- Email: [email protected]
Differences in Inflammation and Bone Resorption between Apical Granulomas, Radicular Cysts, and Dentigerous Cysts
CLINICAL RESEARCH
Differences in Inflammation and Bone Resorption between Apical Granulomas, Radicular Cysts, and Dentigerous Cysts
ABSTRACT Introduction: Dental cysts can be of inflammatory (radicular cysts) or noninflammatory (dentigerous cysts) origin. Apical periodontitis is a necrosis of the pulp and infection of the root canal causing the development of apical granulomas or radicular cysts. The immunology of granuloma and cyst formation is important because modern root filling materials are immunologically active and can contribute to the resolution of apical granulomas. In contrast, radicular cysts often require apicectomy. A better understanding of the pathophysiology of inflammation and bone resorption in apical periodontitis could be the basis for developing new root filling materials with superior immunomodulatory properties. Methods: Forty-one apical granulomas, 23 radicular cysts, and 23 dentigerous cysts were analyzed in this study. A tissue microarray of the 87 consecutive specimens was created, and human leukocyte antigen–DR isotype (HLA-DR)-, CD83-, receptor activator of nuclear factor kappa B ligand–, macrophage colony-stimulating factor (MCSF)-, galectin-3 (Gal3)-, CD4-, and CD8-positive cells were detected by immunohistochemistry. Tissue microarrays were digitized, and the expression of markers was quantitatively assessed. Results: HLA-DR, CD83, MCSF, and Gal3 expression was significantly (P , .05) higher in radicular cysts compared with apical granulomas. HLADR, CD83, MCSF, receptor activator of nuclear factor kappa B ligand, and Gal3 expression in dentigerous cysts was significantly (P , .05) lower than in both periapical lesions (apical granulomas and radicular cysts). CD4 and CD8 infiltration was not statistically different between apical granulomas and radicular cysts. Dentigerous cysts showed a significantly (P , .05) lower T-cell infiltration than apical periodontitis. The CD4/CD8 ratio was not significantly different between the analyzed groups. Conclusions: The development of radicular cysts in apical periodontitis is associated with an increased expression of myeloid inflammatory markers and bone resorption parameters. Antigen-presenting cells and myeloid cells might be more relevant for the pathogenesis of apical periodontitis than T cells. Increased inflammation might promote the formation of radicular cysts and more pronounced bone resorption. (J Endod 2019;-:1–9.)
KEY WORDS Apical granuloma; apical periodontitis; bone resorption; follicular cyst; periapical lesion; radicular cysts Infection and necrosis of the dental pulp causes apical periodontitis, which manifests as apical granulomas or radicular cysts. Both periapical lesions have an inflammatory origin but show a different clinical course1. In contrast to radicular cysts, dentigerous cysts have no inflammatory cause2 and are classified as developmental odontogenic cysts3. In most cases, apical periodontitis is initially treated by orthograde endodontic treatment4. If an apical lesion develops into a cyst, endodontic therapy alone is sometimes not sufficient, and apicectomy or even extraction of the affected teeth is required4.
JOE Volume -, Number -, - 2019
Manuel Weber, MD, DMD,* Jutta Ries, PhD,* €ttner-Herold, MD,† Maike Bu Carol-Immanuel Geppert, MD,‡ Marco Kesting, MD, DMD,* and Falk Wehrhan, MD, DMD*
SIGNIFICANCE Apical granulomas, radicular cysts, and dentigerous cysts show immunologic differences. High infiltration of HLA-DR– and CD83-positive proinflammatory cells is associated with radicular cyst formation. Additionally, bone resorption markers MCSF and Gal3 are significantly increased in radicular cysts compared with apical granulomas. The development of apical periodontists toward granulomas or radicular cysts could be immunologically controlled. Therefore, the use of root filling materials with antiinflammatory properties might be beneficial. From the Departments of *Oral and Maxillofacial Surgery and † Nephropathology and ‡Institute of Pathology, Friedrich-Alexander University €rnberg, Erlangen, Germany Erlangen-Nu Address requests for reprints to Dr Manuel Weber, Department of Oral and Maxillofacial Surgery, Friedrich-Alexander €rnberg, University Erlangen-Nu €ckstraße 11, 91054 Erlangen, Glu Germany. E-mail address: manuel.weber@ uk-erlangen.de 0099-2399/$ - see front matter Copyright © 2019 American Association of Endodontists. https://doi.org/10.1016/ j.joen.2019.06.014
Inflammation and Bone Resorption in Granulomas and Cysts
1
Although it is known that apical periodontitis is caused by inflammatory reactions5, the exact pathogenesis of the different periapical lesions is not yet understood. In a previous study, we analyzed macrophages in apical periodontitis. Tissue macrophages can occur in an M1 or M2 polarized state. M1 macrophages are proinflammatory and can contribute to tissue destruction. In contrast, M2 polarized macrophages act anti-inflammatory and mediate tissue regeneration and wound healing6–9. We could demonstrate that radicular cysts show a significant shift toward proinflammatory, M1 polarized macrophages compared with apical granulomas10. Dentigerous cysts had a significantly lower macrophage infiltration and M1 polarization compared with both periapical pathologies (apical granulomas and radicular cysts)10. These data indicate that radicular cyst formation is associated with M1 polarization of macrophages, and the development of granulomas or cysts might be immunologically determined10. Because apical periodontitis is characterized by inflammation and bone resorption11, additional inflammatory cells and signaling pathways associated with these processes were studied to better understand apical periodontitis. Human leukocyte antigen–DR isotype (HLA-DR) is an antigen presentation receptor expressed on antigen-presenting cells like dendritic cells (DCs) and macrophages12. HLADR–based antigen presentation is essential for the activation of CD4- and CD8-positive T cells and thus for the initiation of specific adaptive immune responses12. Proinflammatory M1 macrophages are characterized by a high HLADR expression, whereas anti-inflammatory M2 macrophages are HLA-DRlow13,14. Consequently, HLA-DR can be regarded as a relevant proinflammatory marker. Besides M1 polarized macrophages, mature myeloid DCs are highly potent antigen-presenting cells with proinflammatory properties15. These DCs show a high expression of CD83 and HLA-DR16. Inflammation is a relevant driver of bone resorption11,17, which is relevant for the formation and growth of cystic jaw bone lesions18. Bone resorption is mediated by osteoclasts19. Receptor activator of nuclear factor kappa B ligand (RANKL) and macrophage colony-stimulating factor (MCSF) signaling are the most relevant pathways for osteoclast differentiation and activation19,20. Antigen presentation in combination with specific coactivating signals leads to adaptive T-cell responses21. In chronic infectious diseases like human immunodeficiency virus infection,
2
Weber et al.
T-cell–mediated inflammation can contribute to bone loss22. In osteoporosis, T cells contribute to bone loss via RANKL signaling23. Galectin-3 (Gal3) is expressed in macrophages and contributes to the regulation of innate and adaptive immune responses. Gal3 is also present in differentiating osteoblasts and osteoclasts and might contribute to the osteoblast-osteoclast cross talk24. Anti-inflammatory as well as proinflammatory effects of Gal3 were described24,25. In rheumatoid arthritis, increased Gal3 levels were detected in inflamed joints24,26. Rats with induced arthritis showed increased Gal3 expression in macrophages in areas with severe bone destruction24. The root filling material most suitable for endodontic treatment is a subject of constant debate27–29. The use of mineral trioxide aggregate (MTA) as root-end filling material during endodontic treatment or apicectomy has shown good clinical results30–32. This might be explained by the immune modulatory properties of MTA because it was shown that MTA induces a shift toward anti-inflammatory M2 polarization of macrophages in vivo33. Thus, M2 polarizing agents, like MTA, might antagonize M1 polarization associated with radicular cysts10 and therefore contribute to the prevention of cyst formation in apical periodontitis. Because MTA also has negative properties including dislocation, discoloration, or difficulties of removal in endodontic retreatment31, a better understanding of the pathophysiologic processes in apical periodontitis could pave the way for the development of superior new apical sealing materials in endodontic treatment. The current study was intended to clarify if the expression of parameters of inflammation (HLA-DR, CD83, CD4, and CD8) and bone resorption (RANKL, MCSF, and Gal3) differ between noncystic periapical lesions (apical granulomas) and cystic periapical lesions (radicular cysts). Additionally, inflammatory odontogenic cysts (radicular cysts) were compared with developmental odontogenic cysts (dentigerous cysts).
with no clinical signs of acute infection. Surgery was performed under local anesthesia or general anesthesia. Complete enucleation/ curettage was performed in all cases. There was no case with large jaw cysts requiring continuity resection included. No surgical procedure specific to this study was performed because tissue samples from routine histologic diagnostics were analyzed. Each specimen included was confirmed to show representative regions of apical granulomas, radicular cysts, and dentigerous cysts. In addition to clinical information, the following histologic criteria were used to differentiate between the pathologies: apical granulomas represent an advanced form of apical periodontitis in which the growth of granulomatous tissue attempts to confine irritating agents escaping from root canal. Granulomatous tissue is composed of acute and chronic inflammation (lymphocytes variably intermixed with neutrophils, plasma cells, histiocytes, mast cells, and eosinophils) in a fibrotic stroma surrounded by a fibrous capsule. In some cases, epithelial rests of Malassez (epithelial layer) can be identified. Dentigerous and radicular cysts have few common histologic features. They are both layered by nonstratified, nonkeratinizing squamous epithelium with scattered mucinous cells. In some cases, radicular cysts can have hyalinized calcification called “Rushton bodies,” even if the certain diagnosis is made on the relation of the cyst with the tooth, because periapical cysts are always related to a nonvital element, whereas follicular cysts are associated with an impacted tooth. Patients with a history of malign or autoimmune diseases as well as irradiated patients were excluded. The study cohort consisted of 41 apical granulomas, 23 radicular cysts, and 23 dentigerous cysts. Apical granulomas were obtained from 41 patients (28 males and 13 females) with a mean age of 57 years, radicular cysts were derived from 21 patients (13 males and 8 females) with a mean age of 52 years, and dentigerous cysts were obtained from 15 patients (10 male and 5 female) with a mean age of 22 years.
