doi:10.1111/j.1365-2591.2012.02086.

Analysis of the frequency and nature of hyaline ring granulomas in inammatory odontogenic cysts

. C. G. Henriques1, J. S. Pereira1, C. F. W. Nonaka2, R. A. Freitas1, L. P. Pinto1 & A M. C. C. Miguel1

Department of Dentistry, Federal University of Rio Grande do Norte, Natal, RN, Brazil; and 2Department of Dentistry, State University of Para ba, Campina Grande, PB, Brazil
1

Abstract
CG, Pereira JS, Nonaka CFW, Freitas RA, Henriques A Pinto LP, Miguel MCC. Analysis of the frequency and nature of hyaline ring granulomas in inammatory odontogenic cysts. International Endodontic Journal, 46, 2029, 2013.

Aim To determine the prevalence of hyaline ring granulomas (HRGs) in a large case series of inammatory odontogenic cysts, and to investigate the nature of these structures. Methodology All records from the patients diagnosed with inammatory odontogenic cysts between January 1970 and April 2009 were reviewed. Histologic sections were evaluated by light microscopy and cases with HRGs for which sufcient biological material was available were submitted to histochemical analysis (Massons trichrome) and immunohistochemistry (CD34, CD68 and collagen IV). Results Twenty-two (3.3%) of the 661 cases of inammatory odontogenic cysts diagnosed during the study period presented HRGs. The relative frequency of HRGs was higher amongst residual radicular cysts (6.1%), followed by paradental cysts (5.6%) and radicular cysts (3.0%). HRGs appeared as roughly

circular homogeneous/brillar masses in 14 (63.6%) cases and as round structures enclosing amorphous material in 3 (13.6%) cases. Most (77.8%) roughly circular homogeneous/brillar masses were positive for collagen, whereas all (100.0%) round structures enclosing amorphous material were negative for this protein. Immunohistochemistry showed that most mononucleated cells and all multinucleated giant cells were positive for CD68, but negative for CD34, in all cases. In addition, collagen IV immunostaining was negative in amorphous structures and weakly positive in homogeneous/brillar masses. Conclusions The present results suggest a very low frequency of HRGs in inammatory odontogenic cysts and support the hypothesis that these structures arise from the implantation of foreign material, most likely food particles of plant or vegetable origin. The diverse microscopic features of HRG possibly represent different developmental stages of this structure. Keywords: histochemistry, hyaline ring granuloma, immunohistochemistry, inammatory odontogenic cysts.
Received 16 October 2011; accepted 19 May 2012

Introduction
Hyaline ring granuloma (HRG) is an uncommon histopathologic nding characterized by hyaline rings or

rcia Cristina da Costa Miguel, UniversidCorrespondence: Ma ade Federal do Rio Grande do Norte, Departamento de Odontologia, Av. Senador Salgado Filho, 1787, Lagoa Nova, Natal, RN, CEP 59056-000 Brasil (Tel./Fax: +55 84 3215 4138; e-mail: mccmiguel@hotmail.com).

ovoid homogeneous/brillar hyaline masses lying within brous connective tissue, which contain variable numbers of inammatory cells and multinucleated giant cells (Talacko & Radden 1988a, Zhai & Maluf 2004). This microscopic nding has been reported in lesions located in the oral cavity, lung, intestine, skin, gallbladder and uterine tube (Knoblich 1969, Zhai & Maluf 2004, Rhee & Wu 2006, Gueiros et al. 2008). In the oral cavity, HRGs have been found in extraosseous lesions such as inammatory

