Ruptured Achilles tendons are significantly more... : Medicine & Science in Sports & Exercise (original) (raw)
Previous studies on the histology of Achilles tendon ruptures have shown that a degenerative process had taken place before the rupture occurred (2,11,12). The histological picture was studied by Kannus and Jozsa (12), when they evaluated specimens obtained from the biopsy of 891 spontaneously ruptured tendons, including 397 Achilles tendons. Using 445 age- and sex-matched controls, they described a variety of degenerative changes within the tendon. Degenerative tendinopathy was found to be the most common disorder in tendons that have ruptured spontaneously, but calcification and mucoid changes have also been described (3,4,10,12). All the ruptured tendons have preexisting histopathological alterations, but such changes were much less frequent in the control tendons (12).
Other studies have shown morphological and metabolism changes with increasing age (9,23). One of these metabolic changes seen is an increase in the production of type III collagen from damaged tenocytes (17) and chondroid metaplasia (22), which results in the tendon becoming cartilage-like, and therefore less able to withstand tensile forces.
Cumulative microtrauma weakens collagen cross-linking, noncollagenous matrix, and vascular elements of the tendon (11). When a tendon has been strained repeatedly to more than 4% of its original length, it is unable to endure any further tension, and injury will occur (7) with a break in collagen structure. Tissue hypoxia and consequent free radical–induced tendon changes resulting from ischemia-reperfusion injury could also be major factors in the pathogenesis of a tendinopathic tendon (1,5,6). Another factor in the development of tendon degeneration could be exercise-induced hyperthermia. Histopathologically, these tendons reveal disordered arrangement of collagen fibrils and an increase in vascularity (15) with an increase in the amount of mucoid ground substance, where tenocytes, if present, are chondroid in appearance (22).
Although preexisting tendinosis has been observed in ruptured tendons (3,8,12), it is still unknown why some patients are symptomatic, and develop tendinopathy, and others are asymptomatic despite the presence of advanced histological changes (13,14). It could be possible that two separate pathological processes occur in patients who rupture their Achilles tendon without any previous pain, and those who suffer from chronic tendinopathy pain.
We therefore sought to ascertain whether there is an association between tendinopathic and ruptured tendons, hypothesizing that the histopathological aspects of tendinosis in tendinopathic tendons are less advanced than those found in a rupture (15). Therefore, we compared nonruptured tendon samples to ruptured and tendinopathic tendon samples.
METHODS
Tendon Samples
All procedures were approved by the Ethical Committee of the Grampian University Hospitals Trust. All patients and, when applicable, their families gave written informed consent that the procedures described in this article could be carried out, as required by British law.
Control (nonruptured) tendons (N = 16; average age 65 ± 19.1; range, 46–82).
Samples were obtained from two sources: 1) patients undergoing amputation for peripheral vascular disease admitted to the department of vascular surgery of Aberdeen Royal Infirmary between April 1996 and December 1998, and 2) patients who died of cardiovascular accidents while inpatients at Aberdeen Royal Infirmary between April 1996 and December 1998. The Achilles tendon was harvested in the post mortem room under sterile conditions through a medial approach. The tendon was freed from surrounding tissue, and as much muscle and fat as possible were removed. The tendon was cut horizontally at the superior and inferior ends. Previous studies (15) showed that there were no statistically significant differences in the histopathological appearance of Achilles tendons from amputated legs and from patients deceased for cardiovascular causes. Therefore, the results from these two groups of patients were grouped together, and such tendons will be referred to as “control tendons.”
Tendinopathic Achilles tendons (N = 13; average age, 35.7 ± 12.9 yr; range, 18–67).
Samples of tendinopathic tendons were obtained from patients undergoing exploration of their Achilles tendons at Woodend Hospital, Aberdeen, during the period June 1999 until November 1999. During the operation, a sample 3 × 3 × 3 mm was removed within the area of degeneration.
Ruptured Achilles tendons (N = 35; average age, 48.4 ± 16.9 yr; range, 26–80).
