Portuguese Version

Year:  2003  Vol. 69   Ed. 3 - (3º)

Artigo Original

Pages: 304 to 310

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Study of the bone structure of etmoidal bulla in chronic rhinosinusitis

Author(s): Washington Luiz Almeida[1],
Perboyre L. Sampaio[2],
Nilvano Andrade[2],
Luiz Ricardo L. Martin[3],
Márcio S. de Carvalho[3]

Keywords: histopathology, ethmoidal bulla, chronic rhinosinusitis.

Abstract:
Several studies have shown a number of histopathological alterations which occur in chronic rhinosinusitis. The nasal mucosa in paranasal sinus has been the primary site of these researches but very little about the bone alterations found in these diseases is known. Aim: Describe the histopathological alterations of the bone structure of the ethmoidal bulla in patients who have chronical rhinosinusitis. Study Design: Clinical prospective. Material and Method: Nineteen patients who had chronic rhinosinusitis were evaluated. Through light microscope, the bone trabeculae of etmoidal bulla were analysed as to the range of osteoid storage, the presence of osteoblasts and osteoclasts on the surface of the trabeculae, reabsorption, apposition lines and fibrosis between the bone trabeculae. The lamina propria was also analysed as to cellular elements, considering that inflammatory cells which release mediators that promote the bone lysis are found on this layer of the mucosa. The bone morphology was analyzed using electronic microscopy. Conclusion: Using scanning electronic microscopy, it was possible to observe the edges of the reabsorbed bone trabeculae more clearly. No case was considered normal, but no bone necrosis was observed. Through light microscope were observed: osteoids storage, osteoblasts agroupment, fibrosis and bone remodeling in about 90% of cases. Although the osteoclasts had not been seen, the bone reabsorption was present in 50% of cases. Deeper studies about inflammatory mediators on lamina propria in their actions on subjacent bone can clear up the physiopathology of chronic rhinosinusitis on the cellular level.

INTRODUCTION

Rhinosinusitis could be clinically defined as an inflammatory response of the lining mucosa of nasal cavity and paranasal sinuses that may be extended to neuroepithelium and underlying bone 1.
In the USA, rhinosinusitis is considered the major cause of medical appointments affecting more than 30 million people every year 2,3.

The obstruction of the ostiomeatal complex (COM), the region of middle meatus in which all maxillary, anterior ethmoidal and frontal sinuses' drainage orifices are located has been considered the most important aspect in the pathophysiology of chronic rhinosinusitis (CR)4-6.

In spite of the advances in research and clinical applications, the clear pathophysiology of Chronic Rhinosinusitis still remains unknown.

There is some experimental and clinical evidence that bone may play a role in CR. Westrin et al. reported in their study that bone synthesis and reabsorption were observed, as well as the presence of inflammatory cells 7. These findings, compared with long bone and mandible osteomyelitis, suggest that subjacent bone works as a rhinosinusitis catalyst 8.

Because of the clinical importance of rhinosinusitis and its high prevalence in the population together with its several etiological and pathogenic mechanisms, and the lack of knowledge about bone alterations found in CR, we designed a study of the bone structure of ethmoidal bulla in CR patients with surgical recommendation.

The objective of this study was to describe the pathophysiology characteristics using light and scan electron microscopy of bone structure of the ethmoidal bulla in CR patients.

MATERIAL AND METHODS

A prospect study was conducted in 19 patients with Chronic Rhinosinusitis, with surgical indication, operated on in Hospital Otorrinos of Feira de Santana, Bahia; in Santa Casa de Misericórdia of Feira de Santana, Bahia and in Hospital Santa Izabel in Salvador, Bahia, from March 2001 through August 2002.

The inclusion criteria defined were as follows:

Patients should be older than 18 years with Chronic Rhinosinusitis with symptoms for more that three months and surgical indication.

The exclusion criteria defined were the following:

- Patients with nasosinusal polyposis, fungal rhinosinusitis and systemic diseases such as diabetes, cystic fibrosis, primary cilia dyskinesia and immune-deficiencies.
- Patients that received antibiotics and/or steroids less than 30 (thirty) days before surgery.
- Patients that have previously undergone nasal or paranasal sinuses surgery.

