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Indian Journal of Otolaryngology and Head & Neck Surgery logoLink to Indian Journal of Otolaryngology and Head & Neck Surgery
. 2024 Jun 11;76(5):4064–4073. doi: 10.1007/s12070-024-04784-8

Evaluation of the Role of Myofibroblast and Fibronectin in the Aetiopathogenesis of Cholesteatoma

Ramya Ravindra Kamath 1,✉, Prahlada Nayaka Basavanthappa 2, B J Bindu 3, C Narayana Murthy 2,3, Gouri Priya Rajeev 2,3, S Raisa 2
PMCID: PMC11455809  PMID: 39376278

Abstract

Background

Primary acquired Cholesteatoma is a complex issue for otolaryngologists, with its development mechanisms still unclear due to the intricate anatomy of this region. It’s aetiopathogenesis remains poorly understood and this aggressive clinical condition often leads to various complications. Recent research explores myofibroblast and fibronectin's potential roles in pathomechanisms of Cholesteatoma.

Objective

To determine and analyze the role of myofibroblast and fibronectin in the aetiopathogenesis of Cholesteatoma.

Methodology

In a cross-sectional study at a tertiary care hospital, 30 patients with chronic suppurative otitis media with cholesteatoma were surgically treated, and intraoperative biopsy specimens were collected. These specimens were processed and subjected to histopathological examination, including immunohistochemical staining with Alpha-smooth muscle actin and anti-fibronectin antibody to identify myofibroblast and fibronectin presence. The data were then analyzed to investigate the aetiopathogenesis of cholesteatoma in this cohort.

Results

On immunostaining, 25 blocks (83.33%) were positively stained for Alpha-SMA (p-value—0.0007), whereas 29 blocks (96.67%) were positively stained for fibronectin (p-value < 0.0001), suggesting a statistically significant association between the presence of both myofibroblast and fibronectin with cholesteatoma perimatrix. Additionally, a statistically significant association was noted between complications and positive staining for myofibroblast (p-value − 0.0415) and positive staining for fibronectin (p-value—0.0254).

Conclusions

Our study indicates that Cholesteatoma retraction and progression are driven by myofibroblast and fibronectin mechanisms, and also links them to disease severity. This understanding opens avenues for innovative diagnostics and treatments targeting these biomarkers.

Keywords: Cholesteatoma, Aetiopathogenesis, Myofibroblast, Fibronectin

Introduction

Acquired cholesteatoma, with an annual incidence of 9–12.6 cases per 100,000 in adults and 3–15 cases per 100,000 in paediatric populations, is a serious health concern due to its proliferation and destruction within the temporal bone [1]. This lesion comprises a central cystic matrix of keratinized stratified squamous epithelium and a perimatrix containing collagen fibres, fibrocytes, and various inflammatory cells [2, 3].

Clinically, cholesteatoma presents with chronically draining ears, malodour, and progressive hearing loss. Complications can include ossicular chain destruction, vestibular dysfunction, facial paralysis, and intracranial issues, often due to osteitis and secondary infections by Pseudomonas aeruginosa and Staphylococcus aureus [2–5]. Diagnosis and post-operative monitoring rely on high-resolution CT and MRI, including diffusion-weighted sequences [6]. Surgical techniques like Canal Wall-Up (CWU) and Canal Wall-Down (CWD) mastoidectomy are employed to clear disease and prevent recurrence [7].

Understanding the pathogenesis of cholesteatoma is critical, yet it remains debated. Theories include squamous metaplasia, epithelial invasion/migration, squamous basal hyperplasia, and retraction pockets/invagination [8–17]. Recent theories by Marchioni et al. and Jackler et al. suggest selective epitympanic dysventilation and mucosal traction, respectively [18, 19].