MATERIALS AND METHODS
Tissue Microarray Construction and Immunohistochemical Staining
Patients and Tissue Harvesting A previously described10 cohort of 87 consecutive specimens of apical granulomas, radicular cysts, and dentigerous cysts was analyzed in this study. The study protocol was approved by the local ethical committee of the University of Erlangen-Nuremberg, Erlangen, Germany (reference number 351_15 Bc). Specimens were obtained during dental surgery procedures in consecutive patients
A tissue microarray (TMA) for apical granulomas, radicular cysts, and dentigerous cysts was assorted as previously described10. For radicular cysts and dentigerous cysts, stroma tissue in direct proximity to the cyst lumen was selected for TMA construction. Each tissue core had a diameter of 1.5 mm. Immunohistochemical staining and quality control were performed as previously described10,34.
JOE Volume -, Number -, - 2019
FIGURE 1 – Virtual microscopy of a TMA slide (1!, 7!, and 35! magnification). The figure shows an overview of a TMA slide stained for CD8-positive cells (1! magnification) and a scheme of the TMA indicating the number of each tissue sample. Samples 5 and 27 are exemplarily selected and magnified (7! and 35! magnification).
The following primary antibodies were used: anti–HLA-DR (M0746, monoclonal mouse, 1:250; Dako Agilent, Santa Clara, CA), anti-CD83 (sc-19677, monoclonal mouse, 1:100; Santa Cruz, Dallas, TX), anti-MCSF (ab-183316, monoclonal rabbit, 1:100; Abcam, Cambridge, UK), anti-RANKL (ab9957, polyclonal rabbit, 1:100, Abcam) anti-Ga13 (sc-20157, clone H-160, polyclonal rabbit, Santa Cruz), anti-CD4 (ORG-8756, clone 1F6, monoclonal mouse, 1:10; Novocastra, Newcastle, UK), and anti-CD8 (IS62330-2, clone c8/144B, monoclonal mouse, 1:100, Dako Agilent).
Quantitative Immunohistochemical Analysis TMAs were completely scanned and digitized using the method of “whole slide imaging” (Fig. 1). The scanning procedure was performed in cooperation with the Institute of Pathology of the University of Erlangen€rnberg using a Pannoramic 250 Flash III Nu Scanner (3D Histech, Budapest, Hungary) in the 40! magnification mode. Scanning and virtual microscopy were performed as previously described10. For each TMA sample, 2 visual fields showing the highest expression rates of each marker were selected (hot spot analysis) (Fig. 2A–E). The area analyzed per tissue sample and marker was 1.4 mm2. Micrographs of the selected areas were imported into Biomas software (MSAB, Erlangen, Germany) for cell counting. Quantitative analysis was performed to determine the number of infiltrating HLA-DR-, CD83-, RANKL-, M-CSF-, Gal3-, CD4-, and
JOE Volume -, Number -, - 2019
CD8-positive cells in all specimens. Assessment of the cell density per mm2 was performed as previously described10,34,35.
Statistical Analysis To analyze the immunohistochemical staining and spatial distribution patterns, the cell count per mm2 was determined. The results are expressed as median and standard deviation. Box plot diagrams represent the median, interquartile range, minimum, and maximum. Two-sided, adjusted P values .05 were considered significant. The analyses were performed using the Mann-Whitney U test with SPSS 22 for Mac OS (IBM Corp, Armonk, NY).
RESULTS Inflammation and Bone Resorption Marker Expression in Apical Granulomas, Radicular Cysts, and Dentigerous Cysts The HLA-DR cell count in radicular cysts was significantly higher than in apical granulomas (median 5 1959 cells/mm2 and 334 cells/ mm2, respectively; P 5 .017) and also significantly higher than in dentigerous cysts (median 5 43 cells/mm2, P , .001; Table 1, Fig. 3A). Compared with dentigerous cysts, apical granulomas showed a significantly higher (P , .001) HLA-DR expression (Table 1, Fig. 3A). Radicular cysts revealed a significantly increased CD83 expression compared with apical granulomas (median 5 43 cells/mm2 and 5 cells/mm2, respectively; P 5 .003) and dentigerous cysts (median 0 cells/mm2,
P , .001; Table 1, Fig. 3B). Moreover, CD83 expression in apical granulomas was significantly higher (P 5 .006) than in dentigerous cysts (Table 1, Fig. 3B). RANKL expression in radicular cysts was significantly higher than in dentigerous cysts (median 5 4841 cells/mm2 and 1464 cells/mm2, respectively; P , .001). The difference in RANKL expression between apical granulomas and radicular cysts was statistically nonsignificant (Table 1, Fig. 3C). Compared with dentigerous cysts, apical granulomas (median 5 4487 cells/mm2) displayed a significantly higher (P , .001) RANKL expression (Table 1, Fig. 3C). MCSF expression in radicular cysts was significantly higher than in apical granulomas (median 5 94 cells/mm2 and 10 cells/mm2, respectively; P , .001) and also significantly higher than in dentigerous cysts (median 5 4 cells/mm2, P , .001; Table 1, Fig. 3D). Apical granulomas had a significantly higher (P 5 .032) MCSF expression than dentigerous cysts (Table 1, Fig. 3D). Radicular cysts displayed a significantly higher density of Gal3-positive cells (median 5 3551 cells/mm2) compared with apical granulomas (median 5 1141 cells/mm2, P , .001) and dentigerous cysts (median 5 291 cells/mm2, P , .001; Table 1, Fig. 3E). Gal3 expression in apical granulomas was significantly higher (P , .001) compared with dentigerous cysts (Table 1, Fig. 3E). In summary, all analyzed inflammation and bone resorption markers showed the highest expression in radicular
Inflammation and Bone Resorption in Granulomas and Cysts
3
A
B
C
D
E
F
G
FIGURE 2 – Typical expression patterns of the analyzed inflammatory, bone resorption, and T-cell markers. Micrographs show the typical expression pattern of all markers (HLA-DR, CD83, RANKL, MCSF, Gal3, CD4, and CD8) in (left column ) apical granulomas, (middle column ) radicular cysts, and (right column ) dentigerous cysts. All micrographs are given in high-power magnification (35! magnification). (A ) HLA-DR and (B ) CD83 show a cytoplasmatic staining with accentuation of the plasma membrane. (C ) RANKL and (D ) MCSF stainings display a cytoplasmatic expression pattern. (E ) Gal3 has a cytoplasmic and nuclear expression. (F ) CD4 and (G ) CD8 reveal a membranous expression pattern. cysts compared with apical granulomas and radicular cysts.
T-cell Infiltration and Expression Ratios in Apical Granulomas, Radicular Cysts, and Dentigerous Cysts Dentigerous cysts showed a significantly lower infiltration by CD4-positive T cells than radicular cysts (median 5 31 cells/
4
Weber et al.
mm2 and 597 cells/mm2, respectively; P , .001) and apical granulomas (median 5 568 cells/mm2, P , .001; Table 2, Fig. 4A). The difference in the CD4 cell count between apical granulomas and radicular cysts was statistically nonsignificant (Table 2, Fig. 4A). Both apical granulomas and radicular cysts showed a significantly higher infiltration of CD8-positive T cells compared with
dentigerous cysts (median 5 298 cells/mm2, 388 cells/mm2, and 63 cells/mm2, respectively; P , .001 for both; Table 2, Fig. 4B). No significant difference in CD8 expression was detected between apical granulomas and radicular cysts (Table 2, Fig. 4B). The CD4/CD8 ratio revealed no significant differences between apical granulomas, radicular cysts, and dentigerous cysts (Table 2, Fig. 4C). There was no difference
JOE Volume -, Number -, - 2019
TABLE 1 - Inflammation and Bone Resorption Marker Expression (Cells/mm2) in Apical Granulomas, Radicular Cysts, and Dentigerous Cysts HLA-DR
Cell count (cells/mm2) Marker Group Apical granuloma Radicular cyst Dentigerous cyst P values Apical granuloma vs radicular cyst Apical granuloma vs dentigerous cyst Radicular cyst vs dentigerous cyst
n 41 23 23
CD83
RANKL
MCSF
Gal3
Median
SD
Median
SD
Median
SD
Median
SD
Median
SD
334 1959 43
4773 2557 109
5 42 0
134 201 17
4487 4841 1464
2012 1687 1382
10 94 4
336 520 25
1141 3551 291
2552 3723 663
.017 ,.001 ,.001
.003 .006 ,.001
.419 ,.001 ,.001
,.001 .032 ,.001
,.001 ,.001 ,.001
SD, standard deviation. Values represent median, SD, and P value (Mann-Whitney U test, SPSS 22).