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brous hyperplasia (Gueiros et al. 2008) and in intraosseous lesions such as periapical granulomas (Pola et al. 2003, Gueiros et al. 2008) and odontogenic cysts (Chen et al. 1981, Talacko & Radden 1988a, Marcussen et al. 1993). Oral HRG was initially described by Lewars (1971) as chronic periostitis, which was characterized by the presence of palely stained eosinophilic structureless material, so-called hyaline rings. Since then, a variety of terms have been used to describe this entity, including giant-cell hyaline angiopathy (Dunlap & Barker 1977, McMillan et al. 1981, Ferguson & Smillie 1986), pulse granuloma (Mincer et al. 1979, McMillan et al. 1981), granuloma tissue with giant cells and hyaline change (McMillan et al. 1981), oral vegetable granuloma (Harrison & Martin 1986), food-induced granuloma (Brown & Theaker 1987) and le granulome alimentaire des maxillares (Iriarte Ortabe et al. 1991). In addition, some authors have caused confusion by mixing up the term hyaline rings with hyaline bodies (Chen et al. 1981, Keirby & Soames 1985, Yang & Barnett 1985). The latter structures, also called Rushton bodies, are morphologically and aetiologically different from hyaline rings (Philipsen & Reichart 2010). Two opposing theories regarding the aetiopathogenesis of HRGs have been proposed (Philipsen & Reichart 2010). The exogenous theory suggests that these structures arise from the implantation of foreign material (food particles of plant or vegetable origin, therapeutic agents) (Lewars 1971, Mincer et al. 1979, Talacko & Radden 1988a, LaMear et al. 1994). The endogenous theory proposes that HRGs represent hyaline degeneration of the walls of blood vessels (Dunlap & Barker 1977). Although the origin of these structures is not clear, the use of the descriptive term HRG has been suggested, which seems more suitable and also avoids misunderstanding (Chou et al. 1990, Gueiros et al. 2008). Data on the occurrence of HRGs in inammatory odontogenic cysts are basically limited to case reports (Chen et al. 1981, Yang & Barnett 1985, Lin et al. 1993, Keskin et al. 2000, Pola et al. 2003), and only a few retrospective studies are available (Talacko & Radden 1988a, Marcussen et al. 1993, Philipsen & Reichart 2010). Therefore, the objective of the present investigation was to determine the frequency of HRGs in a large case series of inammatory odontogenic cysts. In addition, histochemical and immunohistochemical analyses were performed to gain insights into the nature of these structures.

Materials and methods Clinical analysis

After approval of the study by the Research Ethics Committee of the Federal University of Rio Grande do Norte (UFRN), case records from 661 patients with inammatory odontogenic cysts (594 radicular cysts, 49 residual radicular cysts and 18 paradental cysts) diagnosed between January 1970 and April 2009 were retrieved from the archives of the Oral Pathology Service at UFRN. Data regarding age, gender and anatomic location were compiled from the clinical data sent together with the biopsy reports.

Histopathologic analysis
For histopathologic analysis, all slides containing haematoxylin-/eosin-stained 5-lm-thick sections were reassessed. The tissue sections were examined under a light microscope (Olympus CX31, Tokyo, Japan) by three observers, and cases were classied according to the presence or absence of HRGs. In addition, HRGs were classied according to the following morphologic patterns of the hyaline material: round structures enclosing eosinophilic amorphous material (Gueiros et al. 2008) or roughly circular homogeneous/brillar masses (Philipsen & Reichart 2010). The presence of calcication (Talacko & Radden 1988a, Philipsen & Reichart 2010), metaplastic bone formation (Talacko & Radden 1988a) and haemosiderin pigmentation (Chen et al. 1981) were also analysed.

Histochemical and immunohistochemical analysis

To evaluate the nature of the material composing the hyaline rings, 5-lm-thick sections were cut from parafn-embedded tissue blocks and stained with Massons trichrome (EasyPath; Bio-Optica Milano SpA, Milan, Italy). Three observers then examined the reactivity of the hyaline material to Massons trichrome stain under a light microscope (Olympus CX31) according to the morphologic pattern of these structures. Immunohistochemical staining for CD34 and CD68 was used to elucidate the nature of mononucleated cells and multinucleated giant cells associated with HRGs, and staining for collagen IV to gain insights into the nature of the hyaline ring material. Briey, 3-lmthick sections were cut from parafn-embedded tissue blocks, deparafnized and immersed in 3% hydrogen peroxide to block endogenous peroxidase activity. The

