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00005768-200207000-0000100005768_2002_34_1057_maffulli_stainability_7article< 114_0_30_7 >Medicine & Science in Sports & Exercise©2002The American College of Sports MedicineVolume 34(7)July 2002pp 1057-1064Ruptured Achilles tendons show increased lectin stainability[CLINICAL SCIENCES: Clinical Investigations]MAFFULLI, NICOLA; WATERSTON, STUART W.; EWEN, STANLEY W. B.Department of Trauma and Orthopaedic Surgery, Keele University School of Medicine, Stoke on Trent, Staffordshire, UNITED KINGDOM; and Department of Orthopaedic Surgery and Department of Pathology, University of Aberdeen Medical School, Aberdeen, UNITED KINGDOMSubmitted for publication October 2001.Accepted for publication February 2002.AbstractMAFFULLI, N., S. W. WATERSTON, and S. W. B. EWEN. Ruptured Achilles tendons show increased lectin stainability. Med. Sci. Sports Exerc., Vol. 34, No. 7, pp. 1057–1064, 2002.Purpose: To ascertain whether lectins could be a useful tool for investigation of the extracellular matrix of degenerated and normal tendons.Methods: Hematoxylin-eosin–stained slides were assessed blindly using a semiquantitative grading scale for fiber structure, fiber arrangement, rounding of the nuclei, regional variations in cellularity, increased vascularity, decreased collagen stainability, hyalinization, and glycosaminoglycan, with a pathology score giving up to three marks per each of the above variables, with 0 being normal and 3 being maximally abnormal. For lectin staining with Aleuria aurantia, Canavalia ensiformis, Galanthus nivalis, Phaseolus vulgaris, Arachis hypogea, Sambucus nigra, and Triticum vulgaris, assessment of staining on a scale from 0 (no staining) to 5 (strong staining) was performed blindly.Results: The mean pathology sum score of ruptured tendons (N = 14; average age 46.5 yr, range 29–61) was significantly greater than the mean pathology score of the control tendons of Achilles tendons from individuals with no known tendon pathology (N = 16; average age 62.5 yr, range 49–73) (pathology score: 18.5 ± 3.2 vs 6.1 ± 2.3). Four of the seven lectins used exhibited significantly positive results.Conclusions: Ruptured tendons were histologically significantly more degenerated than control tendons. Ruptured tendons showed different lectin staining properties than nonruptured ones. This difference may have resulted from posttranslational changes in the extracellular matrix producing alterations in the biochemistry of the tendon, which might interfere with the interaction with the lateral sugar residues of the collagen molecules or cause steric blockade.Lectins are naturally occurring proteins, found especially in plants (2). They bind selectively and noncovalently to carbohydrate residues, and are not antibodies. Lectins recognize specific monosaccharide residues within sugar chains. The monosaccharides generally found in mammalian sugar chains are mannose, glucose, galactose, fucose, N-acetyl galactosamine, N-acetyl glucosamine, and sialic or neuraminic acids. Lectins are not considered to be specific for single-monosaccharide residues, and lectin binding most likely involves at least three monosaccharides. Lectin binding indicates the main sites of glycoproteins, glycolipids, and glycosaminoglycans (GAGs) in tissue (2). Increases in the quantity of extracellular matrix components have been observed in pathological conditions in tendons (8,20) and under conditions of increased loading (24). Lectin binding is often increased in diseased tissue (2). Lectins have proven useful for the study of the extracellular matrix in a number of tissues (6,18), as lectin binding indicates the main sites of glycoproteins, glycolipids, and GAGs in tissue (2). Recently, we have performed histochemical and immunohistochemical studies on normal and ruptured Achilles tendons, and we wished to extend the scope of our research by using widely available, simple to use, and relatively inexpensive staining materials. An extensive literature search yielded no information on the use of lectins to study the extracellular matrix of tendon. We therefore hypothesized that lectins could be a useful tool for investigation of the extracellular matrix of normal and pathological tendon.MATERIALS AND METHODSAll 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.Tendon SamplesRuptured Achilles tendons (N = 14 tendons).Samples of ruptured Achilles tendons were obtained from patients (N = 14, all men; average age 46.5 yr, SD 13.2, range 29–61) whose unilateral subcutaneous tear of the Achilles tendon was repaired in the Trauma Theater at Aberdeen Royal Infirmary in the period January 1999 to December 1999. The tendon samples consisted of approximately 3 × 3 × 3 mm samples excised from the proximal and distal stumps of the ruptured tendon. Each sample was placed into 6% neutral buffered formalin (NBF, pH 7.4) and left for 24 h to fix.Nonruptured Achilles tendons from deceased individuals (N = 16 tendons).One Achilles tendon was obtained from 16 male patients (mean 62.5 yr, SD 11.2, range 49–73) who had died of cardiovascular accidents while inpatients at Aberdeen Royal Infirmary in the period January 1997 to December 1999. From consulting the hospital notes and direct questioning of the families, no patient had ever sustained an acute or overuse injury to their Achilles tendon, had taken corticosteroids for the past 5 yr, or had taken fluoroquinolones over the course of the 24 months preceding their death. The Achilles tendon was harvested at post mortem under sterile conditions through a medial approach. The post mortem was performed in all cases within 24 h of death. 