A
B
C
D
E
FIGURE 3 – Inflammation and bone resorption marker expression (cells/mm2) in apical granulomas, radicular cysts, and dentigerous cysts. Box plots show the median cell counts (positive cells/mm2) of inflammation and bone resorption markers ([A] HLA-DR, [B] CD83, [C] RANKL, [D] MCSF, and [E] Gal3) in apical granulomas, radicular cysts, and dentigerous cysts. All P values generated by the Mann-Whitney U test are indicated. Expression of the analyzed inflammation and bone resorption markers is significantly increased in radicular cysts compared with apical granulomas and dentigerous cysts, except for (C ) RANKL expression, which shows no significant difference between radicular cysts and apical granulomas.
JOE Volume -, Number -, - 2019
Inflammation and Bone Resorption in Granulomas and Cysts
5
TABLE 2 - T-cell Infiltration (Cells/mm2) and CD4/CD8 Expression Ratio in Apical Granulomas, Radicular Cysts, and Dentigerous Cysts Cell count (cells/mm2) and expression ratios
CD4
Ratio Group Apical granuloma Radicular cyst Dentigerous cyst P values Apical granuloma vs radicular cyst Apical granuloma vs dentigerous cyst Radicular cyst vs dentigerous cyst
n 33 20 13
CD8
CD4/CD8
Median
SD
Median
SD
Median
SD
568 597 31
882 486 208
298 388 34
367 356 63
1.48 1.47 1.34
5.30 0.88 4.92
.849 ,.001 ,.001
.359 ,.001 ,.001
.408 .262 .570
SD, standard deviation. Values represent median, SD, and P value (Mann-Whitney U test, SPSS 22).
in the expression of all analyzed markers between mandibular and maxillary cysts.
DISCUSSION Proinflammatory Antigenpresenting Cells in Apical Periodontitis and Dentigerous Cysts Inflammation of periapical tissue can cause the development of apical granulomas or radicular cysts. We were able to detect significantly increased densities of HLA-DR– and CD83expressing cells in radicular cysts compared with apical granulomas. CD83 is a marker for mature dendritic cells capable of efficient antigen presentation and T-cell activation15,16. HLA-DR is expressed by mature myeloid dendritic cells and M1 polarized,
A
proinflammatory macrophages but also by other cell types capable of antigen presentation15,16. Therefore, the increased expression of HLA-DR and CD83 in radicular cysts indicates that these lesions have a significantly stronger proinflammatory state of immunoreactivity compared with apical granulomas. Previously, we analyzed macrophage infiltration and polarization in apical granulomas and radicular cysts10. Proinflammatory M1 macrophages contribute to inflammation, antigen clearance, and tissue destruction, whereas anti-inflammatory M2 macrophages mediate peripheral immune tolerance, tissue regeneration, and wound healing6–9. Macrophage polarization showed a significant swift toward proinflammatory M1
polarization in radicular cysts compared with apical granulomas10. However, total macrophage density was not significantly different between apical granulomas and radicular cysts10. These results are interesting in the context of the increased HLA-DR and CD83 cell density detected in radicular cysts in the current study. The increased M1 polarization of macrophages in radicular cysts is accompanied by an augmented infiltration of mature CD83-positive dendritic cells and an increased HLA-DR expression and maybe by myeloid cells like proinflammatory macrophages and dendritic cells. Consequently, the development of apical periodontitis into a radicular cyst appears to be associated with a proinflammatory state.
B
C
FIGURE 4 – The T-cell count (cells/mm2) and CD4/CD8 expression ratio in apical granulomas, radicular cysts, and dentigerous cysts. Box plots show the median T-cell counts (positive cells/mm2) of (A ) CD4- and (B ) CD8-positive cells in apical granulomas, radicular cysts, and dentigerous cysts. (C ) The CD4/CD8 ratio. All P values generated by the Mann-Whitney U test are indicated.
6
Weber et al.
JOE Volume -, Number -, - 2019
In contrast, dentigerous cysts showed a significantly lower HLA-DR and CD83 infiltration compared with radicular cysts. This indicates that inflammation is not the driving force in the progression of jaw bone cysts with developmental origin. With regard to apical periodontitis, the results of the current study show that increased inflammation is associated with cyst formation. The results underline the importance of counteracting bacterial infection of the periapical region by sufficient endodontic treatment and thus eliminating the trigger of inflammation36. In addition, there is evidence that root filling materials also influence an immunologic response in periapical tissues33 and may therefore be prognostically relevant. Consequently, the used root filling materials and sealers should be investigated for their immunomodulatory properties. In this regard, materials with antiinflammatory properties33 might be beneficial in the prevention of radicular cyst formation.
T-cell Infiltration in Apical Periodontitis and Dentigerous Cysts In contrast to macrophages and dendritic cells, there was no significant difference in the infiltration of CD4- and CD8-positive T cells between apical granulomas and radicular cysts. Additionally, the CD4/CD8 ratio did not reveal a significant difference between both analyzed periapical lesions. A previous immunohistochemical analysis of apical granulomas and radicular cysts accordingly revealed no significant difference regarding the infiltration of CD8positive T cells37. However, another report describes a significantly increased infiltration of CD8-positive T cells in radicular cysts compared with apical granulomas5. A recent study analyzed programmed cell death 1 (PD1), an immune regulatory receptor expressed predominantly by activated T cells38. PD1 expression was significantly increased in apical granulomas compared with radicular cysts38. The increased expression of the immune-suppressive receptor PD139 in apical granulomas is in line with the results of the current study, which showed a significantly lower expression of the proinflammatory markers HLA-DR and CD83 in apical granulomas compared with radicular cysts. Additionally, a significantly increased
expression of FoxP3-positive regulatory T cells in apical granulomas compared with radicular cysts was described40. These data support the hypothesis that the formation of cystic periapical lesions might be determined by the immune system and associated with increased inflammation. Results of the current study indicate that CD4 and CD8 T-cell infiltration in dentigerous cysts is significantly lower than in apical periodontitis. However, the CD4/CD8 ratio revealed no significant difference between all analyzed lesions. These data suggest that a shift in the CD4/CD8 ratio is not involved in the pathogenesis of cystic jawbone lesions.
Bone Resorption Parameters in Apical Periodontitis and Dentigerous Cysts Osteoclasts are the primary bone-resorbing cells essential for physiologic bone homeostasis41. Analogous to macrophages, osteoclasts derive from mononuclear precursor cells of the myeloid lineage. The main cytokines that promote differentiation and activation of osteoclasts are MCSF and RANKL41. Under physiologic conditions, osteoblasts are the main source of MCSF and RANKL, maintaining the balance between bone formation and bone resorption. However, inflammatory cells can also produce MCSF and RANKL and thus induce bone resorption41. For any kind of expanding pathology in bone, it is essential to activate osteoclasts. For this reason, bisphosphonates or RANKL inhibitors, which block osteoclasts, are used for the treatment of bone metastases42,43. The current study revealed significantly increased MCSF expression in radicular cysts compared with apical granulomas. Elevated MCSF expression might be an indicator of increased activation of osteoclasts and consecutive bone resorption in radicular cysts. In contrast to MCSF, RANKL was not significantly up-regulated in radicular cysts in comparison with apical granulomas. MCSF and RANKL were significantly lower in dentigerous cysts compared with both periapical lesions. This finding is consistent with the lower expression of inflammatory cells in dentigerous cysts and could indicate that inflammation-driven apical periodontitis has a higher bone resorptive potential than developmental cysts, which are associated with a low inflammatory state. Results of the
current study are contrary to previous analyses comparing RANKL expression in radicular and dentigerous cysts, which could detect no significant difference in the expression of the cytokine18,44. However, in contrast to the current report, no quantitative assessment was used. Gal3 is a phylogenetically highly conserved lectin involved in a variety of immunologic and cell biologic processes. In the context of malignant diseases, Gal3 contributes to immunosuppression and tumor progression25. In inflammatory pathologies, Gal3 is involved in tissue remodeling and bone resorption24,26. We could show that Gal3 expression was significantly up-regulated in radicular cysts compared with apical granulomas. In dentigerous cysts, Gal3 was significantly lower than in both periapical pathologies. These data indicate that Gal3 expression in apical periodontitis is associated with inflammation induced bone resorption.
CONCLUSION The development of radicular cysts might be an immunologically controlled process. The increased infiltration of proinflammatory cells in radicular cysts is accompanied by an increased expression of bone resorption– associated cytokines. Antigen-presenting cells seem to be more relevant for the pathophysiology of apical periodontitis than T cells. In contrast to apical periodontitis, the progression of dentigerous cysts seems to be less dependent on immunologic factors.
