Depth profile study of molecular collagen structure in normal human cornea

Purpose To investigate the structure and organisation of corneal collagen as a function of stromal depth. Methods A strip extending from limbus to limbus along the superior to posterior meridian was cut from a normal human cornea and 100µm serial sections, parallel to the surface plane of the tissue, were obtained. Wide angle x-ray diffraction experiments were performed in ID-13 at the European Synchrotron Radiation Facility (ESRF) using a 25µm square beam. Each section was scanned at 450µm or 500µm steps. Additionally, another thin vertical strip (1mm thickness) was mounted in such a way that the beam was scanning edge-on. Two scans were performed (1mm apart, each covering the depth of the cornea) from the anterior to the posterior surface at 25µm intervals. Data analysis provided information on the direction and degree of alignment of preferentially oriented collagen fibrils, as well as collagen intermolecular spacing (IMS). Results IMS is relatively stable throughout the depth of the central cornea, but is decreased in the posterior peripheral cornea. The degree of preferential alignment of fibrillar collagen is lower in the peripheral anterior part. In addition, the previously well-documented tangential or annular collagen structure at the limbus is mainly restricted to the most posterior 100µm of stroma. At more superficial layers collagen preferred orientation tends to obtain an “x” shaped pattern, possibly forming part of the "anchoring fibril" structure alluded to previously in full-thickness averaged x-ray studies. Conclusion The preferred directions of the lamellae and IMS are depth-dependent. The annulus at the limbus, which is thought to be implicated in the maintenance of corneal curvature, resides in the posterior limbus.