The distribution and relationship of the collagen and the proteinpolysaccharide complex in the extracellular matrix of the nucleus pulposus of the young rabbit were studied by electron microscopy. The polysaccharide was demonstrated by treatment of the tissue with either 0.5% bismuth nitrate at pH 2 or a mixture of equal volumes of 1% lead nitrate and 2% osmium tetroxide at pH 5. The protein part of the complex was observed in tissue fixed in osmium tetroxide at pH 7.2 and stained with 1% phosphotungstic acid. The collagen of the tissue was apparent in all preparations. The water-extracted proteinpolysaccharide was studied after coagulation of a concentrated solution in 0.5% bismuth nitrate, whereas the isolated fibrous fraction was examined by negative staining of sprayed material.
Collagen is present in the intact tissues as native collagen fibrils of about 300 Å diameter, and as sheets, 4OO-Å thick, of parallel, separate, filaments of 50-60 Å diameter. Usually several such sheets lie close, and approximately parallel, to one another. After extraction of the proteinpolysaccharide from the tissue with water, all the collagenous components tend to aggregate into large native collagen fibrils.
Some of the proteinpolysaccharide complex of the intact tissue is free in the matrix. The polysaccharide moiety is visualized as 270 Å particles, serially attached to a protein core in the form of beaded filaments about 40Å in diameter. The rest of the complex is attached to the sheets of collagen filaments in regular periodic zones which are 270 Å wide and centred 670 Å apart. None of the complex appears to be associated with the larger collagen fibrils.
The staining reactions of the polysaccharides in several connective tissues are discussed and the relationships of the proteinpolysaccharide complexes to collagen in cartilage, the vitreous body and the nucleus pulposus are compared.