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sections were then washed in phosphate-buffered saline (PBS) and submitted to antigen retrieval (Table 1). After treatment with normal serum, the sections were incubated with the primary anti-CD34, anti-CD68 and anti-collagen IV antibodies in a moist chamber (Table 1). The sections were then washed twice in PBS and incubated at room temperature with the labelled streptavidin biotin complex (LSAB+ System-HRP; Dako, Carpinteria, CA, USA) for anti-CD34 and anti-CD68 antibodies and with a polymer-based complex (AdvanceTM HRP; Dako) for anti-collagen IV antibody. Peroxidase activity was visualized by immersing the tissue sections in diaminobenzidine (Liquid DAB+ Substrate; Dako), which resulted in a brown reaction product. Finally, the sections were counterstained with Mayers haematoxylin and coverslipped. Blood vessels and macrophages present in all specimens served as internal positive controls for CD34 and CD68, respectively. Immunostaining of blood vessels in the basement membrane was used as internal positive control for collagen IV. As negative control, the samples were treated as described above, except that the primary antibody was replaced with a solution of bovine serum albumin in PBS. The slides were analysed by three observers under a light microscope (Olympus CX31). Positive or negative staining for CD34 and CD68 was evaluated in mononucleated cells and multinucleated giant cells present inside and adjacent to HRGs. For collagen IV, positive or negative staining was evaluated in the hyaline material of HRGs. The results were tabulated and analysed by descriptive statistics using the Statistical Package for the Social Sciences, version 17.0 (SPSS, Inc., Chicago, IL, USA).

HRGs were observed in 22 (3.3%) cases. Of these, 18 (81.8%) were radicular cysts, 3 (13.6%) were residual radicular cysts and 1 (4.5%) was a paradental cyst. Analysis of the distribution of cases according to the type of inammatory odontogenic cyst revealed a higher relative frequency of HRGs amongst residual radicular cysts (3/49; 6.1%), followed by paradental cysts (1/18; 5.6%) and radicular cysts (18/594; 3.0%) (Table 2). With respect to gender, cysts containing HRGs were commonly diagnosed in women (63.6%), with a female-to-male ratio of 1.75 : 1. Similarly, most cysts without HRGs were also diagnosed in women (59.8%), with a female-to-male ratio of 1.49 : 1 (Table 2). The mean patient age for cases with and without HRGs was 26.76 years (range 667) and 32.41 years (range 486), respectively (Table 2). Information regarding the anatomic location of cysts containing HRGs was available for 20 cases. Of these, 11 (55.0%) were located in the maxilla and 9 (45.0%) in the mandible (Table 2). Maxillary lesions were commonly located in the anterior region (81.8%), whereas mandibular lesions were usually found in the posterior region (88.9%) (Table 2).

Histopathologic analysis
Both morphologic patterns of hyaline material could be observed in the present series of inammatory odontogenic cysts: round structures enclosing eosinophilic amorphous material consistent with degenerated starch cells (Fig. 1a,b) and roughly circular homogeneous or brillar masses exhibiting a corrugated border (Fig. 1c,d). In both morphologic patterns, the hyaline material was found lying in chronically and, less often, acutely inamed brous connective tissue. In addition, mononucleated cells and multinucleated giant cells were observed inside and adjacent to the hyaline material (Fig. 1ad). Three (13.6%) cases exhibited only round structures enclosing amorphous material, 14 (63.6%) cases showed only roughly circular homogeneous or brillar masses and both morphologic patterns were

Results Clinical analysis

A total of 661 cases of inammatory odontogenic cysts were identied during the study period. Of these, 594 (89.9%) were radicular cysts, 49 (7.4%) were residual radicular cysts and 18 (2.7%) were paradental cysts.

Table 1 Manufacturer, clone, antigen retrieval, dilution and incubation period of the primary antibodies
Antibody CD68 CD34 Collagen IV Manufacturer Dako Dako Dako Clone KP1 QBEnd10 CIV22 Antigen retrieval Citrate, pH 6.0, Pascal, 121 C, 3 min Tris/EDTA, pH 9.0, Pascal, 121 C, 3 min Citrate, pH 6.0, Pascal, 121 C, 3 min Dilution 1 : 50 1 : 50 1 : 25 Incubation Overnight Overnight Overnight

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Table 2 Number of cases, gender, age and anatomic location according to the presence or absence of hyaline ring granulomas in inammatory odontogenic cysts
Gender Anatomic location Maxilla Group n (%) Female Male Age (years); mean (range) A P A/P A Mandible P A/P