Myofibroblasts and fibronectin play a role in cholesteatoma pathogenesis. Fibronectin, a crucial extracellular matrix component, influences keratinocyte migration and differentiation, impacting epithelial gap closure and terminal differentiation [20–22]. Studies by Clark et al. and Sundqvist et al. link fibronectin to delayed-type hypersensitivity reactions and lymphocyte-tissue interactions [24–31]. Imbalances in matrix metalloproteinase activity, as well as overexpression of TGF-beta, lead to abnormal fibronectin deposition and pathological conditions [20, 37–47]. Myofibroblasts contribute to fibronectin production and tissue remodelling, with disruptions leading to fibrosis and conditions like hypertrophic scars and chronic otitis media [23, 48–56].

Given the incomplete understanding of cholesteatoma pathogenesis, comprehensive patient management remains challenging. This study aims to elucidate the roles of myofibroblasts and fibronectin in cholesteatoma by examining their presence and intensity in biopsy samples. This could propose a novel enteropathogenic theory, offering new therapeutic insights for this aggressive disease.

Materials and Methods

Study Design and Setting: This cross-sectional study was conducted at the Department of Otorhinolaryngology, Basaveshwara Medical College Hospital, Chitradurga, spanning from March 1, 2021, to September 1, 2022, after obtaining clearance from the Institutional Ethics Committee with reference no BMC & H/IEC/2020–2021/99, dated 16/01/2021.

Study Participants: The study comprised 30 patients diagnosed with chronic suppurative otitis media complicated by cholesteatoma and aged 18 or above. Exclusion criteria encompassed patients deemed unfit for surgery or those expressing reluctance towards the procedure. Patients were enrolled upon obtaining written informed consent, and a pre-structured questionnaire was used to compile their clinical profiles.

Diagnostic Procedures: Patients underwent a thorough ENT examination with a specific emphasis on otological examination and Pure Tone Audiometry (PTA) to evaluate their hearing. The extent of the disease, its anatomical variations, and any associated complications were assessed using HRCT of the temporal bone.

Surgical Procedure: Depending on the individual case, patients were subjected to either Canal-wall-up (combined approach tympanoplasty) or Canal-wall-down surgical procedures under appropriate anaesthesia. During surgery, cholesteatoma biopsy samples, including the adjacent diseased tympanic membrane and middle ear mucosa, were harvested for histopathological analysis.

Histopathological and Immunohistochemical Evaluation: Biopsy specimens were preserved in 10% neutral buffered formalin (NBF) and submitted to the hospital's Pathology Department. The tissues were processed into formalin-fixed paraffin-embedded (FFPE) blocks, subsequently stained with hematoxylin and eosin (H&E), and analyzed for cholesteatoma and its constituent elements.

Immunohistochemical (IHC) analysis utilized the same FFPE blocks. For myofibroblast identification, Alpha-smooth muscle actin (Alpha-SMA) [Clone – Alpha actin – EP118, Manufacturer – Path & Situ, USA] served as the marker. Leiomyoma tissue was employed as the positive control. Meanwhile, fibronectin was detected using an anti-fibronectin antibody [Clone-Rabbit polyclonal antibody, Manufacturer –DBS, USA], with skin tissue acting as the control.

Staining Evaluation: Staining intensities for Alpha-SMA positive cells, which were myofibroblasts, were assessed in line with Etemad-Moghadam et al.'s protocol and graded from 1 to 457,58. Concurrently, fibronectin immunostaining was categorized into strong, intermediate, or weak intensities.

Statistical Analysis: Data collated on Microsoft Excel was analyzed using IBM SPSS Statistics for Windows, Version 20.0 (IBM Corp., Armonk, NY, USA). Categorical variable outcomes were described through frequencies and percentages, with associations gauged using the Chi-square test. Continuous variables were expressed as mean ± standard deviation (SD) and analysed with the T-test or Mann–Whitney U test based on data distribution. Statistical significance was ascertained at a p-value < 0.05, wit.

Results

In this study, 30 patients were evaluated, ranging from 18 to 66 years, with an average age of 32.133 ± 13.97 SD years. Both genders were equally represented in the cohort. The universal complaint was ear discharge, with 96.67% reporting decreased hearing. Other notable symptoms included ear pain (26.67%), tinnitus (20%), and giddiness (10%). Only one patient had a prior history of myringotomy with grommet insertion.

