Morphology and biochemistry of the tympanic membrane in relation to retraction pathology

Abstract

Otitis media is very common during childhood and may predispose for tympanic membrane (TM) pathology later in life. Therefore, it is important to investigate the possible changes in TMs subjected to otitis media.

There is evidence of a relationship between previous otitis media and TM retraction disease. A weakening of the collagen fibers, that form the backbone of the TM, is a prerequisite for the genesis of a retraction. Secretory otitis media results in a TM stiffness loss in animals but no loss of collagen fibers has been found. Different collagen types have different tensile strength. An altered collagen type distribution due to otitis media could possibly explain the stiffness loss.The relative distribution of the different collagens in the TM in normal and pathologic states needs further clarification. Secretory otitis media also results in a thickening of the outer keratinizing epithelium. In cholesteatomas, as well, the outer keratinizing epithelium is thickened but to a much greater extent.

The overall aim of the study was to further elucidate the possible relationship between otitis media and retraction pathology. The specific aims were to identify the different collagen types in the normal TM in rats and humans. Thereafter, the collagens and the morphology were investigated in cholesteatomas and human TMs subjected to secretory otitis media. Furthermore, the human keratinizing epithelium was investigated regarding the presence of possible stem cells.

This was done with the use of healthy TMs from rats and humans, biopsies from human TMs subjected to long-standing secretory otitis media and cholesteatomas.

The methods used were immunohistochemistry with DAB and immunofluorescent techniques using antibodies against the four most common collagen types and different stem cell markers. Visualization was achieved using light microscopy, laser confocal microscopy and transmission electron microscopy.

Collagen type II was found to be the predominant collagen type in the lamina propria of TMs from both rats and humans. In the human TM, a distinction could be made between the outer and inner layers of the lamina propria. The inner layer proved to contain collagen type III to a large extent. Collagen type IV was found in the basal laminas. In the TM biopsies, all four types of collagens could be identified but no quantification could be performed. The cholesteatomas proved to contain remnants of collagens and were positive for all collagen types except for collagen type III.

The outer keratinizing epithelium displayed thickness variations in the normal human TMs, especially regarding the basal layer, which was markedly enlarged in the umbo, along the handle of the malleus and in the annular region. In these areas the basal layer cells were elongated perpendicularly to the basal lamina, a finding contrasting to the findings in the adjacent regions. The basal layer cells in the thicker parts of the TM biopsies and the cholesteatomas were perpendicularly elongated in a similar fashion. It is hypothesized that this is a sign of an increased cellular proliferation. The healthy human TMs were investigated for stem cell markers that proved to be positive in the areas with a thickening of the basal layer of the keratinizing epithelium.