Samples of ruptured tendons were obtained from patients who sustained a unilateral subcutaneous tear of the Achilles tendon repaired in the trauma theater at Aberdeen Royal Infirmary in the period January 1997 to December 1998. During surgical repair of the ruptured tendon, performed within 48 h of the injury and without using a tourniquet, two samples measuring approximately 3 × 3 × 3 mm were removed from both the proximal and distal stumps of the tendon.
Preparation of Slides
The specimens obtained were placed in 20 mL of sterile 10% formalin and fixed in 10% neutral buffered formalin (10% NBF) for 24–48 h and processed to paraffin wax; 5-μm sections were then mounted onto 3-aminopropyltriethoxysilane (APES) coated slides and dried at 37°C overnight.
Sections were dewaxed in two 10-min changes of xylene, followed by one change in absolute alcohol, 95% alcohol, for 10 min each to rehydrate the sections. The sections were then rinsed under running tap water. Sections were stained using hematoxylin and eosin, and using the alcian blue (pH 2.5)/periodic acid–Schiff (AB/PAS) method for the detection of glycosaminoglycan (GAG) rich areas.
Assessment of Tendon Degeneration
Per each tendon sample and per each staining technique, three slides were randomly selected and examined using a light microscope (×600, SM-LUX, Leitz, Wetzlar, Germany). The identification number on each slide was covered with a removable sticker, and each slide was numbered using randomly generated numbers. After one of the authors (C.T.) interpreted all the slides once, the stickers were removed, a new sticker was applied, and the slides were renumbered using a new series of randomly generated numbers. The degree of staining was reassessed by the same author, and the two results were compared. If an inconsistency (more than one grade on the scoring system described in Table 1) existed between the two results, the slides were reassessed with the help of a consultant pathologist (S.W.B.E.) with a special interest in musculoskeletal pathology. The area of each specimen showing the most advanced pathological changes was selected, and the worst possible results for each slide were used in this study.
Median values for each semiquantitative histopathological criterion in each group.
The criteria used to score the slides were adapted from a semiquantitative grading scale described by Movin (20). Using this method, we assessed 1) fiber structure, 2) fiber arrangement, 3) rounding of the nuclei, 4) regional variations in cellularity, 5) increased vascularity, 6) decreased collagen stainability, 7) hyalinization, and 8) GAG content. The hematoxylin and eosin stained slides were used to assess the first seven variables, using a four-point scoring system, where 0 indicates a normal appearance, 1 is slightly abnormal, 2 is moderately abnormal, and 3 is markedly abnormal.
The sections stained with AB/PAS were examined stereologically to assess the GAG-rich areas. The areas were examined using a square lattice of 36 points placed on the projection screen. The intersection points on the GAG-rich areas (stained blue) were recorded 10 times for each slide, making a total maximum score of 360 points for each specimen. By taking quartiles after all the slides had been scored, these scores were then converted to the previous four-point scoring system using the following groups: 0 = 0 to 10, 1 = 11 to 51, 2 = 51 to 206, and 3 = 207 to 360. Overall, the total score for a given slide could vary between 0 (normal tendon) and 24 (most severe degeneration detectable).
Statistical Analysis
Nonparametric statistical methods were used for ordinal data and for continuous variables. Kappa statistics was used to analyze the intraobserver reproducibility of the classification of the tendon appearance. Differences in the pathological variables, comparing all groups, were analyzed using the chi-square test. As the total scores were not normally distributed, the Mann-Whitney U test was used to determine whether the difference between the two independent tendon groups was statistically significant, and the Kruskal-Wallis test was used to test the three sample groups together. The SPSS (release 9.0.1. standard version, SPSS, Inc., Chicago, IL) statistical package was used to analyze the results. A probability level of P < 0.05 was considered significant.
RESULTS
The distribution of the duplicate scores is shown in Table 2, and the median values for each criterion in each group are shown in Table 1. The mean values for the control tendons was 5.9 ± 7.4 and 5.1 ± 6.8 for the first and second reading, respectively. The mean values for the tendinopathic tendons was 10.5 ± 6.1 and 11.2 ± 5.8 for the first and second reading, respectively. The mean values for the ruptured tendons was 17.4 ± 4.9 and 15.9 ± 5.9 for the first and second reading, respectively.