Patients selected were then subjected to nasosinusal endoscope surgery under general anesthesia with only their medial wall of ethmoidal bulla removed with scissors or cutting forceps. The ethmoidal bulla was divided in two fragments: one fragment was placed in a container with Formaldehyde at 10% for light microscopy (LM) and the other one was placed in Karnovsky solution (Paraformolaldehyde at 2% plus gluraldehyde at 5% and phosphate buffer 0.1 molar) for scan electron microscopy (SEM).
Sections were dyed with Masson and Hematoxylin-eosin (HE) for light microscopy of lamina propria and bone structures.
Soft tissues were removed with Sodium Hypochloride at 1% for visualization of bone tissue.

RESULTS

Light Microscopy

# Lamina propria

The pathological and histological analysis of lamina propria of the ethmoidal bulla mucosa in 19 cases of Chronic Rhinosinusitis as to inflammatory infiltrate and cell composition has revealed the following. (Table 1):
1. In all cases a mild inflammatory infiltrate was observed (Figure 1).
2. Only lymphocytes and plasmocytes were present in 100% of the studied cases. (Figure 2).
3. Lymphocytes were present in large quantity in only 3 cases (15.79%), whereas they were moderate in the other cases (84.21%).
4. Plasmocytes were found in moderate quantity in 15 cases (78.95%) (Figure 2) and in small amounts in the other 4 studied cases (Figure 1).
5. Hystiocyte were absent in all cases.
6. Neutrophils were present in 6 cases (31.58%), but in small amount relative to the inflammatory cells present.
7. Eosinophils were found in 8 cases (42.11%), all in small quantity relative to the inflammatory cells present.

# Bone Trabeculae

The histopathological analysis of bone trabeculae of ethmoidal bulla in 19 cases of chronic rhinosinusitis (Table 2) revealed that:
8. In 17 cases (89.47%), osteoid storage was observed on the surface of bone trabeculae, but in most of them (63.16%), rare focuses were found. Multiple focuses were found in only 1 case (5.26%) in more than 50% of bone trabeculae.
9. Osteoblast clusters were also detected in 17 cases (89.47%). In two of them (10.53%), these clusters were present in more than 50% of the trabecular surface and in the 15 remaining cases (78.95%) they present in less than 50% of the trabecular surface.
10. Osteoclasts were not observed in any case.
11. Bone reabsorption occurred in 9 cases (47.37%). In 6 of them (31.58%), bone reabsorption was present with multiple focuses (Figure 3) in up to 50% of the bone trabeculae, and in other 3 cases (15.79%), in small quantity and rare focuses.
12. Bone apposition lines were present in all cases (Figure 4), and in general were parallel to the trabecular surface and in most of the cases (13 cases [68.42%[) they were rare (one line).
13. Fibrosis was present in 17 cases (89.47%), hypocellular fibroid tissue was found between bone trabeculae, nonetheless significant inflammatory tissue permeable infiltrate was not found in any of them.
14. No inflammatory cells were observed between bone trabeculae.
15. Bone remodeling was observed in only 2 cases (10.53%) with extensive activity; it was moderate in 8 (42.11%) and mild in 7 (36.84%). Bone remodeling was not found in 2 cases in our study.

Scanning Electron Microscopy

# Bone Tissue

In panoramic images in which mucosa and submucosa were removed it was possible to see the distribution of collagen tissue (periostium) in nasal cavity occupied by ethmoidal sinus (ethmoidal bulla) with view of sinus cells and signs of erosion in bone structure (Figure 5).

Connective tissue that was in contact with bone, submucosa and periostium was distributed in collagen strands of well defined orientation, many times forming helicoid paths (Figure 6). The tissue was dense in nature and highly adhered to bone, and in spite of the methods used in the study, it was difficult to remove it.

We clearly observed in bone trabeculae the bone edges that presented irregular contours with invaginated aspect (Figure 7). In other areas, the surface of bone trabecula was perforated.