Examination revealed various tympanic membrane findings, with posterosuperior retraction pocket (PSRP) being the most common at 46.67%. Additionally, 96.67% of the patients displayed attic erosion, an early cholesteatoma indicator. Other findings, like granulation tissue and aural polyps, were less frequent. Hearing loss (HL) was a universal complication, with 86.67% experiencing conductive hearing loss. Following ASHA guidelines, most patients had moderate (40%) or mild (30%) hearing loss, with fewer in the more severe categories [59].

The most frequently conducted surgeries were modified radical mastoidectomy with ossiculoplasty (73.33%). Additional procedures like labyrinthine fistula repair were also utilized in 23.33% of cases. Cholesteatoma predominantly occurred in the epitympanum (96.67%), and involvement of the mastoid antrum was universal. All biopsy specimens were confirmed to contain cholesteatoma debris. Microscopic evaluation revealed dense granulation tissue in 60% of cases and dense chronic inflammatory infiltrate in 80% of cases. Half of the cases exhibited abundant keratin debris.

Cholesteatoma has innermost cystic content or keratin mass, enclosed by a matrix layer of stratified squamous epithelium, as depicted in Fig. 1 & 2. The surrounding perimatrix consists of fibrous tissue and inflammatory infiltrate [4].

Fig. 1.

Fig. 1

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Hematoxylin and eosin stained slide depicting cholesteatoma

Fig. 2.

Fig. 2

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Immunohistochemical staining negative for Alpha-SMA and fibronectin in the matrix and cystic content of cholesteatoma

The study identified positive immunohistochemical (IHC) staining for Alpha-SMA and fibronectin solely in the cholesteatoma's perimatrix. Specifically, 83.33% (n = 25) of the examined blocks displayed positive Alpha-SMA staining (Fig. 3 & 4), suggesting a significant correlation between myofibroblast immunostaining and cholesteatoma perimatrix (p = 0.0007).

Fig. 3.

Fig. 3

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Distribution of Immunohistochemical Staining for Alpha SMA staining

Fig. 4.

Fig. 4

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Grades of intensity for immunohistochemical staining of Alpha SMA

The positive immunostaining for Alpha-SMA appeared as membranous staining. A 33.33% (n = 10) of cases had a grade 2 staining percentage for Alpha-SMA with 34 to 66% positive cells visible on microscopy, followed by 26.67% of cases (n = 8) had a grade 3 staining percentage with 67 to 100% positive cells whereas 23.33% of cases (n = 7) had grade 1 staining percentage with only 1 to 33% positive cells noted. The remaining 16.67% of cases (n = 5) were negative for Alpha-SMA staining, i.e., zero positive cells were noted (Fig. 4 & 5).

Fig. 5.

Fig. 5

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Immunohistochemical golden brown membranous staining of myofibroblasts for Alpha-SMA in perimatrix of cholesteatoma. A. Grade 0 negative staining intensity (40x). B. Grade 1 staining intensity (40x). C. Grade 2 staining intensity (10x). D. Grade 3 intensity (10x)

Concerning fibronectin, 96.67% (n = 29) of blocks were positively stained, and there was a significant association between fibronectin immunostaining and cholesteatoma perimatrix (p < 0.0001), as depicted in Fig. 6.

Fig. 6.

Fig. 6

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Distribution of Immunohistochemical Staining for Fibronectin

A 46.67% (n = 14) of cases had intermediate staining for fibronectin with staining visible at 40 × on microscopy, followed by 33.33% of cases (n = 10) had strong staining that was visible at 10 × on microscopy, whereas 16.67% of cases (n = 5) had weak staining, which was visible only on oil immersion (100x). One case (3.33%) was negative for fibronectin staining, i.e., no staining was noted even on 100x (Fig. 7 & 8).

Fig. 7.

Fig. 7

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Grades of intensity for immunohistochemical staining of Fibronectin

Fig. 8.

