplasm and nuclear condensation; F = Squamous epithelium exhibiting metaplastic cells close to cystic lumen.

Figure 3 .
Figure 3. Radicular Cyst.A = Hyaline bodies of Rushton composed of lamellar structures surrounded by epithelial cells; B = Cystic wall showing marked accumulation of foamy macrophages; C = Epithelial remnants resembling squamous odontogenic tumorlike; D = Exogenous material and foreign body reaction with multinucleated giant cells and focal calcification; E = Cholesterol crystals and foreign body-type reaction with giant cells.

Table 1 .
Distribution of morphological alterations observed in radicular cysts from the pathology archives of the Dental School of the Federal University of Bahia, diagnosed between 2001 and 2008 (Salvador, BA, 2009).