Radicular cystsa Absence 576 (97.0) Presence 18 (3.0) Total 594 (100.0) Residual radicular cysta Absence 46 (93.9) Presence 3 (6.1)b Total 49 (100.0) Paradental cysta Absence 17 (94.4)c Presence 1 (5.6) Total 18 (100.0) All cysts Absence 639 (96.7) Presence 22 (3.3) Total 661 (100.0)

349 11 360 20 2 22 13 1 14 382 14 396

227 7 234 26 1 27 3 0 3 256 8 264

31.59 (486) 24.78 (660) 31.38 (486) 44.23 (1678) 47.50 (2867) 44.37 (1678) 28.07 (1768) 27.63 (1768) 32.41 (486) 26.76 (667) 32.22 (486)

186 9 195 14 0 14 0 0 0 200 9 209

137 1 138 9 1 10 1 0 1 147 2 149

7 0 7 2 0 2 0 0 0 9 0 9

34 0 34 6 0 6 0 0 0 40 0 40

152 7 159 10 0 10 14 1 15 176 8 184

6 1 7 2 0 2 0 0 0 8 1 9

A, anterior; P, posterior; A/P, anterior and posterior. a Information regarding anatomic location was not available for 54 radicular cysts, ve residual radicular cysts and two paradental cysts. b Information regarding age was not available for one case. c Information regarding gender was not available for one case.

present in 5 (22.7%) cases (Table 3). One case (4.5%) presented calcications in the form of small, coalesced basophilic droplets inside the hyaline material (Fig. 1e, Table 3). In one case (4.5%), haemosiderin pigmentation was found scattered within the hyaline material and in the cytoplasm of mononucleated and multinucleated giant cells (Fig. 1f, Table 3). None of the cases showed metaplastic bone formation.

Histochemical and immunohistochemical analysis

Analysis of tissue sections stained with Massons trichrome revealed the presence of hyaline material in the form of roughly circular homogeneous or brillar masses in nine cases and in the form of round structures enclosing amorphous material in three cases. Seven (77.8%) of the cases of roughly circular homogeneous or brillar masses exhibited variable amounts of collagen (Fig. 2a,b), and 2 (22.8%) were negative for collagen (Fig. 2c). On the other hand, all HRGs characterized by round structures enclosing eosinophilic amorphous material were negative for collagen (Fig. 2d). All cysts containing HRGs were submitted to immunohistochemistry. However, because of the small size

of some HRGs, these structures could be identied in only 15 of the 22 cases treated with the anti-CD68 antibody, in 12 of the 22 cases treated with the antiCD34 antibody and in eight of the 22 cases treated with the anti-collagen IV antibody. Most mononucleated cells and all multinucleated giant cells inside and adjacent to HRGs were positive for CD68 in all cases (Fig. 3a). In contrast, most mononucleated cells and all multinucleated giant cells inside and adjacent to HRGs were negative for CD34 (Fig. 3b). In fact, CD34 highlighted the presence of small blood vessels near the HRGs. No signicant difference in the immunoexpression of CD68 or CD34 was observed between the two morphologic patterns of hyaline material (Fig. 3c,d). Furthermore, all HRGs appearing in the form of roughly circular homogeneous or brillar masses were weakly positive for collagen IV (Fig. 3e). On the other hand, all HRGs characterized by round structures enclosing eosinophilic amorphous material were negative for this protein (Fig. 3f).

Discussion
HRGs have been described in both extraosseous and intraosseous oral lesions (Lewars 1971, Dunlap &

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(a)

(b)

(c)

(d)

(e)

(f)

Figure 1 (a) Hyaline ring granuloma (HRG) appearing as round structure enclosing eosinophilic amorphous material (HE, 9400). (b) HRGs lying in brous connective tissue densely inltrated by acute and chronic inammatory cells (HE, 9400). (c) HRGs in the form of roughly circular homogeneous or brillar masses exhibiting a corrugated border (HE, 9400). (d) Multinucleated giant cells inside and adjacent to hyaline material (HE, 9400). (e) Calcication in the form of small, coalescing basophilic droplets inside the hyaline material (HE, 9400). (f) Haemosiderin pigmentation scattered within the hyaline material and in the cytoplasm of mononucleated cells and multinucleated giant cells (HE, 9400).

