Electron microscopic quantification of collagen fibril diameters in the rabbit medial collateral ligament: A baseline for comparison

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Abstract
To establish a normal baseline for comparison, thirty-one thousand collagen fibril diameters were measured in calibrated transmission electron (TEM) photomicrographs of normal rabbit medial collateral ligaments (MCL's). A new automated method of quantitation was used to compare statistically fibril minimum diameter distributions in one midsubstance location in both MCL's from six animals at 3 months of age (immature) and three animals at 10 months of age (mature). Pooled results demonstrate that rabbit MCL's have statistically different (p < 0.001) mean minimum diameters at these two ages. Interanimal differences in mean fibril minimum diameters were also significant (p < 0.001) and varied by 20% to 25% in both mature and immature animals. Finally, there were significant differences (p < 0.001) in mean diameters and distributions from side-to-side in all animals. These mean left-to-right differences were less than 10% in all mature animals but as much as 62% in some immature animals. Statistical analysis of these data demonstrate that animal-to-animal comparisons using these protocols require a large number of animals with appropriate numbers of fibrils being measured to detect small intergroup differences. With experiments which compare left to right ligaments, far fewer animals are required to detect similarly small differences. These results demonstrate the necessity for rigorous control of sampling, an extensive normal baseline and statistically confirmed experimental designs in any TEM comparisons of collagen fibril diameters.