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Collagenous components were isolated chemically from fetal bovine or guinea pig cochlear bone and human stapes after stapedectomy, and the purified protein was characterized by immunoblot assay and amino acid analysis. The results of this study suggest that these are mixtures of type I and type II collagens. The presence of type II collagen in the human stapes also was demonstrated by immunohistologic methods using monoclonal antibody. The presence of type II collagen in these tissues is significant, since it has been postulated as an autoantigen in autoimmune inner ear disease.

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Immunohistochemical studies of the chick columella have shown that the extracellular matrix of this ossicular cartilage template is composed largely of type II collagen. As development proceeds, synthesis of type X collagen, a hypertrophic cartilage-specific molecule, is initiated by endochondral chondrocytes within the zone of cartilage cell hypertrophy. Subsequently, these cells and their surrounding extracellular matrix are removed, resulting in marrow cavity formation. We have examined which of these processes are programmed within the columella chondrocytes themselves, and which require involvement of exogenous factors. Prehypertrophic columella from 12-day chick embryos were grown either in organ culture on Nuclepore filters or as explants on the chorioallantoic membrane of host embryos. Chondrocytes from the same source were grown in monolayer cell cultures. In both organ culture and cell culture, chondrocytes developed to the stage at which some of them entered the hypertrophic program and initiated the production of type X collagen as determined by immunofluorescence histochemistry with a monoclonal antibody specific for that collagen type. The organ cultures, however, did not progress to the next stage, in which detectable removal of the type X collagen-containing matrix occurs. When identical columella were grown on the chorioallantoic membrane of host chicks, the type X collagen-containing matrix which formed was rapidly removed, resulting in the formation of a marrow cavity. Thus, progression of endochondral chondrocytes to the deposition of type X collagen-containing matrix seems to be programmed within the cells themselves. Subsequent removal of this matrix requires the involvement of exogenous factors.

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The underlying mechanism responsible for bone resorption in chronic otitis media with or without cholesteatoma is not sufficiently well documented. Certain areas in the middle ear are affected more often than others. The most frequent site of bony erosion in the ossicular chain is the long process of the incus. The mineral content of the ossicular chain was investigated with a clinical radiologic technique (polytomography). The findings were correlated with chemical measurements of the mineral content in normal and diseased ossicles. A poor correlation between the radiologic findings and the chemical assessment was found. The mineral content of ossicles obtained from ears with chronic otitis was similar to that of specimens obtained from healthy middle ears. The findings indicate that the demineralization process seems to be localized to a narrow zone of the ossicle and that this zone cannot be adequately visualized by standard radiologic techniques.

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The purpose of the present experiment was to look at the dissolution time in physiological sodium chloride (0.9% NaCl) and sodium sulfide (Na2S) of any mercury (Hg) contained in human auditory ossicles and cartilage after preservation in Cialit and Merthiolate. Both homograft tissues produced almost identical graph complexes. Under static conditions 10 micrograms Hg could be found in NaCl after 15 min, whereas there was complete demercurization after 3 min in Na2S. The dissolution time in NaCl subjected to constant stirring was about 10 min and was less than 1 min in Na2S. For the rapid elimination of Hg from auditory ossicles stored in preservatives containing Hg, our studies show that the use of a flowing solvent is advisable. Further, the extraction of Hg is quicker in sodium sulfide than in an isotonic solution of sodium chloride.

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Otosclerotic stapes footplates, superstructures and temporal cortical bones were extracted with 0.25 M guanidine X HCl 0.5 M EDTA (pH 7.4) solution. The extracted non-collagenous peptides/proteins were separated chromatographically on a Sephadex G-25 microcolumn. The peptide composition of the bone samples were compared by capillary analytical isotachophoresis (ITP) in the molecular mass range 0.3-5 kD. The otosclerotic stapes footplate contained 13 ITP subfractions, while the stapes superstructures and cortical bone contained only 9 and 10, respectively. An otosclerosis-specific ITP subfraction was also detected in the stapes footplate, but not in the stapes superstructure or cortical bone. This subfraction was previously demonstrated in the otosclerotic perilymph as well. Four ITP subfractions occurred common in the otosclerotic stapes footplate, the superstructure and the cortical bone. Two of these common subfractions were not found in the cortical bone peptide extract, but all of them revealed higher than normal levels in the otosclerotic perilymph.

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The presence of the amino acid gamma-carboxyglutamate (GLA) was established in human middle ear bones. Proteins containing GLA have been described as being associated with normal as well as pathological calcifications. The GLA content of human incus, malleus, and stapes with 1-2 nmol/mg bone is in the range previously reported for a variety of bone. A limited number of samples with middle ear pathology, including otosclerosis, did not show altered GLA levels.

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We have differentially precipitated 2 fractions of noncollagen proteins extracted from separately pooled superstructure and footplate of surgically removed stapes of patients from both sexes, who have suffered from otosclerosis. The two fractions were: EDTA-soluble, CaCl2 precipitable (Fraction-1) and EDTA-soluble, CaCl2 non-precipitable proteins. The protein pattern of these two fractions was compared by isoelectric focusing and two-dimensional gel electrophoresis. The phosphoprotein fraction (Fraction-1) of the otosclerotic stapes footplate contained acidic (pI 3-6), low molecular weight (20-40 kD) proteins, which were not detected in the superstructure and temporal cortical bone. Two-dimensional mapping of the Fraction-2 showed predominantly acidic proteins in the footplate and some basic minor components in the superstructure. The lack of the low molecular weight proteins in the superstructure proves the localisation of the otosclerotic process only in the footplate.

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With increased use of human otologic homograft tissue for implantation, different preservation techniques and products are used for storing and preserving the grafts before surgery. Preserved ossicles may, according to some authors, already exhibit signs of decalcification after 2 weeks. Therefore, the mineral (calcium, phosphorus) and collagen content of human ossicles (incus) and mastoid cortex and the influence of different storage techniques was biochemically investigated. No significant mineral or collagen-concentration changes were observed after a 6-week storage in pH-7 formaldehyde or in cialit, in either the ossicles or in the cortical bone. These biochemical findings are in agreement with histologic observations and with similar experiments using the photon absorptiometry technique.

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