Results of semiquantitative histopathological assessment.
Within each specific category of tendon pathology, the chi-square test showed no association between control, tendinopathic, and ruptured tendons. All variables were significantly different (Mann-Whitney U test, 0.05 <P < 0.001). The distribution of the scores for each category marked is shown in Table 1.
Kappa statistics.
The intraobserver reproducibility of the scores was evaluated using kappa statistics. The results are shown in Table 3, ranging from 0.170 (poor agreement when assessing hyalinization) to 0.750 (good to excellent agreement when assessing GAG content).
Results of kappa statistics assessing intraobserver reproducibility of the semiquantitative histopathological scores.
Chi-square test.
The chi-square test was performed to ascertain whether there was any difference within the criteria scored, comparing sample groups. The results of each set of blinded scores are shown in Table 4.
Results of chi-square test to ascertain whether there was any difference within the criteria scored, comparing sample groups (P values are shown).
There was a significant difference in all criteria except hyalinization, which was rarely seen in either control, tendinopathic, or ruptured specimens. When comparing the control group with the tendinopathic group, five of the criteria were seen to be significantly different. Fiber structure, fiber arrangement, and vascularity were significantly different when comparing the ruptured and tendinopathic groups.
Mann-Whitney U test.
This test compared the differences in the total scores in two independent, nonnormally distributed sample groups.
All of these groups were significantly different, showing that all of the samples are from separate populations. The higher rank means, in both scores, showed that the ruptured tendons were significantly more degenerate than the tendinopathic tendon specimens, and these were, in turn, more degenerate than the controls (Table 5).
Results of Mann-Whitney U test to compare the differences in the total scores in two independent, nonnormally distributed sample groups.
Kruskal-Wallis test.
This tested the total scores, comparing all groups at once. Comparing all of the groups in the same analysis, it was again seen that the mean rank of the ruptured group was higher than that of the tendinopathic group, which was greater than that of the control group (Table 6).
Kruskal-Wallis test, testing the total histopathological scores, comparing all groups at the same time.
Histopathological Appearance
The mean pathology sum score of ruptured tendons was significantly greater than the mean pathology score of the tendinopathic tendons, and this, in turn, was significantly greater than the mean pathology score of the control tendons (20.5 ± 3.6 vs 6.5 ± 2.1).
Fiber structure.
In the control specimens, the fibers were arranged close and parallel to each other (Fig. 1). In the ruptured specimens, the fibers showed increased waviness, separation and, in some cases, a complete loss of structure and hyalinization. The tendinopathic specimens tended to show generally less waviness than the ruptured specimens.
Hematoxylin and eosin stain of a control Achilles tendon in a 59-yr-old man who underwent amputation for peripheral vascular disease. There are closely packed, lightly stained parallel bundles of collagen fibers that contain the flattened nuclei of tenocytes. Original magnification, ×150.
Fiber arrangement.
In the control tendons, the fibers were arranged parallel to each other. In ruptured and tendinopathic samples, this parallel arrangement was lost and haphazard (Fig. 2).
Hematoxylin and eosin stain of Achilles tendon adjacent to the rupture in a 51-yr-old male squash player. Note the disorganization within the tissue, with loss of the normal collagen fiber distribution, hypercellularity, increased waviness, separation, with nearly complete loss of the tendon structure. The whole area is hypercellular. Original magnification, ×150.
Cell nuclei.
Normally, the tenocyte nuclei were flattened and spindle shaped, sometimes arranged in rows. In the ruptured and tendinopathic samples, the tenocytes first decreased in number; then, as the pathologic changes progressed, the nuclei became progressively rounded. In some instances, these tenocytes resembled chondrocytes (Fig. 3).
Hematoxylin and eosin stain of the tendinopathic area of Achilles tendon in a 32-yr-old female triathlete. Slight hypercellularity, with rounding of the tenocyte nuclei. Original magnification, ×150.