Barker 1977, Chen et al. 1981, Talacko & Radden 1988a, Marcussen et al. 1993, Pola et al. 2003, Gueiros et al. 2008). Despite these reports, the aetiopathogenesis of these unusual histopathologic ndings remains a matter of discussion and data regarding their prevalence in inammatory odontogenic cysts are scarce. Therefore, this study determined the frequency of HRGs in a large case series of inammatory odontogenic cysts. In addition, histochemical and immunohistochemical analysis was performed to gain insights into the nature of these structures. In a retrospective study of oral HRGs, Talacko & Radden (1988a) only found minimal evidence of these structures in odontogenic cysts and periapical lesions and suggested that HRGs in these lesions might have

been overlooked in the past. In the present study, HRGs were usually small and inconspicuous. Nevertheless, even after careful and thorough microscopic examination, HRGs were found in only 22 (3.3%) of the 661 cases of inammatory odontogenic cysts studied, suggesting a very low frequency of these microscopic features in these lesions. The age range of affected individuals was 667 years in the present series and in 36 previous reports of HRGs in inammatory odontogenic cysts (Table 4), with a slight predominance amongst females (54.8%). In a review of 173 cases of oral HRGs, Philipsen & Reichart (2010) observed that more than two-thirds of the lesions occurred in the mandible, particularly in the posterior region where food stagnation is

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Table 3 Microscopic features of hyaline ring granulomas according to the type of inammatory odontogenic cyst
Cyst type Radicular cyst Residual radicular cyst Paradental cyst

Microscopic features Morphological pattern Round structures enclosing eosinophilic amorphous material Roughly circular homogeneous or brillar masses Round structures enclosing eosinophilic amorphous material and roughly circular homogeneous or brillar masses Calcication Presence Absence Haemosiderin pigmentation Presence Absence Metaplastic bone formation Presence Absence

3 11 4

0 2 1

0 1 0

1 17 1 17 0 18

0 3 0 3 0 3

0 1 0 1 0 1

(a)

(b)

(c)

(d)

Figure 2 (a) Focal positivity for collagen in hyaline ring granuloma (HRG) presenting as roughly circular homogeneous mass (Massons trichrome, 9400). (b) Diffuse positivity for collagen in HRGs presenting as roughly circular homogeneous masses (Massons trichrome, 9400). (c) Negativity for collagen in HRGs appearing as roughly circular homogeneous masses (Massons trichrome, 9400). (d) Negativity for collagen in HRG appearing as round structure enclosing amorphous material (Massons trichrome, 9400).

common. However, when only inammatory odontogenic cysts are analysed (Table 4), there is a slightly higher percentage of HRGs in the maxilla (55.4%). Nevertheless, in the present sample, most (88.9%) of the inammatory odontogenic cysts with HRGs located in the mandible were found in the posterior

region, in agreement with Philipsen & Reichart (2010). Amongst the 22 inammatory odontogenic cysts with HRGs found in this study, 18 (81.8%) were radicular cysts, 3 (13.6%) were residual radicular cysts and 1 (4.5%) was a paradental cyst. As radicu-

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(a)

(b)

(c)

(d)

(e)

(f)

Figure 3 (a) Positivity for CD68 in mononucleated and multinucleated giant cells inside and adjacent to hyaline ring granulomas (HRGs) in the form of roughly circular homogeneous masses (LSAB method, 9400). (b) Negativity for CD34 in mononucleated and multinucleated giant cells inside and adjacent to HRGs in the form of roughly circular homogeneous masses (LSAB method, 9400). (c) Positivity for CD68 in mononucleated and multinucleated giant cells inside and adjacent to HRG appearing as round structure enclosing amorphous material (LSAB method, 9400). (d) Negativity for CD34 in mononucleated and multinucleated giant cells inside and adjacent to HRG appearing as round structure enclosing amorphous material (LSAB method, 9400). (e) Weak positivity for collagen IV in HRGs in the form of roughly circular homogeneous masses (Advance method, 9400). (f) Negativity for collagen IV in HRG in the form of round structure enclosing amorphous material (Advance method, 9400).

