Calcium deposition and expression of bone modelling markers in the tympanic membrane following acute otitis media

BACKGROUND AND OBJECTIVES: In accordance with clinical findings, myringosclerosis develops after otitis media (OM) and paracentesis in an experimental setting. The pathogenesis of this phenomenon of calcification is poorly understood. As the calcification process and the sclerotic plaques of the drum mimics features of bone tissue, this study explores tympanic membrane calcium deposition in association with the expression of three bone modelling markers: osteopontin (OPN), osteoprotegerin (OPG) and osteonectin (ON). OPN is secreted by osteoblasts and is found at calcification sites, e.g. during pathological calcification in chronic OM. The cytokine OPG is an inhibitor of bone resorption and consequently bone remodelling. ON is a calcium binding glycoprotein necessary for the maintenance of bone mass and remodelling. It is found in bone matrix and synthesized by osteoblasts. METHOD: A rat model of acute otitis media (AOM) caused by non-typeable Haemophilus influenzae was used. Four days following middle ear inoculation, a myringotomy was performed in six animals. Another group of ten animals was inoculated only. The drum was dissected in two animals from each group on day 4, 7, 14 and 28 post-inoculation, and the expression of OPN, OPG and ON was determined by immunohistochemistry. von Kossa staining determined the deposition of calcium and immune staining for CD68 identified macrophages. RESULTS: Calcium depositions were initially accumulated in the cytoplasm of macrophages and dispersed in the connective tissue layers of the pars flaccida and tensa. Late accumulation occurred in the lamina propria of pars tensa, more extensively in myringotomized ears. OPN expression was found early in inflammatory cells including especially macrophages and late in pars tensa fibrocytes. OPG expression was initially located to inflammatory cells and late to pars tensa fibrocytes and the inner basal membrane of pars flaccida. Some ears displayed a marked pars flaccida expression of ON in the connective tissue matrix on early days and at the inner basal membrane on later days. The latter cases were from myringotomized ears. Otherwise, no apparent differences of marker expression occurred between myringotomized and non-myringotomized animals. CONCLUSION: We conclude that osteopontin, osteoprotegerin and osteonectin are expressed by different cell types in the tympanic membrane during calcification in association with AOM, with or without myringotomy. These molecules may accordingly play a role in the pathogenesis of myringosclerosis, in which macrophages and fibrocytes appear as potential major players.