Localization of osteopontin at calcification sites of cholesteatoma: possible role as a regulator of deposition of calcium phosphate in the middle ear

Objective: Recently, we showed that osteopontin (OPN), a major acidic phosphorylated glycoprotein of bone, participates in the pathological calcification that occurs as a result of chronic otitis media. To investigate the possibility of OPN as a common regulator of the pathological calcification in the middle ear, we here examined whether or not OPN is localized at the calcification sites of cholesteatoma, which is clearly different from chronic otitis media in the pathogenesis. Methods: Middle ear tissues including cholesteatoma were obtained from 32 cases who underwent tympanoplasty. The tissues of 29 cases were used for the immunohistochemistry of OPN and CD68, the marker of macrophages, and in situ hybridization of OPN mRNA. And those of the other three for reverse transcriptase polymerase chain reaction (RT-PCR). Results: Middle ear tissues including cholesteatoma were histologically classified as to the degree of calcification. In hyalinized tissues with macroscopic calcification, OPN was immunohistochemically found at the calcification sites. In inflammatory tissues with microscopic calcification, OPN was also found in the calcifying foci, and many OPN mRNA-expressing cells, as determined by in situ hybridization, were located near the foci. Moreover, immunohistochemical double staining for OPN and CD68 showed that the OPN-expressing cells were CD68-positive, indicating the cells were macrophages. In inflammatory tissues without calcification, immunohistochemistry of CD68 and in situ hybridization of OPN mRNA revealed that most OPN mRNA-expressing cells were CD68-positive. The expression of OPN mRNA in cholesteatoma tissues was also demonstrated by means of RT-PCR. Conclusion: OPN secreted by exudate macrophages was localized in the pathological calcification that occurs in association with cholesteatoma. These results are consistent with the observations in chronic otitis media. Therefore, it is suggested the possibility that OPN might contribute as a common regulator of the deposition of calcium phosphate in the middle ear.