Fig. 8

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Immunohistochemical golden brown staining for fibronectin (10x). A. Negative-No staining in perimatrix or epidermal–dermal junction. B. Weak staining. C. Intermediate staining noted in perimatrix. D. Strong staining at perimatrix (band pattern)

Out of the 30 cases with complications, Alpha-SMA staining was positive in 83.33% (n = 25) of the cases (Fig. 9), with a p-value of 0.0415 obtained on Chi-square test, suggesting a statistically significant association between the presence of complications and positive staining for myofibroblast.

Fig. 9.

Fig. 9

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Among 30 cases with complications, 83.33% (n = 25) showed positive Alpha-SMA staining. A Chi-square test yielded a p-value of 0.0415, indicating a statistically significant association between complications and positive myofibroblast staining

Similarly, fibronectin staining was positive in the 30 cases, with complications in 96.67% (n = 29) (Fig. 10). A p-value of 0.0254 obtained on the Chi-square test pointed towards a statistically significant association between the presence of complications and staining for fibronectin.

Fig. 10.

Fig. 10

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In the 30 cases with complications, 96.67% (n = 29) exhibited positive fibronectin staining. A Chi-square test resulted in a p-value of 0.0254, suggesting a significant correlation between complications and fibronectin staining

We further investigated the correlation between preoperative complications and the intensities of two IHC markers, Alpha SMA (mMFB) and Fibronectin with Pearson correlation coefficients. The results showed a weak positive correlation (0.0873) between preoperative complications and IHC-Alpha SMA (mMFB) intensity, indicating a slight increase in marker intensity with more complications. In contrast, there was a moderate positive correlation (0.5086) between preoperative complications and IHC-Fibronectin intensity, suggesting a more noticeable increase in marker intensity with more complications. The multiple linear regression analysis aimed to understand the relationship between preoperative complications and the intensities of two IHC markers, Alpha SMA (mMFB) and Fibronectin. The model used the number of preoperative complications as the response variable and the intensities of the IHC markers as predictors. The results indicated that both IHC-Alpha SMA and IHC-Fibronectin had positive coefficients (0.605 and 0.540, respectively), suggesting that higher intensities of these markers were associated with an increase in the number of preoperative complications. However, the relationships were not statistically significant, with p-values of 0.232 and 0.308, respectively. The model's R-squared value was 0.308, meaning that approximately 30.8% of the variability in preoperative complications could be explained by the intensities of the IHC markers. This suggests a moderate fit, but also indicates that other factors likely play a significant role.

Overall, these results emphasize the intricate relationship between cholesteatoma's pathological development based on myofibroblast and fibronectin-based mechanisms and the associated propensity for complications in those patients with positive immunostaining.

Discussion

Cholesteatoma is a progressive lesion of the temporal bone with a propensity for bone erosion and complications with no effective non-surgical treatments. Thus, it is vital to know the mechanisms underlying its development and progression [2, 3, 8]. Several theories on aetiopathogenesis of acquired cholesteatoma, like squamous metaplasia theory, epithelial invasion/migration theory, and Retraction pockets/invagination theory, among others, have been proposed, which emphasize the role of the squamous layer of the tympanic membrane. They are based on a similar concept: a deep retraction with narrow opening forms, followed by sustenance of further cholesteatoma expansion by the pressure effect from the impacted keratin [8, 19].

Recently, Marchioni et al. introduced the concept of selective epitympanic dysventilation syndrome, while Jackler et al. put forward the mucosal traction theory, both aiming to elucidate the pathogenesis of cholesteatoma [18, 19].

Squamous metaplasia Theory [9, 10]

Currently, histological or experimental evidence of squamous metaplasia resulting in cholesteatoma does not exist, and it has been supported by evidence that the squamous epithelium in cholesteatoma has an ectodermal origin. This is the drawback of the squamous metaplasia theory [11].

Squamous Immigration / Invagination Theory [11, 12].

Though squamous epithelium can be made to migrate medially through a marginal perforation in animal models, the drawback of the squamous immigration or the invagination theory is that despite the higher incidence of tympanic membrane perforation, cholesteatomas are relatively infrequent. Conversely, cholesteatomas have also been noted to develop behind an intact tympanic membrane [11].

