Scanning electron microscopic characterization of healing and normal rat ligament microstructure under slack and loaded conditions

The objective of this study was to observe and compare behavior of the collagen fiber microstructure in normal and healing ligaments, both in situ and ex vivo, in order to add insight into the structure-function relationship in normal and healing ligaments. Fifty-two ligaments from 26 male rats were investigated. Eleven animals underwent surgical transection of both medial collateral ligaments (MCLs) (22 ligaments), which were allowed to heal for a period of 2 weeks. An additional 15 animals (30 ligaments) were used as normals. Ligaments were placed into six groups: Slack (n = 6 control, n = 6 healing), Reference (n = 4 control, n = 4 healing), Loaded (n = 4 control, n = 4 healing), 15° Flexion (n = 4 control, n = 4 healing), 120° Flexion (n = 4 control, n = 4 healing), and Tissue Strain vs. Flexion Angle (n = 8 normals). All ligaments, except those in the Tissue Strain vs. Flexion Angle group, were prepared for scanning electron microscopy. Tissues were harvested, mounted in a load frame, and chemically fixed in one of five states: (1) slack, (2) reference (onset of loading), (3) loaded, (4) 15° knee flexion, or (5) 120° knee flexion. After fixation the tissues were prepared for electron microscopy (SEM). The micrographs from the slack, reference, and loaded groups show fiber straightening with loading in normal ligaments as well as in both scar and "retracted" regions of healing ligaments. Collagen fibers' diameter and crimp patterns were dramatically changed in the scar region of healing ligaments: Width decreased from 19.4±1.7 μm to 6.5±2.1 μm (p<.000001), period from 51.4± 15.1 μm to 11.0±2.4 μm (p<.000001), and amplitude from 9.8± 0.8 μm to 3.9±0.8 μm (p<.000001). Normal ligaments fixed in situ show wavy regions at 120° but less so at 15° flexion. Healing ligaments fixed in situ show regions of fiber waviness in the scar region at 120° and also at 15° flexion, indicating ligament laxity persists toward both extremes of the range of motion. The data suggest that straightening of crimped fibers is a functionally relevant phenomenon, not only in normal but also in healing ligaments.