ACKNOWLEDGMENTS The authors thank Rudolf Jung for manufacturing the tissue microarrays and Susanne Schoenherr and Elke Diebel for technical assistance. We would also like to thank the dental students Sabrin Abu-Hossin, €ring for Franziska Gerhardt, and Lisa Go processing the tissue specimens and operating the immunohistochemistry autostainer apparatus; Tilo Schlittenbauer for collecting the tissue samples; and Luitpold Distel for his assistance in the cell counting procedure. The authors deny any conflicts of interest related to this study.
REFERENCES 1.
JOE Volume -, Number -, - 2019
Lin LM, Huang GT, Rosenberg PA. Proliferation of epithelial cell rests, formation of apical cysts, and regression of apical cysts after periapical wound healing. J Endod 2007;33:908–16.
Inflammation and Bone Resorption in Granulomas and Cysts
7
2.
de Moraes M, da Rocha Neto PC, de Matos FR, et al. Immunoexpression of transforming growth factor beta and interferon gamma in radicular and dentigerous cysts. J Endod 2014;40:1293–7.
3.
Wright JM, Odell EW, Speight PM, Takata T. Odontogenic tumors, WHO 2005: where do we go from here? Head Neck Pathol 2014;8:373–82.
4.
Del Fabbro M, Corbella S, Sequeira-Byron P, et al. Endodontic procedures for retreatment of periapical lesions. Cochrane Database Syst Rev 2016:CD005511.
5.
Rodini CO, Lara VS. Study of the expression of CD681 macrophages and CD81 T cells in human granulomas and periapical cysts. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2001;92:221–7.
6.
Mantovani A, Sica A, Locati M. New vistas on macrophage differentiation and activation. Eur J Immunol 2007;7:14–6.
7.
Hirata Y, Tabata M, Kurobe H, et al. Coronary atherosclerosis is associated with macrophage polarization in epicardial adipose tissue. J Am Coll Cardiol 2011;58:248–55.
8.
Mantovani A, Biswas SK, Galdiero MR, et al. Macrophage plasticity and polarization in tissue repair and remodelling. J Pathol 2013;229:176–85.
9.
€ttner-Herold M, Hyckel P, et al. Small oral squamous cell carcinomas with nodal Weber M, Bu lymphogenic metastasis show increased infiltration of M2 polarized macrophages–an immunohistochemical analysis. J Craniomaxillofac Surg 2014;42:1087–94.
10.
Weber M, Schlittenbauer T, Moebius P, et al. Macrophage polarization differs between apical granulomas, radicular cysts, and dentigerous cysts. Clin Oral Investig 2018;22:385–94.
11.
Hienz SA, Paliwal S, Ivanovski S. Mechanisms of bone resorption in periodontitis. J Immunol Res 2015;2015:615486.
12.
Zhuang Y, Peng H, Chen Y, et al. Dynamic monitoring of monocyte HLA-DR expression for the diagnosis, prognosis, and prediction of sepsis. Front Biosci (Landmark Ed) 2017;22:1344–54.
13.
Shimada S, Ebina Y, Iijima N, et al. Decidual CD68(1) HLA-DR(1) CD163(-) M1 macrophages increase in miscarriages with normal fetal chromosome. Am J Reprod Immunol 2018;79. https:// doi.org/10.1111/aji.12791.
14.
Lin R, Zhang J, Zhou L, Wang B. Altered function of monocytes/macrophages in patients with autoimmune hepatitis. Mol Med Rep 2016;13:3874–80.
15.
Seldon TA, Pryor R, Palkova A, et al. Immunosuppressive human anti-CD83 monoclonal antibody depletion of activated dendritic cells in transplantation. Leukemia 2016;30:692–700.
16.
Maney NJ, Reynolds G, Krippner-Heidenreich A, Hilkens CM. Dendritic cell maturation and survival are differentially regulated by TNFR1 and TNFR2. J Immunol 2014;193:4914–23.
17.
Nogueira AV, de Molon RS, Nokhbehsaim M, et al. Contribution of biomechanical forces to inflammation-induced bone resorption. J Clin Periodontol 2017;44:31–41.
18.
de Moraes M, de Lucena HF, de Azevedo PR, et al. Comparative immunohistochemical expression of RANK, RANKL and OPG in radicular and dentigerous cysts. Arch Oral Biol 2011;56:1256–63.
19.
Adamopoulos IE. Inflammation in bone physiology and pathology. Curr Opin Rheumatol 2018;30:59–64.
20.
Li ZH, Si Y, Xu G, et al. High-dose PMA with RANKL and MCSF induces THP1 cell differentiation into human functional osteoclasts in vitro. Mol Med Rep 2017;16:8380–4.
21.
Murphy K, Weaver C. Janeway’s Immunobiology. Boca Raton, FL: CRC Press; 2016.
22.
Weitzmann MN, Vikulina T, Roser-Page S, et al. Homeostatic expansion of CD41 T cells promotes cortical and trabecular bone loss, whereas CD81 T cells induce trabecular bone loss only. J Infect Dis 2017;216:1070–9.
23.
Weitzmann MN. T-cells and B-cells in osteoporosis. Curr Opin Endocrinol Diabetes Obes 2014;21:461–7.
24.
Iacobini C, Fantauzzi CB, Pugliese G, Menini S. Role of galectin-3 in bone cell differentiation, bone pathophysiology and vascular osteogenesis. Int J Mol Sci 2017;18:E2481.
25.
€ttner-Herold M, Distel L, et al. Galectin 3 expression in primary oral squamous cell Weber M, Bu carcinomas. BMC Cancer 2017;17:906.
26.
he -Okouma M, Ea HK, et al. Galectin-3: a key player in arthritis. Joint Bone Spine Hu Y, Y ele 2017;84:15–20.
8
Weber et al.
JOE Volume -, Number -, - 2019
JOE Volume -, Number -, - 2019
27.
Ma X, Li C, Jia L, et al. Materials for retrograde filling in root canal therapy. Cochrane Database Syst Rev 2016;12:CD005517.
28.
Ay H, Duane B. Limited evidence on best material for retrograde root fillings. Evid Based Dent 2018;19:8–9.
29.
Tomson RM, Polycarpou N, Tomson PL. Contemporary obturation of the root canal system. Br Dent J 2014;216:315–22.
30.
Alsulaimani RS. Single-visit endodontic treatment of mature teeth with chronic apical abscesses using mineral trioxide aggregate cement: a randomized clinical trial. BMC Oral Health 2016;16:78.
31.
Torabinejad M, Parirokh M, Dummer PM. Mineral trioxide aggregate and other bioactive endodontic cements: an updated overview - part II: other clinical applications and complications. Int Endod J 2018;51:284–317.
32.
Torabinejad M, Parirokh M. Mineral trioxide aggregate: a comprehensive literature review–part II: leakage and biocompatibility investigations. J Endod 2010;36:190–202.
33.
Ito T, Kaneko T, Yamanaka Y, et al. M2 macrophages participate in the biological tissue healing reaction to mineral trioxide aggregate. J Endod 2014;40:379–83.
34.
Weber M, Iliopoulos C, Moebius P, et al. Prognostic significance of macrophage polarization in early stage oral squamous cell carcinomas. Oral Oncol 2016;52:75–84.
35.
€ttner-Herold M, et al. Macrophage polarisation changes within the time Weber M, Moebius P, Bu between diagnostic biopsy and tumour resection in oral squamous cell carcinomas–an immunohistochemical study. Br J Cancer 2015;113:510–9.
36.
Kim DS, Shin MR, Kim YS, et al. Anti-inflammatory effects of glutamine on LPS-stimulated human dental pulp cells correlate with activation of MKP-1 and attenuation of the MAPK and NF-kappaB pathways. Int Endod J 2015;48:220–8.
37.
MA, Melo RA, et al. Analysis of CD571 natural killer cells and CD81 T lymphocytes in Silva L, Sa periapical granulomas and radicular cysts. Braz Oral Res 2017;31:e106.
38.
Wang HS, Yang FH, Li Y, et al. The expression of PD-1 and LAG-3 in periapical lesions. Am J Transl Res 2018;10:2677–84.
39.
Sharpe AH, Pauken KE. The diverse functions of the PD1 inhibitory pathway. Nat Rev Immunol 2018;18:153–67.
40.
Peixoto RF, Pereira Jdos S, Nonaka CF, et al. Immunohistochemical analysis of FoxP31 cells in periapical granulomas and radicular cysts. Arch Oral Biol 2012;57:1159–64.
41.
Lampiasi N, Russo R, Zito F. The alternative faces of macrophage generate osteoclasts. Biomed Res Int 2016;2016:9089610.
42.
€lscher T, Hakenberg OW, Wirth MP. Treatment of bone metastases in urologic Froehner M, Ho malignancies. Urol Int 2014;93:249–56.
43.
Jacobs C, Amir E, Paterson A, et al. Are adjuvant bisphosphonates now standard of care of women with early stage breast cancer? A debate from the Canadian Bone and the Oncologist New Updates meeting. J Bone Oncol 2015;4:54–8.
44.
de Moraes M, de Matos FR, de Souza LB, et al. Immunoexpression of RANK, RANKL, OPG, VEGF, and vWF in radicular and dentigerous cysts. J Oral Pathol Med 2013;42:468–73.
