Spatial distribution and orientation of dermatan sulfate in human medial collateral ligament.

The proteoglycan decorin and its associated glycosaminoglycan (GAG), dermatan sulfate (DS), regulate collagen fibril formation, control fibril diameter, and have been suggested to contribute to the mechanical stability and material properties of connective tissues. The spatial distribution and orientation of DS within the tissue are relevant to these mechanical roles, but measurements of length and orientation from 2D transmission electron microscopy (TEM) are prone to errors from projection. The objectives of this study were to construct a 3D geometric model of DS GAGs and collagen fibrils, and to use the model to interpret TEM measurements of the spatial orientation and length of DS GAGs in the medial collateral ligament of the human knee. DS was distinguished from other sulfated GAGs by treating tissue with chondroitinase B, an enzyme that selectively degrades DS. An image processing pipeline was developed to analyze the TEM micrographs. The 3D model of collagen and GAGs quantified the projection error in the 2D TEM measurements. Model predictions of 3D GAG orientation were highly sensitive to the assumed GAG length distribution, with the baseline input distribution of 69+/-23 nm providing the best predictions of the angle measurements from TEM micrographs. The corresponding orientation distribution for DS GAGs was maximal at orientations orthogonal to the collagen fibrils, tapering to near zero with axial alignment. Sulfated GAGs that remained after chondroitinase B treatment were preferentially aligned along the collagen fibril. DS therefore appears more likely to bridge the interfibrillar gap than non-DS GAGs. In addition to providing quantitative data for DS GAG length and orientation in the human MCL, this study demonstrates how a 3D geometric model can be used to provide a priori information for interpretation of geometric measurements from 2D micrographs.