lar cysts are the most frequent type of odontogenic cyst and residual radicular cysts and paradental cysts are relatively uncommon (de Souza et al. 2010), the frequency of HRGs was determined according to the type of cyst to avoid bias. There was a higher relative frequency of HRGs amongst residual radicular cysts (6.1%), followed by paradental cysts (5.6%) and radicular cysts (3.0%). As extraction sites and pericoronitis around the lower third molars, associated with food stagnation in the area, are possible pathways for the implantation of foreign bodies, especially food particles, the present ndings support the concept of an exogenous origin of oral HRGs (Lewars 1971, Mincer

et al. 1979, Talacko & Radden 1988a, LaMear et al. 1994). Moreover, in view of the low relative frequency of HRGs in radicular cysts, it may be suggested that carious teeth or root canals left open to the oral cavity are less efcient portals of entry for food particles than extraction sites and pericoronitis. Nevertheless, further studies including large series of residual radicular cysts and paradental cysts are needed to conrm this hypothesis. Microscopically, oral HRGs can manifest as round structures enclosing eosinophilic amorphous material (Gueiros et al. 2008) or as roughly circular homogeneous/brillar masses (Philipsen & Reichart

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Table 4 Distribution of the cases of inammatory odontogenic cysts with hyaline ring granulomas reported in the literature according to cyst type, age, gender and anatomic location
Gender Author Chen et al. (1981) Yang & Barnett (1985) Talacko & Radden (1988a) No. of cases 1 1 27 Cyst type Residual radicular cyst Radicular cyst Radicular cyst (13) Residual radicular cyst (12) Paradental cyst (2) Residual radicular cyst Residual radicular cyst Radicular cyst Radicular cyst Radicular cyst (18) Residual radicular cyst (3)a Paradental cyst (1) Age (years) 59 67 NS NS NS 20 30, 34 and 57 18 and 38 16 660 28 and 67 21 Male 1 NS NS NS 1 2 1 7 1 1 1 NS NS NS 1 2 11 2 10 5 1 2 1 1 10 1 Female Anatomic location Maxilla Mandible 1 1 3 7 2 1 1 8 1

Lin et al. (1993) Marcussen et al. (1993) Keskin et al. (2000) Pola et al. (2003) Current study

1 3 2 1 22

NS, not stated. a Information regarding anatomic location was not available for two cases. Information regarding age was not available for one case.

2010). Both patterns may be found in the same tissue section (Talacko & Radden 1988a). The present results suggest that roughly circular homogeneous or brillar masses are the most common morphologic pattern of oral HRGs in inammatory odontogenic cysts. According to Talacko & Radden (1988a), the hyaline material can also present a rod-like shape or appear in the form of long, branching laments. None of these features were observed in the HRGs studied here. Calcications are relatively uncommon in oral HRGs and appear in the form of small basophilic droplets either inside the hyaline material or inside the almost empty circular structures (Dunlap & Barker 1977, Chen et al. 1981, Ide et al. 1982, Talacko & Radden 1988a). Occasionally, calcication of the entire hyaline structure is observed (McMillan et al. 1981, Ide et al. 1982, Talacko & Radden 1988a, Philipsen & Reichart 2010). In the present series, calcication was seen in only one case (4.5%) of radicular cyst, conrming the low frequency of this microscopic feature in oral HRGs. Haemosiderin pigmentation was found scattered within the hyaline material and in the cytoplasm of mononucleated and multinucleated giant cells in only one case (4.5%) of radicular cyst. These ndings are in agreement with those reported by Chen et al. (1981). Metaplastic bone formation is very uncommon in oral HRGs, with only one case described in the literature (Ide et al. 1982). Coherently, this feature was not observed in the present series of HRGs.