Inflammation and Bone Resorption in Granulomas and Cysts
9
Differences in Inflammation and Bone Resorption between Apical Granulomas, Radicular Cysts, and Dentigerous Cysts
ABSTRACT Introduction: Dental cysts can be of inflammatory (radicular cysts) or noninflammatory (dentigerous cysts) origin. Apical periodontitis is a necrosis of the pulp and infection of the root canal causing the development of apical granulomas or radicular cysts. The immunology of granuloma and cyst formation is important because modern root filling materials are immunologically active and can contribute to the resolution of apical granulomas. In contrast, radicular cysts often require apicectomy. A better understanding of the pathophysiology of inflammation and bone resorption in apical periodontitis could be the basis for developing new root filling materials with superior immunomodulatory properties. Methods: Forty-one apical granulomas, 23 radicular cysts, and 23 dentigerous cysts were analyzed in this study. A tissue microarray of the 87 consecutive specimens was created, and human leukocyte antigen–DR isotype (HLA-DR)-, CD83-, receptor activator of nuclear factor kappa B ligand–, macrophage colony-stimulating factor (MCSF)-, galectin-3 (Gal3)-, CD4-, and CD8-positive cells were detected by immunohistochemistry. Tissue microarrays were digitized, and the expression of markers was quantitatively assessed. Results: HLA-DR, CD83, MCSF, and Gal3 expression was significantly (P , .05) higher in radicular cysts compared with apical granulomas. HLADR, CD83, MCSF, receptor activator of nuclear factor kappa B ligand, and Gal3 expression in dentigerous cysts was significantly (P , .05) lower than in both periapical lesions (apical granulomas and radicular cysts). CD4 and CD8 infiltration was not statistically different between apical granulomas and radicular cysts. Dentigerous cysts showed a significantly (P , .05) lower T-cell infiltration than apical periodontitis. The CD4/CD8 ratio was not significantly different between the analyzed groups. Conclusions: The development of radicular cysts in apical periodontitis is associated with an increased expression of myeloid inflammatory markers and bone resorption parameters. Antigen-presenting cells and myeloid cells might be more relevant for the pathogenesis of apical periodontitis than T cells. Increased inflammation might promote the formation of radicular cysts and more pronounced bone resorption. (J Endod 2019;-:1–9.)
KEY WORDS Apical granuloma; apical periodontitis; bone resorption; follicular cyst; periapical lesion; radicular cysts Infection and necrosis of the dental pulp causes apical periodontitis, which manifests as apical granulomas or radicular cysts. Both periapical lesions have an inflammatory origin but show a different clinical course1. In contrast to radicular cysts, dentigerous cysts have no inflammatory cause2 and are classified as developmental odontogenic cysts3. In most cases, apical periodontitis is initially treated by orthograde endodontic treatment4. If an apical lesion develops into a cyst, endodontic therapy alone is sometimes not sufficient, and apicectomy or even extraction of the affected teeth is required4.
JOE Volume -, Number -, - 2019
Manuel Weber, MD, DMD,* Jutta Ries, PhD,* €ttner-Herold, MD,† Maike Bu Carol-Immanuel Geppert, MD,‡ Marco Kesting, MD, DMD,* and Falk Wehrhan, MD, DMD*
SIGNIFICANCE Apical granulomas, radicular cysts, and dentigerous cysts show immunologic differences. High infiltration of HLA-DR– and CD83-positive proinflammatory cells is associated with radicular cyst formation. Additionally, bone resorption markers MCSF and Gal3 are significantly increased in radicular cysts compared with apical granulomas. The development of apical periodontists toward granulomas or radicular cysts could be immunologically controlled. Therefore, the use of root filling materials with antiinflammatory properties might be beneficial. From the Departments of *Oral and Maxillofacial Surgery and † Nephropathology and ‡Institute of Pathology, Friedrich-Alexander University €rnberg, Erlangen, Germany Erlangen-Nu Address requests for reprints to Dr Manuel Weber, Department of Oral and Maxillofacial Surgery, Friedrich-Alexander €rnberg, University Erlangen-Nu €ckstraße 11, 91054 Erlangen, Glu Germany. E-mail address: manuel.weber@ uk-erlangen.de 0099-2399/$ - see front matter Copyright © 2019 American Association of Endodontists. https://doi.org/10.1016/ j.joen.2019.06.014
Inflammation and Bone Resorption in Granulomas and Cysts
1
Although it is known that apical periodontitis is caused by inflammatory reactions5, the exact pathogenesis of the different periapical lesions is not yet understood. In a previous study, we analyzed macrophages in apical periodontitis. Tissue macrophages can occur in an M1 or M2 polarized state. M1 macrophages are proinflammatory and can contribute to tissue destruction. In contrast, M2 polarized macrophages act anti-inflammatory and mediate tissue regeneration and wound healing6–9. We could demonstrate that radicular cysts show a significant shift toward proinflammatory, M1 polarized macrophages compared with apical granulomas10. Dentigerous cysts had a significantly lower macrophage infiltration and M1 polarization compared with both periapical pathologies (apical granulomas and radicular cysts)10. These data indicate that radicular cyst formation is associated with M1 polarization of macrophages, and the development of granulomas or cysts might be immunologically determined10. Because apical periodontitis is characterized by inflammation and bone resorption11, additional inflammatory cells and signaling pathways associated with these processes were studied to better understand apical periodontitis. Human leukocyte antigen–DR isotype (HLA-DR) is an antigen presentation receptor expressed on antigen-presenting cells like dendritic cells (DCs) and macrophages12. HLADR–based antigen presentation is essential for the activation of CD4- and CD8-positive T cells and thus for the initiation of specific adaptive immune responses12. Proinflammatory M1 macrophages are characterized by a high HLADR expression, whereas anti-inflammatory M2 macrophages are HLA-DRlow13,14. Consequently, HLA-DR can be regarded as a relevant proinflammatory marker. Besides M1 polarized macrophages, mature myeloid DCs are highly potent antigen-presenting cells with proinflammatory properties15. These DCs show a high expression of CD83 and HLA-DR16. Inflammation is a relevant driver of bone resorption11,17, which is relevant for the formation and growth of cystic jaw bone lesions18. Bone resorption is mediated by osteoclasts19. Receptor activator of nuclear factor kappa B ligand (RANKL) and macrophage colony-stimulating factor (MCSF) signaling are the most relevant pathways for osteoclast differentiation and activation19,20. Antigen presentation in combination with specific coactivating signals leads to adaptive T-cell responses21. In chronic infectious diseases like human immunodeficiency virus infection,
2
Weber et al.
T-cell–mediated inflammation can contribute to bone loss22. In osteoporosis, T cells contribute to bone loss via RANKL signaling23. Galectin-3 (Gal3) is expressed in macrophages and contributes to the regulation of innate and adaptive immune responses. Gal3 is also present in differentiating osteoblasts and osteoclasts and might contribute to the osteoblast-osteoclast cross talk24. Anti-inflammatory as well as proinflammatory effects of Gal3 were described24,25. In rheumatoid arthritis, increased Gal3 levels were detected in inflamed joints24,26. Rats with induced arthritis showed increased Gal3 expression in macrophages in areas with severe bone destruction24. The root filling material most suitable for endodontic treatment is a subject of constant debate27–29. The use of mineral trioxide aggregate (MTA) as root-end filling material during endodontic treatment or apicectomy has shown good clinical results30–32. This might be explained by the immune modulatory properties of MTA because it was shown that MTA induces a shift toward anti-inflammatory M2 polarization of macrophages in vivo33. Thus, M2 polarizing agents, like MTA, might antagonize M1 polarization associated with radicular cysts10 and therefore contribute to the prevention of cyst formation in apical periodontitis. Because MTA also has negative properties including dislocation, discoloration, or difficulties of removal in endodontic retreatment31, a better understanding of the pathophysiologic processes in apical periodontitis could pave the way for the development of superior new apical sealing materials in endodontic treatment. The current study was intended to clarify if the expression of parameters of inflammation (HLA-DR, CD83, CD4, and CD8) and bone resorption (RANKL, MCSF, and Gal3) differ between noncystic periapical lesions (apical granulomas) and cystic periapical lesions (radicular cysts). Additionally, inflammatory odontogenic cysts (radicular cysts) were compared with developmental odontogenic cysts (dentigerous cysts).
with no clinical signs of acute infection. Surgery was performed under local anesthesia or general anesthesia. Complete enucleation/ curettage was performed in all cases. There was no case with large jaw cysts requiring continuity resection included. No surgical procedure specific to this study was performed because tissue samples from routine histologic diagnostics were analyzed. Each specimen included was confirmed to show representative regions of apical granulomas, radicular cysts, and dentigerous cysts. In addition to clinical information, the following histologic criteria were used to differentiate between the pathologies: apical granulomas represent an advanced form of apical periodontitis in which the growth of granulomatous tissue attempts to confine irritating agents escaping from root canal. Granulomatous tissue is composed of acute and chronic inflammation (lymphocytes variably intermixed with neutrophils, plasma cells, histiocytes, mast cells, and eosinophils) in a fibrotic stroma surrounded by a fibrous capsule. In some cases, epithelial rests of Malassez (epithelial layer) can be identified. Dentigerous and radicular cysts have few common histologic features. They are both layered by nonstratified, nonkeratinizing squamous epithelium with scattered mucinous cells. In some cases, radicular cysts can have hyalinized calcification called “Rushton bodies,” even if the certain diagnosis is made on the relation of the cyst with the tooth, because periapical cysts are always related to a nonvital element, whereas follicular cysts are associated with an impacted tooth. Patients with a history of malign or autoimmune diseases as well as irradiated patients were excluded. The study cohort consisted of 41 apical granulomas, 23 radicular cysts, and 23 dentigerous cysts. Apical granulomas were obtained from 41 patients (28 males and 13 females) with a mean age of 57 years, radicular cysts were derived from 21 patients (13 males and 8 females) with a mean age of 52 years, and dentigerous cysts were obtained from 15 patients (10 male and 5 female) with a mean age of 22 years.
