Diameter Distribution, Number Density, and Area Fraction of Fibrils

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Abstract

The purpose of this investigation is to show microstructural information at various regions within the rabbit patellar tendon. The properties of the rabbit patellar tendon are well documented mechanically, but detailed information at them icroscopic level is not available. Increasing attention has been directed to soft tissue microscopy as the demand for development of biologically inspired materials increases. Microstructural examination of the tendon fibrils is performed to provide further insight into understanding of the structure to function relations within the rabbit patellar tendon. Limited studies on rabbit patellar tendon collagen fibrils at the microscopic level have been computed. Furthermore, evaluation of structure–function relations in multiple regions of any given specimen of a particular tissue type has not been conducted. In this study the number density, area fraction, and diameter distribution of collagen fibrils have been determined. Overall, this examination showed considerable variation within each section of the tendon.Correlating these structural results with mechanical tests of the tendon portions in the various regions could provide additional information on the mechanics of the rabbit tendon as well as insight into development of artificial tissue constructs.[1]

Methods

Four skeletally mature white New Zealand male rabbits were euthanized as part of a separate and unrelated IACUC approved protocol. The rabbits weighed between 3 and 4 kg. Patellar tendons wereimmediately resected from each animal with asharp scalpel blade. The desired section wa sremoved from the whole tendon and placed in afixative of 1/2 strength Karnovsky’s solution (0.1Mcacodylate buffer pH 7.2) in preparation for TEM.Each section was approximately 3mm by 3mm in size. Care was taken to avoid the thin edges of thepatellar tendon and as a method of standardization each of the sections was cut from all four tendons,at the same time from its respective location.However, due to the small size of the sections someof the sections were distorted and did not undergo the TEM process.

Immediately after the tissue was removed via sharp dissection with a scalpel, it was placed in a fixative solution in preparation for TEM. Tendons were sectioned in the central center, central proximal, central distal, medial center, and lateral distal sections. Orientation of specimens was maintained by tying a suture to one end. The patellar tendon tissue pieces were fixed in strength Karnovsky's solution (in 0.1 M cacodylate buffer, pH 7.2) for 7 days in a 4 °C environment. The tendon specimens were rinsed in a 0.1 M sodium cacoldylate buffer (pH 7.2). After rinsing, they were fixed in 1% osmium tetroxide (in 0.1 M sodium cacoldylate buffer, pH 7.2) for 2 h, and then placed in 1% (aqueous) tannic acid for 1 h. Thereafter, they were placed in 1% osmium tetra oxide (in 0.1 M sodium cacodylate buffer, pH 7.2) for 2 h. The tissue was rinsed twice in buffer followed by further twice rinsing in water. Then it was dehydrated in a graded ethanol series. The tissue was infiltrated and embedded in Spur's resin. The sections were cut perpendicular to the longitudinal axis at 75 nm thickness on a Reichert Jung Ultra cut E ultra microtome and viewed using a JOEL JEM 100CXII (JEOL USA, Peabody, MA.) transmission electron microscope at 60 kV. Electron micrographs of 25 equally sized fibril fields within the fascicles of each tendon segment were obtained (20,000×magnification).

The fibrils in each image were approximately circular in shape. The image areas were 6.4×4.3 μ2. The digitized TEM images were analyzed using Image J Software (National Institutes of Health, Baltimore, MD). Image J is a public domain software (inspired by NIH Image) used for image processing and analysis. Image J can display, animate, enhance, analyze, and edit images.

Using Image J, four images (n=5) were analyzed from the central center portion, three (n=5) from the medial center, two images (n=5) from the central proximal, two images (n=5) from the central distal, and two images (n=5) from the lateral distal portion. A total of 25 images were processed, and the fibril numberdensity, area fraction, and diameter distributions were determined from each image. The collagen fibril number density (ND) was calculated using Eq. (1); the area fraction of the fibrils was calculated using Eq. (2), and the mean fibril diameter was noted as the average size of the fibril diameters within each section. The number density units are fibrils per micrometer squared. The area fraction denotes the percent of fibers within the area of the image analyzed, and the diameter distribution is demonstrated by histograms of frequency versus diameter size (nanometers).

1.gif (1)

2.gif (2)

Results

TEM micrographs revealed slight structural differences between the various areas of the tendon. Table 1 lists the fibril number density, area fractions, and mean fibrildiameterdistributions for each examined section. The results show the overall fibril diameter distribution to range in fibrils sizes from as small as 45 nm to fibril sizes greater than 500 nm. Although the mean fibril diameters are close in value, the results do not show any of the locations to hold exactly the same trend for diameter distribution.

Calculations of the standard error values show a significant difference in number density between the lateral distal section and the four other sections. Very little difference in number density exists between the number density of the medial center and central proximal. Otherwise, no other significant differences were measured in number density. Significant fibril area fraction differences were noticed between the central center section and medial center and central proximal sections. Also, differences were found between the lateral distal section and medial center and central proximal sections. The area fraction between the central distal section and the medial center and central proximal sections demonstrated significant differences. A morphometric analysis shows a statistically significant difference between the mean fibril diameters of each section, with the central center section having the largest mean fibril diameter. The medial center, central proximal and lateral distal sections fibril frequency were mostly in the small-diameter class (diameter <200 nm), while most of the central center and central distal values were in the large-diameter class.

Table 1. Mean and standard deviations of number density, area fraction and fibril diameter within the analyzed sections of the tendon
Tendon section (n=5) Number density (fibrils/μ2) Area fraction (%) Mean fibril diameter (nm)
Central center 14.8±1.9 79±4 244±3
Medial center 13.5±1.4 60±9 215±3
Central proximal 15.5±1.7 65±6 210±2
Central distal 16.9±4.2 76±3 225±2
Lateral distal 25.8±7.0 79±4 176±2

References

  1. Williams, L.N., Elder, S.H., Harbarger, D., Horstemeyer, M.F., “Variation of diameter distribution, number density, and area fraction of fibrils within five areas of the Rabbit Patellar Tendon,” Annals of Anatomy. 190, 2008: 442-451.
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