Table 1. Histopathology analysis of the lamina propria of the mucosa of the ethmoidal bulla concerning the inflammatory infiltrate and cell composition. Cases Lymphocytes Plasmocytes Hystiocytes Neutrophils Eosinophils Inflammatory infiltrate

(-) absent; (+) up to 25% inflammatory cells; (++) from 25.1% to 50%; (+++) from 50.1% to 100%. D= mild.

Tabela 2

Table 2. Histopathology analysis of bone trabeculae of the ethmoidal bulla. Cases Osteoid on the surface of TO Osteoblast on the surface of TO Osteoclast on the surface of TO Bone apposition lines Fibrosis between TO Inflammatory cells between TO Bone reabsorption Bone remodeling

- Osteoid on bone trabecular surface (-) absent, (+) small amount and rare focuses, (++) multiple focuses in up to 50% of bone trabeculae, (+++) multiple focuses in more than 50 % of bone trabeculae.

- Osteoblasts and osteoclasts on bone trabecula surface (-) absent, (+) small number of cells and rare clusters, (++) cellular clusters in up to 50 % of bone trabeculae, (+++) cellular clusters in more than 50% of bone trabeculae.

- Bone apposition lines (-) absent, (+) rare, (++) moderate quantity of lines, (+++) frequent lines.

- Fibrosis between bone trabeculae A = absent, P = present.

- Inflammatory cells between bone trabeculae (BT) A = absent, P = present.

- Bone reabsorption (-) absent, (+) small quantity and rare focuses, (++) multiple focuses in up to 50 % of bone trabeculae, (+++) multiple focuses in more than 50% of bone trabeculae.

- Bone remodeling: (+++) extensive osteoid build up or large quantity of osteoblasts in more than 50% of trabecular surface; (++) moderate osteoid build up or moderate quantity of osteoblasts in up to 50% of trabecular surface; (+) mild osteoid build up or small quantity of osteoblasts in rare focuses of trabecular surface and (-) absent.

DISCUSSION

Many times, endoscope nasosinusal surgeries performed in patients with CR fail to control the disease or prevent its recurrence which leads us to believing that there might be a factor sustaining the inflammatory process in CR. Nasal and paranasal sinuses mucosa have been the primary site in studies, however very little is known about bone alterations in chronic rhinosinusitis.

Ethmoidal bulla is usually the largest and most consistent cell in the anterior ethmoidal labyrinth. 9,10. According to several authors 11,12, ethmoidal sinuses play the most significant role in nasosinusal infectious process. Since ethmoidal bulla is inserted in the ostiomeatal complex, it has an indirect or direct role in nasal-sinusal infectious processes, and it is easily obtained during surgery for histological and ultra-structural exams, therefore justifying the choice of such structure in our study.

Bone lysis that occurs in Chronic Otitis Media similarly to CR is caused by enzyme effect of inflammatory cells (lymphocytes, plasmocytes, hystiocytes, neutrophils and eosinophils) found in sub mucosa layers of the middle ear and paranasal sinuses 13,14. There was a predominant presence of eosinophils among such inflammatory cells 15,16. In our study, lymph-plasmocitary prevalence was fount in all cases (Figure 2). Eosinophils were found in only 8 cases in our study, still in small quantities against the other inflammatory cells found.

Enzyme action of inflammatory cells may stimulate bone cells increasing its proteolytic enzyme production17. The combination of destructive action of inflammatory cells with osteocystic osteolysis could explain bone lysis in the absence of osteoclasts. In our case, we were able to assume that bone reabsorption had been caused by the action of mediators of inflammatory cells present in the lamina propria since osteoclasts were not found in the reabsorption focus. Nonetheless, osteoclasts between bone cells have shorter life, and could have already performed their reabsorption function and suffered apoptosis.

Cytokines, osteoclast activating factors, leukotrienes and prostaglandins may stimulate osteoclasts to take part in bone remodeling, therefore playing a key role in pathophysiology of Chronic Rhinosinusitis14.