Basal Cell Hyperplasia/Papillary Ingrowth Theory [13, 14].

Though processes like inflammation-driven basal keratinocyte proliferation and their subsequent penetration of the basement membrane to extend into the subepithelial space can be induced in animal models and have also been demonstrated in histopathological specimens of human cholesteatoma, currently no evidence exists to prove the presence of a driving force for the basal cells to migrate medially rather than laterally. In order for this to be plausible, there should also exist simultaneous weakening of the supporting structures and exertion of inward traction on the basal keratinocytes [11].

Squamous Obstruction-Vacuum-Retraction Theory / Retraction Pocket Theory [15–17].

This currently widely accepted theory focuses on the Eustachian tube dysfunction inducing a vacuum in the middle ear cavity, resulting in a retraction pocket. However, it has been noted that several animal models of Eustachian tube dysfunction did not induce cholesteatoma. Also, several ears with cholesteatoma have been found to have no evidence of Eustachian tube pathology on endoscopy. Though this theory states that Eustachian tube obstruction must induce cholesteatoma, it has been observed that ears with tubal occlusion develop cholesteatoma infrequently. Additionally, tympanostomy tubes did not have any role in preventing cholesteatoma formation. These observations provide evidence against the squamous obstruction-vacuum-retraction or retraction pocket theory [11].

Selective Epitympanic Dysventilation Syndrome [18].

Marchioni et al. proposed Selective Epitympanic Dysventilation Syndrome as the cause of cholesteatoma, requiring four conditions: an attic retraction pocket or cholesteatoma, normal tubal function tests, an intact epitympanic diaphragm, and an isthmus blockage. However, Jackler et al. suggest this theory is a variation of the obstruction-vacuum retraction theory, focusing on hypoventilation in the epitympanum creating negative pressure. While this explains epitympanic retraction with normal eustachian tube function, it fails to address cholesteatoma expansion into fluid-filled areas or the development of other types post-surgery, highlighting its limitations [60].

Mucosal Traction Theory [20].

In 2015, Jackler et al. conducted a study to evaluate the migratory behaviour of the medial mucosal surface of the tympanic membrane, which needs to be better understood, unlike the well-described migration property of the squamous outer surface. He proposed the mucosal traction theory, which emphasizes that the tractional force generated by mucosal migration in opposing directions provides the basis for the properties of acquired cholesteatoma [11]. However, Pauna et al. conducted a study in 2018. They found that ciliated cells in the middle ear cleft are plentiful in the tympanum and hypotympanum. However, they are sparse on the medial surface of the tympanic membrane and epitympanum and over the lateral surface of the ossicles in the epitympanum. In cases of chronic otitis media with and without cholesteatoma and in cases of retraction pockets, these ciliated cells were even rare. The results of their study do not support the concept that the epithelium of the epitympanum and ossicles undergoes metaplasia to become ciliated cells [52, 61]. Thus, it was found that the earlier theories on the aetiopathogenesis of cholesteatoma have several drawbacks.

Several recent studies support the observations made in our study. On analyzing the ultrastructure of cholesteatoma, Franzer et al. (2010) identified certain similarities between the basal lamina of cholesteatoma and normal skin. A marked fibronectin expression was observed in the basal membrane zone of the cholesteatoma. This is consistent with our observations where we found positive fibronectin immunostaining in the cholesteatoma, specifically at the epidermal-stromal junction and perimatrix. Our study revealed a distinct pattern of cholesteatoma showing a dysregulated cell–matrix interaction, which can potentially explain its hyperproliferative epithelial nature [62].

Similar patterns of fibronectin staining have been noted in studies by Lang et al. and Sudhoff et al. [50, 51], corroborating our findings. The role of fibronectin, periostin, and tenascin in cholesteatoma formation was also suggested by Birinci et al. [63], supporting our observation regarding the immunostaining patterns of fibronectin in cholesteatoma. Furthermore, Sudhoff et al. noted the presence of inflammatory reactions and increased vascularization within the cholesteatoma perimatrix, supporting our observations of chronic inflammatory infiltrate in the biopsy specimens [51]. Our findings were also mirrored in studies conducted by Schilling et al., Gantz BJ, Hart MJ, Palva T, and Taskinen E, which pointed to the role of fibronectin in cell–matrix interactions during inflammatory reactions [20, 24–26].