MATERIALS AND METHODS
Tissue Microarray Construction and Immunohistochemical Staining
Patients and Tissue Harvesting A previously described10 cohort of 87 consecutive specimens of apical granulomas, radicular cysts, and dentigerous cysts was analyzed in this study. The study protocol was approved by the local ethical committee of the University of Erlangen-Nuremberg, Erlangen, Germany (reference number 351_15 Bc). Specimens were obtained during dental surgery procedures in consecutive patients
A tissue microarray (TMA) for apical granulomas, radicular cysts, and dentigerous cysts was assorted as previously described10. For radicular cysts and dentigerous cysts, stroma tissue in direct proximity to the cyst lumen was selected for TMA construction. Each tissue core had a diameter of 1.5 mm. Immunohistochemical staining and quality control were performed as previously described10,34.
JOE Volume -, Number -, - 2019
FIGURE 1 – Virtual microscopy of a TMA slide (1!, 7!, and 35! magnification). The figure shows an overview of a TMA slide stained for CD8-positive cells (1! magnification) and a scheme of the TMA indicating the number of each tissue sample. Samples 5 and 27 are exemplarily selected and magnified (7! and 35! magnification).
The following primary antibodies were used: anti–HLA-DR (M0746, monoclonal mouse, 1:250; Dako Agilent, Santa Clara, CA), anti-CD83 (sc-19677, monoclonal mouse, 1:100; Santa Cruz, Dallas, TX), anti-MCSF (ab-183316, monoclonal rabbit, 1:100; Abcam, Cambridge, UK), anti-RANKL (ab9957, polyclonal rabbit, 1:100, Abcam) anti-Ga13 (sc-20157, clone H-160, polyclonal rabbit, Santa Cruz), anti-CD4 (ORG-8756, clone 1F6, monoclonal mouse, 1:10; Novocastra, Newcastle, UK), and anti-CD8 (IS62330-2, clone c8/144B, monoclonal mouse, 1:100, Dako Agilent).
Quantitative Immunohistochemical Analysis TMAs were completely scanned and digitized using the method of “whole slide imaging” (Fig. 1). The scanning procedure was performed in cooperation with the Institute of Pathology of the University of Erlangen€rnberg using a Pannoramic 250 Flash III Nu Scanner (3D Histech, Budapest, Hungary) in the 40! magnification mode. Scanning and virtual microscopy were performed as previously described10. For each TMA sample, 2 visual fields showing the highest expression rates of each marker were selected (hot spot analysis) (Fig. 2A–E). The area analyzed per tissue sample and marker was 1.4 mm2. Micrographs of the selected areas were imported into Biomas software (MSAB, Erlangen, Germany) for cell counting. Quantitative analysis was performed to determine the number of infiltrating HLA-DR-, CD83-, RANKL-, M-CSF-, Gal3-, CD4-, and
JOE Volume -, Number -, - 2019
CD8-positive cells in all specimens. Assessment of the cell density per mm2 was performed as previously described10,34,35.
Statistical Analysis To analyze the immunohistochemical staining and spatial distribution patterns, the cell count per mm2 was determined. The results are expressed as median and standard deviation. Box plot diagrams represent the median, interquartile range, minimum, and maximum. Two-sided, adjusted P values .05 were considered significant. The analyses were performed using the Mann-Whitney U test with SPSS 22 for Mac OS (IBM Corp, Armonk, NY).
RESULTS Inflammation and Bone Resorption Marker Expression in Apical Granulomas, Radicular Cysts, and Dentigerous Cysts The HLA-DR cell count in radicular cysts was significantly higher than in apical granulomas (median 5 1959 cells/mm2 and 334 cells/ mm2, respectively; P 5 .017) and also significantly higher than in dentigerous cysts (median 5 43 cells/mm2, P , .001; Table 1, Fig. 3A). Compared with dentigerous cysts, apical granulomas showed a significantly higher (P , .001) HLA-DR expression (Table 1, Fig. 3A). Radicular cysts revealed a significantly increased CD83 expression compared with apical granulomas (median 5 43 cells/mm2 and 5 cells/mm2, respectively; P 5 .003) and dentigerous cysts (median 0 cells/mm2,
P , .001; Table 1, Fig. 3B). Moreover, CD83 expression in apical granulomas was significantly higher (P 5 .006) than in dentigerous cysts (Table 1, Fig. 3B). RANKL expression in radicular cysts was significantly higher than in dentigerous cysts (median 5 4841 cells/mm2 and 1464 cells/mm2, respectively; P , .001). The difference in RANKL expression between apical granulomas and radicular cysts was statistically nonsignificant (Table 1, Fig. 3C). Compared with dentigerous cysts, apical granulomas (median 5 4487 cells/mm2) displayed a significantly higher (P , .001) RANKL expression (Table 1, Fig. 3C). MCSF expression in radicular cysts was significantly higher than in apical granulomas (median 5 94 cells/mm2 and 10 cells/mm2, respectively; P , .001) and also significantly higher than in dentigerous cysts (median 5 4 cells/mm2, P , .001; Table 1, Fig. 3D). Apical granulomas had a significantly higher (P 5 .032) MCSF expression than dentigerous cysts (Table 1, Fig. 3D). Radicular cysts displayed a significantly higher density of Gal3-positive cells (median 5 3551 cells/mm2) compared with apical granulomas (median 5 1141 cells/mm2, P , .001) and dentigerous cysts (median 5 291 cells/mm2, P , .001; Table 1, Fig. 3E). Gal3 expression in apical granulomas was significantly higher (P , .001) compared with dentigerous cysts (Table 1, Fig. 3E). In summary, all analyzed inflammation and bone resorption markers showed the highest expression in radicular
Inflammation and Bone Resorption in Granulomas and Cysts
3
A
B
C
D
E
F
G
FIGURE 2 – Typical expression patterns of the analyzed inflammatory, bone resorption, and T-cell markers. Micrographs show the typical expression pattern of all markers (HLA-DR, CD83, RANKL, MCSF, Gal3, CD4, and CD8) in (left column ) apical granulomas, (middle column ) radicular cysts, and (right column ) dentigerous cysts. All micrographs are given in high-power magnification (35! magnification). (A ) HLA-DR and (B ) CD83 show a cytoplasmatic staining with accentuation of the plasma membrane. (C ) RANKL and (D ) MCSF stainings display a cytoplasmatic expression pattern. (E ) Gal3 has a cytoplasmic and nuclear expression. (F ) CD4 and (G ) CD8 reveal a membranous expression pattern. cysts compared with apical granulomas and radicular cysts.
T-cell Infiltration and Expression Ratios in Apical Granulomas, Radicular Cysts, and Dentigerous Cysts Dentigerous cysts showed a significantly lower infiltration by CD4-positive T cells than radicular cysts (median 5 31 cells/
4
Weber et al.
mm2 and 597 cells/mm2, respectively; P , .001) and apical granulomas (median 5 568 cells/mm2, P , .001; Table 2, Fig. 4A). The difference in the CD4 cell count between apical granulomas and radicular cysts was statistically nonsignificant (Table 2, Fig. 4A). Both apical granulomas and radicular cysts showed a significantly higher infiltration of CD8-positive T cells compared with
dentigerous cysts (median 5 298 cells/mm2, 388 cells/mm2, and 63 cells/mm2, respectively; P , .001 for both; Table 2, Fig. 4B). No significant difference in CD8 expression was detected between apical granulomas and radicular cysts (Table 2, Fig. 4B). The CD4/CD8 ratio revealed no significant differences between apical granulomas, radicular cysts, and dentigerous cysts (Table 2, Fig. 4C). There was no difference
JOE Volume -, Number -, - 2019
TABLE 1 - Inflammation and Bone Resorption Marker Expression (Cells/mm2) in Apical Granulomas, Radicular Cysts, and Dentigerous Cysts HLA-DR
Cell count (cells/mm2) Marker Group Apical granuloma Radicular cyst Dentigerous cyst P values Apical granuloma vs radicular cyst Apical granuloma vs dentigerous cyst Radicular cyst vs dentigerous cyst
n 41 23 23
CD83
RANKL
MCSF
Gal3
Median
SD
Median
SD
Median
SD
Median
SD
Median
SD
334 1959 43
4773 2557 109
5 42 0
134 201 17
4487 4841 1464
2012 1687 1382
10 94 4
336 520 25
1141 3551 291
2552 3723 663
.017 ,.001 ,.001
.003 .006 ,.001
.419 ,.001 ,.001
,.001 .032 ,.001
,.001 ,.001 ,.001
SD, standard deviation. Values represent median, SD, and P value (Mann-Whitney U test, SPSS 22).