Osteoclasts are the cells responsible for bone reabsorption. After some period of bone reabsorption, osteoclasts were arranged in small depressions on bone surface called Howship's lacunae. Bone reabsorption was present in less than half of our cases, and most of it happened due to bone irregularities. No Howship's lacunae were found in the cases evaluated in this study. Although in his study Rodrigues13 found bone erosion in patients with Chronic Otitis Media and Cholesteatomatous Otitis Media, no osteoclast erosion focus was observed. Likewise, no osteoclast was found in bone reabsorption focuses.

Trial studies with rhinosinusitis induction in rabbits identified inflammatory processes in underlying bone with massive fibrosis and bone reabsorption by osteoclasts 7. In the case studied, fibrosis was present in almost all of them; however, inflammatory infiltrate was mildly intense. Bone reabsorption occurred in 9 out of 19 cases, with no osteoclasts in the reabsorption focuses. It is worthy mentioning that results of the studies quoted by the authors were obtained through sinusitis induction in lab animals, in which histopathological findings could be considerably more intense than those found during the normal course of chronic rhinosinusitis in human beings.

In the histopathological studies of diffuse chronic osteomyelitis of the mandible, sclerotic bone was found with narrowing or occlusion of Harvesian canal 18. In his study about chronic rhinosinusitis induced in rabbits Khalid19 observed multifocal osteonecrosis, massive bone remodeling and fibrosis inside the Harvesian System. Kennedy et al. found massive sclerosis, fibrosis and bone remodeling in human ethmoidal bone with CR inflammatory process with histopathological results similar to those found in osteomyelitis. Fibrosis was present in almost all cases of this study (89.47%), being that hypo cellular fibrous tissue was observed amidst bone trabeculae; however, permeable inflammatory infiltrate was not found in this tissue. No case in our study was considered normal, yet no cases with osteomyelitis sign were reported.

Nonetheless Hwang et al.20 observed massive ethmoidal bone remodeling in CR, which was histologically similar to that found in osteomyelitis, no bacteria were found in subjacent bone as commonly found in osteomyelitis. No bacteria were found with the method used in this study. Additionally, no inflammatory cells were found in bone trabeculae in the studied cases.

Bone remodeling is a phenomenon that occurs at an ongoing basis during all life. In such phenomenon, the bone changes and replaces its structure and is more intense both in Chronic Otitis Media and Chronic Rhinosinusitis 8. In this study, bone remodeling occurred in a mild and moderate fashion in most of the cases, and they were more intense in only two cases. This remodeling was classified according to osteoid storage and quantity of osteoblasts in bone trabeculae. Since we did not find excavated areas like Howship's lacunae or osteoclasts, the reabsorption areas considered were the bone irregularities suggesting that previous reabsorption and osteoid storage occurred first, followed by osteoblasts.

The use of scan electron microscopy in the study of chronic rhinosinusitis has been employed by several authors 21,22, but the target of such studies has been the respiratory epithelium. We did not find articles in the literature about morphological characteristics of bone tissue in chronic rhinosinusitis with use of SEM that would enable us to compare them with our results.

CONCLUSION

Light Microscopy findings were as follows: osteoid storage, osteoblasts clusters and fibrosis in approximately 90% of the cases; the presence of bone reabsorption in approximately 50% of the cases, in spite of osteoclasts absence; bone remodeling occurred in approximately 90% of the cases.

Through SEM we could more clearly observe the following: the 3D aspect of the periosteum formed by dense collagen tissue distributed in beams of well defined orientation, many times forming helicoid paths and the bone trabecula edges, which presented irregular contours.

Most certainly, future studies on the action of inflammatory mediators present in the lamina propria on subjacent bone will be able to clarify the pathophysiology of chronic rhinosinusitis at cellular level.

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1 - Ph.D. in Medicine, Medical School, University of São Paulo. Clinical Director of Clinica Otorrinos.
2 - Ph.D. in Medicine, Medical School, University of São Paulo.
3 - Resident in Otorhinolaryngology, Clinica Otorrinos.

Study conducted at Clinica Otorrinos, Feira de Santana BA.

Address correspondence to: Washington Luiz Almeida - Rua Barão de Cotegipe, 1141 Centro 44025-030 Feira de Santana BA - Tel/Fax (55 75) 623-4455/ 223-4117 - E-mail: otorrinos@uol.com.br

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