Regional variations in cellularity.
The whole area of the slide was assessed for these variations in cellularity. The control specimens showed little variation in cellularity, unlike the ruptured and tendinopathic specimens. In those specimens with the highest evidence of degeneration, there were areas of densely packed cells compared with the surrounding area. In both the tendinopathic and ruptured specimens, there was mostly a generalized increase in cellularity as a whole, but also focal areas of cellular proliferation were seen.
Vascularity.
In some cases, the rupture samples showed random blood vessel formation throughout the section, increasing with the degeneration of the tendon (Fig. 4). These blood vessels were less common in the tendinopathic samples. Normally, these vascular bundles run parallel to the collagen fibers.
Hematoxylin and eosin stain of the tendinopathic area of Achilles tendon in a 29-yr-old male middle-distance runner. Marked random blood vessel formation. The collagen fibers have a disorganized appearance, and the whole section is hypercellular. Original magnification, ×150.
Collagen stainability.
Collagen fibers stain a deep color, as seen mostly in the control specimens. The ruptures and tendinopathic samples appeared paler pink in color, showing decreased collagen stainability.
Hyalinization.
Very few specimens showed any evidence of hyalinization, and analytical statistics showed that this histopathological criterion was poorly reproducible (Table 3).
Glycosaminoglycans.
The GAG-rich areas stain blue with AB/PAS. Areas of blue staining occurred in the pathologic tendons (Fig. 5), whereas a pink staining was observed in the control specimens (Fig. 6).
AB/PAS stain of a control Achilles tendon in a 76-yr-old farmer, who ruptured his Achilles tendon while plowing a field. There is increased GAG-containing matrix, and collagen fiber disruption. Original magnification, ×150.
AB/PAS stain of a control Achilles tendon in a 62-yr-old man, who died of a cardiovascular accident. GAG-containing matrix is minimal and hardly appreciable as foci between the collagen fibers. Original magnification, ×150.
DISCUSSION
This study has shown that samples of ruptured and tendinopathic tendons both show profound histopathological changes, that these changes are more pronounced in ruptured than tendinopathic tendons, and that nonruptured, aged tendon samples have little evidence of histopathology.
Histopathological findings.
The histopathological findings described in the present study are consistent with those described by other authors in Achilles tendon ruptures (12) and chronic Achilles tendinopathy (18,19).
The most prominent features seen in the samples from ruptured Achilles tendons were marked collagen degeneration and disorganization, increased cellularity and rounding of nuclei and, in some specimens, hypervascularity. There were similar areas of degeneration within the tendinopathic tendons. However, these changes were not as pronounced, as confirmed by the lower median scores obtained (11 compared with 17). There was an increased content of GAG seen in both the ruptured and the tendinopathic tendons (median scores of 2). The increase in extracellular matrix, coupled with the decrease in collagen fibers, shows an imbalance between the two structural components of the tendon tissue. It is not certain which process precedes the other. It has been suggested that the increase in GAG content may be a result of mechanical overloading, and this, in its turn, may affect the fiber structure and arrangement, leading to a reparative response with neovascularization. This imbalance between injury and repair leads to tissue damage (18). Although a similar histopathological picture is seen in the ruptured and tendinopathic tendons, it remains unclear why these processes present so differently in a clinical situation (13,14). The chi-square test confirmed that the fiber structure, fiber arrangement, and vascularity were all significantly different (P < 0.05), and the Mann-Whitney U test showed that the total scores for the criteria assessed were significantly different when comparing the ruptured and tendinopathic groups (P < 0.01). The present study therefore shows that the tendinopathic tendons are significantly less degenerated than the ruptured tendons.
The control specimens had a parallel and organized arrangement of collagen fibers and elongated tenocytes, despite coming from patients up to 30 yr older than those in the other two groups. The control specimens exhibited significantly less degeneration in all criteria except hyalinization (which we were able to identify in only a very few specimens) when compared with the ruptured group, and significantly less degeneration in all criteria when compared with the tendinopathic group. Therefore, these aged tendons exhibited little evidence of degeneration. Merkel et al. (18) proposed that normal aging of connective tissue is morphologically different from degeneration, and our data would confirm their statement. Aging tissue has a low rate of metabolism and exhibits a progressive decrease in elasticity and tensile strength.