The aetiopathogenesis of oral HRG is still unclear (Gueiros et al. 2008). Dunlap & Barker (1977) suggested that oral HRGs represent hyaline degeneration of the walls of blood vessels precipitated by localized acute vasculitis. According to other studies, oral HRGs may be formed by pooling and coagulation of extravasated serum proteins (Chen et al. 1981) or degenerated collagen (El-Labban & Kramer 1981). In contrast, evidence from an experimental animal model (Talacko & Radden 1988b) and the results of histopathologic (Marcussen et al. 1993, Sato et al. 2005, Gueiros et al. 2008) and ultrastructural (Harrison & Martin 1986, Pola et al. 2003, Sato et al. 2005, Gueiros et al. 2008) studies support the viewpoint that exogenous foreign material, especially from leguminous food, is responsible for the formation of oral HRGs. As the cellulose part of plant foods is indigestible, it may persist in human tissues in the form of hyaline material that elicits a chronic granulomatous response (Talacko & Radden 1988a, Philipsen & Reichart 2010). In the present study, most mononucleated cells and all multinucleated giant cells inside and adjacent to HRGs were positive for CD68 in all cases, regardless of the morphologic pattern of the hyaline material. On the other hand, these cell types were negative for CD34 in all cases, irrespective of the morphologic pattern of the hyaline material. This negative staining for CD34 near the hyaline structure rules out a possible endogenous origin as a result of hyaline degeneration of the vessel wall. Positive staining for CD68 indicates the presence of cells of the macrophage lineage, which is

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compatible with a foreign body granulomatous reaction. In fact, CD34 highlighted the presence of small blood vessels near the HRGs. These small vascular structures found near the hyaline ring, but not inside, would participate in the granulomatous reaction together with macrophages. Taken together, these ndings do not lend support to the endogenous theory proposed by Dunlap & Barker (1977), but rather reinforce the exogenous theory, which suggests that oral HRGs arise from the implantation of foreign material, most likely leguminous foods (Lewars 1971, Mincer et al. 1979, Talacko & Radden 1988a, LaMear et al. 1994, Philipsen & Reichart 2010). The different microscopic features of oral HRGs have been suggested to be related to distinct evolutionary stages (Gueiros et al. 2008). In this respect, the round structures enclosing eosinophilic amorphous material may represent a more recent stage in the development of oral HRGs when compared with the roughly circular homogeneous/brillar masses (Gueiros et al. 2008). The present histochemical ndings support this suggestion because all HRGs characterized by round structures enclosing eosinophilic amorphous material were negative for collagen, whereas most HRGs in the form of roughly circular homogeneous or brillar masses exhibited variable amounts of this protein. According to Harrison & Martin (1986), hyaline rings sometimes contain both vegetable cell wall and collagen, a fact that might explain the variable staining of these structures with different collagen stains (e.g. van Giesons and Mallorys) seen in previous studies (Chen et al. 1981, Harrison & Martin 1986, Talacko & Radden 1988a) and in the present investigation. Pola et al. (2003) detected no immunoreactivity to basement membrane proteins, such as laminin or type IV collagen, in an HRG present in the wall of a radicular cyst. Coherently, all HRGs characterized by round structures enclosing eosinophilic amorphous material were negative for collagen IV in the present study. However, all HRGs that appeared as roughly circular homogeneous or brillar masses were weakly positive for collagen IV, suggesting that small amounts of this protein might be incorporated into HRGs during their development. The fact that weak collagen IV staining was only observed in structures that represent a more advanced developmental stage of oral HRG does not support the endogenous theory proposed by Dunlap & Barker (1977). In the case series of Gueiros et al. (2008), HRGs appearing as round structures enclosing eosinophilic

amorphous material exhibited stronger periodic acid Schiff and diastase-resistant staining than those appearing in the form of roughly circular homogeneous/brillar masses. Taken together, these ndings and the present histochemical and immunohistochemical results suggest that vegetable material undergoes progressive degenerative changes, whereas collagen is deposited during the progression of oral HRGs. Gueiros et al. (2008) highlighted that inammation can be responsible for a distinct and persistent evolution of HRGs, especially at an intraosseous site. According to these authors, chronic exposure to inammatory enzymes probably modies the morphologic aspects of the hyaline rings without compromising their antigenic potential.

Conclusion
The present results suggest a very low frequency of HRGs in inammatory odontogenic cysts and support the hypothesis that these structures arise from the implantation of foreign material, most likely food particles of plant or vegetable origin. In addition, the diverse microscopic features of HRG possibly represent different developmental stages of this structure.

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