A
B
C
D
E
FIGURE 3 – Inflammation and bone resorption marker expression (cells/mm2) in apical granulomas, radicular cysts, and dentigerous cysts. Box plots show the median cell counts (positive cells/mm2) of inflammation and bone resorption markers ([A] HLA-DR, [B] CD83, [C] RANKL, [D] MCSF, and [E] Gal3) in apical granulomas, radicular cysts, and dentigerous cysts. All P values generated by the Mann-Whitney U test are indicated. Expression of the analyzed inflammation and bone resorption markers is significantly increased in radicular cysts compared with apical granulomas and dentigerous cysts, except for (C ) RANKL expression, which shows no significant difference between radicular cysts and apical granulomas.
JOE Volume -, Number -, - 2019
Inflammation and Bone Resorption in Granulomas and Cysts
5
TABLE 2 - T-cell Infiltration (Cells/mm2) and CD4/CD8 Expression Ratio in Apical Granulomas, Radicular Cysts, and Dentigerous Cysts Cell count (cells/mm2) and expression ratios
CD4
Ratio Group Apical granuloma Radicular cyst Dentigerous cyst P values Apical granuloma vs radicular cyst Apical granuloma vs dentigerous cyst Radicular cyst vs dentigerous cyst
n 33 20 13
CD8
CD4/CD8
Median
SD
Median
SD
Median
SD
568 597 31
882 486 208
298 388 34
367 356 63
1.48 1.47 1.34
5.30 0.88 4.92
.849 ,.001 ,.001
.359 ,.001 ,.001
.408 .262 .570
SD, standard deviation. Values represent median, SD, and P value (Mann-Whitney U test, SPSS 22).
in the expression of all analyzed markers between mandibular and maxillary cysts.
DISCUSSION Proinflammatory Antigenpresenting Cells in Apical Periodontitis and Dentigerous Cysts Inflammation of periapical tissue can cause the development of apical granulomas or radicular cysts. We were able to detect significantly increased densities of HLA-DR– and CD83expressing cells in radicular cysts compared with apical granulomas. CD83 is a marker for mature dendritic cells capable of efficient antigen presentation and T-cell activation15,16. HLA-DR is expressed by mature myeloid dendritic cells and M1 polarized,
A
proinflammatory macrophages but also by other cell types capable of antigen presentation15,16. Therefore, the increased expression of HLA-DR and CD83 in radicular cysts indicates that these lesions have a significantly stronger proinflammatory state of immunoreactivity compared with apical granulomas. Previously, we analyzed macrophage infiltration and polarization in apical granulomas and radicular cysts10. Proinflammatory M1 macrophages contribute to inflammation, antigen clearance, and tissue destruction, whereas anti-inflammatory M2 macrophages mediate peripheral immune tolerance, tissue regeneration, and wound healing6–9. Macrophage polarization showed a significant swift toward proinflammatory M1
polarization in radicular cysts compared with apical granulomas10. However, total macrophage density was not significantly different between apical granulomas and radicular cysts10. These results are interesting in the context of the increased HLA-DR and CD83 cell density detected in radicular cysts in the current study. The increased M1 polarization of macrophages in radicular cysts is accompanied by an augmented infiltration of mature CD83-positive dendritic cells and an increased HLA-DR expression and maybe by myeloid cells like proinflammatory macrophages and dendritic cells. Consequently, the development of apical periodontitis into a radicular cyst appears to be associated with a proinflammatory state.
B
C
FIGURE 4 – The T-cell count (cells/mm2) and CD4/CD8 expression ratio in apical granulomas, radicular cysts, and dentigerous cysts. Box plots show the median T-cell counts (positive cells/mm2) of (A ) CD4- and (B ) CD8-positive cells in apical granulomas, radicular cysts, and dentigerous cysts. (C ) The CD4/CD8 ratio. All P values generated by the Mann-Whitney U test are indicated.
6
Weber et al.
JOE Volume -, Number -, - 2019
In contrast, dentigerous cysts showed a significantly lower HLA-DR and CD83 infiltration compared with radicular cysts. This indicates that inflammation is not the driving force in the progression of jaw bone cysts with developmental origin. With regard to apical periodontitis, the results of the current study show that increased inflammation is associated with cyst formation. The results underline the importance of counteracting bacterial infection of the periapical region by sufficient endodontic treatment and thus eliminating the trigger of inflammation36. In addition, there is evidence that root filling materials also influence an immunologic response in periapical tissues33 and may therefore be prognostically relevant. Consequently, the used root filling materials and sealers should be investigated for their immunomodulatory properties. In this regard, materials with antiinflammatory properties33 might be beneficial in the prevention of radicular cyst formation.
T-cell Infiltration in Apical Periodontitis and Dentigerous Cysts In contrast to macrophages and dendritic cells, there was no significant difference in the infiltration of CD4- and CD8-positive T cells between apical granulomas and radicular cysts. Additionally, the CD4/CD8 ratio did not reveal a significant difference between both analyzed periapical lesions. A previous immunohistochemical analysis of apical granulomas and radicular cysts accordingly revealed no significant difference regarding the infiltration of CD8positive T cells37. However, another report describes a significantly increased infiltration of CD8-positive T cells in radicular cysts compared with apical granulomas5. A recent study analyzed programmed cell death 1 (PD1), an immune regulatory receptor expressed predominantly by activated T cells38. PD1 expression was significantly increased in apical granulomas compared with radicular cysts38. The increased expression of the immune-suppressive receptor PD139 in apical granulomas is in line with the results of the current study, which showed a significantly lower expression of the proinflammatory markers HLA-DR and CD83 in apical granulomas compared with radicular cysts. Additionally, a significantly increased
expression of FoxP3-positive regulatory T cells in apical granulomas compared with radicular cysts was described40. These data support the hypothesis that the formation of cystic periapical lesions might be determined by the immune system and associated with increased inflammation. Results of the current study indicate that CD4 and CD8 T-cell infiltration in dentigerous cysts is significantly lower than in apical periodontitis. However, the CD4/CD8 ratio revealed no significant difference between all analyzed lesions. These data suggest that a shift in the CD4/CD8 ratio is not involved in the pathogenesis of cystic jawbone lesions.
Bone Resorption Parameters in Apical Periodontitis and Dentigerous Cysts Osteoclasts are the primary bone-resorbing cells essential for physiologic bone homeostasis41. Analogous to macrophages, osteoclasts derive from mononuclear precursor cells of the myeloid lineage. The main cytokines that promote differentiation and activation of osteoclasts are MCSF and RANKL41. Under physiologic conditions, osteoblasts are the main source of MCSF and RANKL, maintaining the balance between bone formation and bone resorption. However, inflammatory cells can also produce MCSF and RANKL and thus induce bone resorption41. For any kind of expanding pathology in bone, it is essential to activate osteoclasts. For this reason, bisphosphonates or RANKL inhibitors, which block osteoclasts, are used for the treatment of bone metastases42,43. The current study revealed significantly increased MCSF expression in radicular cysts compared with apical granulomas. Elevated MCSF expression might be an indicator of increased activation of osteoclasts and consecutive bone resorption in radicular cysts. In contrast to MCSF, RANKL was not significantly up-regulated in radicular cysts in comparison with apical granulomas. MCSF and RANKL were significantly lower in dentigerous cysts compared with both periapical lesions. This finding is consistent with the lower expression of inflammatory cells in dentigerous cysts and could indicate that inflammation-driven apical periodontitis has a higher bone resorptive potential than developmental cysts, which are associated with a low inflammatory state. Results of the
current study are contrary to previous analyses comparing RANKL expression in radicular and dentigerous cysts, which could detect no significant difference in the expression of the cytokine18,44. However, in contrast to the current report, no quantitative assessment was used. Gal3 is a phylogenetically highly conserved lectin involved in a variety of immunologic and cell biologic processes. In the context of malignant diseases, Gal3 contributes to immunosuppression and tumor progression25. In inflammatory pathologies, Gal3 is involved in tissue remodeling and bone resorption24,26. We could show that Gal3 expression was significantly up-regulated in radicular cysts compared with apical granulomas. In dentigerous cysts, Gal3 was significantly lower than in both periapical pathologies. These data indicate that Gal3 expression in apical periodontitis is associated with inflammation induced bone resorption.
CONCLUSION The development of radicular cysts might be an immunologically controlled process. The increased infiltration of proinflammatory cells in radicular cysts is accompanied by an increased expression of bone resorption– associated cytokines. Antigen-presenting cells seem to be more relevant for the pathophysiology of apical periodontitis than T cells. In contrast to apical periodontitis, the progression of dentigerous cysts seems to be less dependent on immunologic factors.