Reliability of histopathological investigations.
In this study, each slide was scored twice by the same investigator with the help of a consultant pathologist with a special interest in the musculoskeletal system. The kappa statistics assessed the measure of agreement between the two scores. When comparing all groups, the scores varied from fair (0.289) to good (0.668), showing how difficult it is to recognize specific patterns in histology and the importance of having well-trained individuals to interpret the slides. To improve on these kappa statistics, the assessment could be repeated several more times. Also, using another observer would decrease observer bias.
It is possible that these changes seen in the ruptured tendons could be secondary abnormalities occurring after the rupture. However, all patients were operated on within 48 h from the time of the rupture, and it is unlikely that such profound histopathological change could occur in such a time frame.
Limitations of the present study.
There are several limitations to this study. For example, our study population of ruptured, tendinopathic, and control tendons is relatively small, and our control tendons came from patients with various degrees of vascular disease. However, the Achilles tendon is normally a relatively avascular structure (17). It is therefore likely that our tendon samples were representative of normality, given the age of the patients. A possible solution could have been to use ultrasonography-guided percutaneous biopsy to obtain samples of tendons in live healthy individuals (18), or to use tendons from younger patients undergoing traumatic amputations. However, for ethical and practical reasons, neither of these alternatives was possible, and the differences between the control and ruptured tendons are strong enough to justify our conclusions. Also, as aging causes at least some morphological changes in the tendons, and given that our control tendons were harvested from donors at least 20 yr older than patients with a ruptured Achilles tendon, the use of an age-matched control population would have further highlighted the histopathological differences that we have described.
When interpreting the results of the present study, it should be considered that we only used two staining methods. Obviously, extra lipids, calcium deposits, collagen denaturation, pathological tenocyte metabolism, collagen types, and foreign materials in the control group could have been detected using more advanced histochemical and immunohistochemical techniques. However, the staining techniques used in the present study have many advantages: they are widely available, cost-effective, and require little technical abilities, and most pathologists are familiar with them and are accustomed to interpreting a variety of specimens stained in this fashion.
Finally, we have no reference data on the level of tendon degeneration in the general Scottish adult population. Although we have reported the epidemiological characteristics of a cohort of 4201 patients with Achilles tendon rupture in the last 15 years (17), we are not aware of any study detailing the histopathological appearance of Achilles tendon degeneration in this populace.
CONCLUSION
Ruptured and tendinopathic tendons are histologically significantly more degenerated than control tendons. The general pattern of degeneration was common to the ruptured and tendinopathic tendons, but there was a statistically more advanced degree of degeneration in the ruptured tendons. It is therefore possible that there is a common, as yet unidentified, pathological mechanism that has acted on both of these tendon populations. We have recently shown that tenocytes from ruptured Achilles tendons produce greater quantities of type III collagen than tenocytes from normal Achilles tendons (16,21). This altered production of collagen may be one reason for the histopathological alterations described in this study, and may result in the tendon being less resistant to tensile forces, and thus at increased risk of micro- and macroscopic changes. It remains unclear why tendons that are histologically less degenerated cause marked pain, whereas tendons that rupture show a greater histopathological degree of degeneration despite not producing symptoms before the rupture (13,14).
Many thanks are given to Miss Linda Lothian for her help with the manuscript.
Address for correspondence: Nicola Maffulli, Department of Trauma and Orthopaedic Surgery, Keele University School of Medicine, North Staffordshire Hospital, Thornburrow Drive, Hartshill, Stoke on Trent, Staffordshire, ST4 7QB, ENGLAND; E-mail: [email protected].
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Keywords:
TENDINOSIS; ETIOLOGY; SURGERY; HISTOPATHOLOGY
© 2001 Lippincott Williams & Wilkins, Inc.