A study by Berlinger et al. highlighted myofibroblasts' potential role in cholesteatoma development [54]. Similarly, we observed the presence of myofibroblasts in a statistically significant proportion of examined blocks. Moreover, Raffa et al. noted altered fibroblast properties in cholesteatoma samples, pointing to the need to understand epithelial-mesenchymal interactions in various disease states [55]. Similar observations were made in our study.

Moreover, the research conducted by Lang et al. centred on the disrupted interactions between cells and the matrix in cholesteatoma cases [50]. This aligns with our findings and reflects Schilling et al.'s observation of fibronectin's role in aural cholesteatoma [20, 37]. Hence, our observations and similar earlier studies provide valuable evidence corroborating our hypothesis about the role of myofibroblasts and fibronectins in cholesteatoma development.

Additionally, our study postulates a possible relationship between the presence of myofibroblasts and fibronectin and the severity of the clinical disease. Understanding these myofibroblast- and fibronectin–based pathomechanisms of cholesteatoma development can significantly transform this insidious disease's diagnostic and treatment modalities.

Moreover, recent studies demonstrated that fibroblast stimulation depends on the deposition of extracellular domain fibronectin (ED-A FN) [47], blockade of TGF-beta demonstrated benefit in some fibrotic diseases [64] and TLR4 signalling pathway enhanced fibroblast proliferation [62], thus allowing novel therapeutic approaches to cholesteatoma by targeting these biomarkers [65–70].

In summary, the immunohistochemical presence of myofibroblasts and fibronectin in the majority of our biopsy specimens suggests a significant cellular and molecular involvement in the aetiopathogenesis of cholesteatoma. Furthermore, the statistically significant association of myofibroblasts and fibronectin with severe cases implies that these cellular and molecular elements could underlie the aggressiveness of the disease.

Nevertheless, we acknowledge the limitations of our study, including a smaller sample size and the absence of specific analytical techniques due to equipment constraints. Given the paucity of studies on the role of myofibroblast and fibronectin in cholesteatoma, there exists a scope for future research.

Conclusion

In conclusion, our research contributes to understanding cholesteatoma's aetiopathogenesis, emphasizing the roles of myofibroblasts and fibronectin. While existing theories present several limitations, our findings reveal a distinct pattern of cell–matrix interaction, aligning with recent studies. This insight could revolutionize cholesteatoma's diagnostics and treatment, especially considering the potential of targeting specific biomarkers for therapeutic intervention. However, our study's scope is limited by its sample size and analytical capabilities, highlighting the need for further research in this area.

Acknowledgements

Hereby, we acknowledge the Head of ENT, Dr. Manjunath Rao S.V, Dean, Dr. Prashanth G, and the Medical Superintendent, Dr. Palakshaiah L, at Basaveshwara Medical College & Hospital, Chitradurga, Karnataka, for supporting the study and waiving the cost of evaluation and treatment of all study subjects.

Funding

“We, the authors also declare that this study did not receive any external funding. All aspects of this research, including its conception, execution, data analysis, and manuscript preparation, were conducted independently and without financial support from any external sources.”

Declarations

Conflict of interest

“We, the authors hereby declare that there are no conflicts of interest regarding the publication of this paper. There have been no financial or personal relationships with other people or organizations that could inappropriately influence or bias our work. This includes, but is not limited to, patent or stock ownership, membership of a company board of directors, advisory roles, consultancy, speaker's fees, or any form of external funding. As the corresponding author, I have discussed and reviewed this policy with all co-authors, and we collectively affirm that no relevant commercial or other relationships exist.”

Ethical Approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Informed Consent

Informed consent was obtained from all individual participants included in the study.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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