ACKNOWLEDGMENTS The authors thank Rudolf Jung for manufacturing the tissue microarrays and Susanne Schoenherr and Elke Diebel for technical assistance. We would also like to thank the dental students Sabrin Abu-Hossin, €ring for Franziska Gerhardt, and Lisa Go processing the tissue specimens and operating the immunohistochemistry autostainer apparatus; Tilo Schlittenbauer for collecting the tissue samples; and Luitpold Distel for his assistance in the cell counting procedure. The authors deny any conflicts of interest related to this study.
REFERENCES 1.
JOE Volume -, Number -, - 2019
Lin LM, Huang GT, Rosenberg PA. Proliferation of epithelial cell rests, formation of apical cysts, and regression of apical cysts after periapical wound healing. J Endod 2007;33:908–16.
Inflammation and Bone Resorption in Granulomas and Cysts
7
2.
de Moraes M, da Rocha Neto PC, de Matos FR, et al. Immunoexpression of transforming growth factor beta and interferon gamma in radicular and dentigerous cysts. J Endod 2014;40:1293–7.
3.
Wright JM, Odell EW, Speight PM, Takata T. Odontogenic tumors, WHO 2005: where do we go from here? Head Neck Pathol 2014;8:373–82.
4.
Del Fabbro M, Corbella S, Sequeira-Byron P, et al. Endodontic procedures for retreatment of periapical lesions. Cochrane Database Syst Rev 2016:CD005511.
5.
Rodini CO, Lara VS. Study of the expression of CD681 macrophages and CD81 T cells in human granulomas and periapical cysts. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2001;92:221–7.
6.
Mantovani A, Sica A, Locati M. New vistas on macrophage differentiation and activation. Eur J Immunol 2007;7:14–6.
7.
Hirata Y, Tabata M, Kurobe H, et al. Coronary atherosclerosis is associated with macrophage polarization in epicardial adipose tissue. J Am Coll Cardiol 2011;58:248–55.
8.
Mantovani A, Biswas SK, Galdiero MR, et al. Macrophage plasticity and polarization in tissue repair and remodelling. J Pathol 2013;229:176–85.
9.
€ttner-Herold M, Hyckel P, et al. Small oral squamous cell carcinomas with nodal Weber M, Bu lymphogenic metastasis show increased infiltration of M2 polarized macrophages–an immunohistochemical analysis. J Craniomaxillofac Surg 2014;42:1087–94.
10.
Weber M, Schlittenbauer T, Moebius P, et al. Macrophage polarization differs between apical granulomas, radicular cysts, and dentigerous cysts. Clin Oral Investig 2018;22:385–94.
11.
Hienz SA, Paliwal S, Ivanovski S. Mechanisms of bone resorption in periodontitis. J Immunol Res 2015;2015:615486.
12.
Zhuang Y, Peng H, Chen Y, et al. Dynamic monitoring of monocyte HLA-DR expression for the diagnosis, prognosis, and prediction of sepsis. Front Biosci (Landmark Ed) 2017;22:1344–54.
13.
Shimada S, Ebina Y, Iijima N, et al. Decidual CD68(1) HLA-DR(1) CD163(-) M1 macrophages increase in miscarriages with normal fetal chromosome. Am J Reprod Immunol 2018;79. https:// doi.org/10.1111/aji.12791.
14.
Lin R, Zhang J, Zhou L, Wang B. Altered function of monocytes/macrophages in patients with autoimmune hepatitis. Mol Med Rep 2016;13:3874–80.
15.
Seldon TA, Pryor R, Palkova A, et al. Immunosuppressive human anti-CD83 monoclonal antibody depletion of activated dendritic cells in transplantation. Leukemia 2016;30:692–700.
16.
Maney NJ, Reynolds G, Krippner-Heidenreich A, Hilkens CM. Dendritic cell maturation and survival are differentially regulated by TNFR1 and TNFR2. J Immunol 2014;193:4914–23.
17.
Nogueira AV, de Molon RS, Nokhbehsaim M, et al. Contribution of biomechanical forces to inflammation-induced bone resorption. J Clin Periodontol 2017;44:31–41.
18.
de Moraes M, de Lucena HF, de Azevedo PR, et al. Comparative immunohistochemical expression of RANK, RANKL and OPG in radicular and dentigerous cysts. Arch Oral Biol 2011;56:1256–63.
19.
Adamopoulos IE. Inflammation in bone physiology and pathology. Curr Opin Rheumatol 2018;30:59–64.
20.
Li ZH, Si Y, Xu G, et al. High-dose PMA with RANKL and MCSF induces THP1 cell differentiation into human functional osteoclasts in vitro. Mol Med Rep 2017;16:8380–4.
21.
Murphy K, Weaver C. Janeway’s Immunobiology. Boca Raton, FL: CRC Press; 2016.
22.
Weitzmann MN, Vikulina T, Roser-Page S, et al. Homeostatic expansion of CD41 T cells promotes cortical and trabecular bone loss, whereas CD81 T cells induce trabecular bone loss only. J Infect Dis 2017;216:1070–9.
23.
Weitzmann MN. T-cells and B-cells in osteoporosis. Curr Opin Endocrinol Diabetes Obes 2014;21:461–7.
24.
Iacobini C, Fantauzzi CB, Pugliese G, Menini S. Role of galectin-3 in bone cell differentiation, bone pathophysiology and vascular osteogenesis. Int J Mol Sci 2017;18:E2481.
25.
€ttner-Herold M, Distel L, et al. Galectin 3 expression in primary oral squamous cell Weber M, Bu carcinomas. BMC Cancer 2017;17:906.
26.
he -Okouma M, Ea HK, et al. Galectin-3: a key player in arthritis. Joint Bone Spine Hu Y, Y ele 2017;84:15–20.
8
Weber et al.
JOE Volume -, Number -, - 2019
JOE Volume -, Number -, - 2019
27.
Ma X, Li C, Jia L, et al. Materials for retrograde filling in root canal therapy. Cochrane Database Syst Rev 2016;12:CD005517.
28.
Ay H, Duane B. Limited evidence on best material for retrograde root fillings. Evid Based Dent 2018;19:8–9.
29.
Tomson RM, Polycarpou N, Tomson PL. Contemporary obturation of the root canal system. Br Dent J 2014;216:315–22.
30.
Alsulaimani RS. Single-visit endodontic treatment of mature teeth with chronic apical abscesses using mineral trioxide aggregate cement: a randomized clinical trial. BMC Oral Health 2016;16:78.
31.
Torabinejad M, Parirokh M, Dummer PM. Mineral trioxide aggregate and other bioactive endodontic cements: an updated overview - part II: other clinical applications and complications. Int Endod J 2018;51:284–317.
32.
Torabinejad M, Parirokh M. Mineral trioxide aggregate: a comprehensive literature review–part II: leakage and biocompatibility investigations. J Endod 2010;36:190–202.
33.
Ito T, Kaneko T, Yamanaka Y, et al. M2 macrophages participate in the biological tissue healing reaction to mineral trioxide aggregate. J Endod 2014;40:379–83.
34.
Weber M, Iliopoulos C, Moebius P, et al. Prognostic significance of macrophage polarization in early stage oral squamous cell carcinomas. Oral Oncol 2016;52:75–84.
35.
€ttner-Herold M, et al. Macrophage polarisation changes within the time Weber M, Moebius P, Bu between diagnostic biopsy and tumour resection in oral squamous cell carcinomas–an immunohistochemical study. Br J Cancer 2015;113:510–9.
36.
Kim DS, Shin MR, Kim YS, et al. Anti-inflammatory effects of glutamine on LPS-stimulated human dental pulp cells correlate with activation of MKP-1 and attenuation of the MAPK and NF-kappaB pathways. Int Endod J 2015;48:220–8.
37.
MA, Melo RA, et al. Analysis of CD571 natural killer cells and CD81 T lymphocytes in Silva L, Sa periapical granulomas and radicular cysts. Braz Oral Res 2017;31:e106.
38.
Wang HS, Yang FH, Li Y, et al. The expression of PD-1 and LAG-3 in periapical lesions. Am J Transl Res 2018;10:2677–84.
39.
Sharpe AH, Pauken KE. The diverse functions of the PD1 inhibitory pathway. Nat Rev Immunol 2018;18:153–67.
40.
Peixoto RF, Pereira Jdos S, Nonaka CF, et al. Immunohistochemical analysis of FoxP31 cells in periapical granulomas and radicular cysts. Arch Oral Biol 2012;57:1159–64.
41.
Lampiasi N, Russo R, Zito F. The alternative faces of macrophage generate osteoclasts. Biomed Res Int 2016;2016:9089610.
42.
€lscher T, Hakenberg OW, Wirth MP. Treatment of bone metastases in urologic Froehner M, Ho malignancies. Urol Int 2014;93:249–56.
43.
Jacobs C, Amir E, Paterson A, et al. Are adjuvant bisphosphonates now standard of care of women with early stage breast cancer? A debate from the Canadian Bone and the Oncologist New Updates meeting. J Bone Oncol 2015;4:54–8.
44.
de Moraes M, de Matos FR, de Souza LB, et al. Immunoexpression of RANK, RANKL, OPG, VEGF, and vWF in radicular and dentigerous cysts. J Oral Pathol Med 2013;42:468–73.
Inflammation and Bone Resorption in Granulomas and Cysts
9