Using a freeze substitution fixation technique and histological crimp analysis for characterizing regions of strain in ligaments loaded in situ.

Type I collagen fibrils in tendons and ligaments assume a sinusoidal wave shape, or crimp, which straightens only with tensile load. The load response of crimp has been studied primarily in isolated subunits and not in complex, intact structures. The purpose of our study was to determine if freeze substitution fixation of an entire ligament could preserve changes in crimp morphology induced by functionally relevant loading conditions. We hypothesized that, in ligaments prepared by freeze-substitution fixation under load, crimp would progressively extinguish with increasing loads, and nonuniform strain following partial section could be detected from crimp morphology. Tensile loads ranging from 0 to 220 N were applied to patellar ligaments of 16 fresh rabbit stifle joints using simulated isometric quadriceps pull through the patella. The loaded joints were flash frozen with isopentane cooled in liquid nitrogen, then fixed using freeze substitution. Another six ligaments were loaded to 150 N following incision of the anterior third and evaluated under polarized light microscopy for crimp distribution. Ligaments with no or low loads could be identified by the presence of crimp on mid-sagittal sections. Strain distribution was inhomogeneous, in that the ligament displayed a consistent pattern of collagen fiber recruitment among three morphologically distinct bands seen on coronal sections. At very low loads (about 18 N), the fibers in a central band were uncrimped; anterior and deep bands uncrimped at higher loads. The crimp in the entire specimen was extinguished at about 67 N, which correlates closely with the previously reported toe-region of the stress-strain curve of the rabbit patellar ligament. When the anterior third was transected, fibers within that segment retained a crimp in ligaments prepared under loads that ordinarily would ablate all crimp. These findings suggest that freeze fixation could be used to map the functional microstructure of ligaments or tendons. Copyright 2006 Orthopaedic Research Society