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. Both Achilles tendons were removed, and the specimen was pinned to a piece of cork to prevent contraction and distortion of the tissue during the fixation process (10% NBF for 24 h). For this study, a 3 × 3 × 3 mm sample was excised from the main body of the tendon 4 cm proximal to the distal end of the tendon (i.e., in the region where most tendon ruptures occur).Staining Procedures and Light MicroscopyHalf of each sample was used for hematoxylin and eosin staining, and the other half for lectin staining. After fixation, the sections were placed in tissue cassettes that had each been given a unique identification number. The sections were then placed in 10% NBF for a further 2 h to ensure that the fixation process was complete. The tissue then underwent six dehydration steps in separate containers, two steps in 95% alcohol for 1 h each, then a further four steps in pure alcohol for 1 h each. Sections were then cleared of any residual alcohol by three immersions in xylene, each step lasting 1 h each. After clearing, the tissue was embedded in paraffin wax (melting point 62°C). Longitudinal sections 4 μm thick were then cut using a LEICA 2065 microtome, and stained with hematoxylin and eosin (21).Digoxigenin Conjugated Lectin: Antidigoxigenin/Horseradish PeroxidaseAfter fixation, tissue blocks were subsequently processed to paraffin wax and sectioned at a thickness of 4 λm onto 3-aminopropyltriethoxysilane (APES) -coated glass slides and dried at 37°C overnight. Sections were then de-waxed in xylene for 20 min and rinsed in clean absolute ethanol for 1 min. Any endogenous peroxidase activity in the tissue was blocked by immersion in a freshly prepared solution consisting of 180 mL of methanol and 6 mL of hydrogen peroxide (BDH Chemicals, Dorset, U.K.) for 15 min. The sections were rinsed for 5 min in running water, then placed into a preprepared trypsin solution at 37°C for 16 min. The activity of the trypsin solution used varies according to the length of time since its preparation. Pilot studies showed that this potential variable was eliminated by preparing the trypsin solution while the sections were being de-waxed in xylene for 20 min. The trypsin solution was prepared by adding 0.8 g of trypsin (DIFCO trypsin 1:250, DIFCO Laboratories, Detroit, MI) and 0.4 g of calcium chloride (BDH Chemicals, Dorset, U.K.) to 400 mL of deionized water preheated to 37°C. The pH of the solution was adjusted to 7.8 by addition of 0.1 M sodium hydroxide. The solution was then placed in a waterbath at 37°C until it was required, the pH being readjusted to 7.8 just before use. After immersion in the trypsin solution, the sections were washed in 0.005% Tris-buffered saline (TBS) for 10 min, then ringed carefully using a Dako immunohistochemistry pen (Dako Corporation, Carpiteria, CA). Sections were then incubated for 15 min with 100 λL of normal sheep serum (NShS; SAPU, Carluke, U.K.) prepared to a dilution of 1 in 5 with TBS. After incubation, the NShS was tapped off onto a paper towel, and 100 λL of the appropriate digoxigenin conjugated lectin (Boehringer Mannheim, GmbH, Mannheim, Germany) was applied. The lectins used, their source, and the dilutions at which they were used are outlined in Table 1. Sections were left to incubate with the lectin for 30 min.TABLE 1. Properties of the digoxigenin labelled lectins used in this study (Man-Mannose, GLcNAc-β-D-N-acetylglucosamine).After incubation with the appropriate digoxigenin-conjugated lectin, sections were washed in TBS for three 10-min periods; 100 λL of sheep antidigoxigenin horseradish peroxidase (Boehringer Mannheim) diluted to 1 in 200 with TBS was added and left for 30 min. Sections were then washed again in TBS for three 2-min periods, then developed in a solution consisting of 200 mL of 0.05 M Tris/hydrochloric acid buffer and 2 mL of preprepared diaminobenzidine solution (DAB) (Sigma, St. Louis, MO) for 10 min. Sections were removed from the DAB solution and rinsed with a 0.5% solution of cupric sulfate in saline. The rinse was repeated, and the sections left in the cupric sulfate solution for 5 min. After washing well in running water, sections were counterstained with hematoxylin and Scott’s tap water substitute, dehydrated in alcohols, cleared in xylene, then mounted using DPX synthetic mountant (CellPath, Herts, U.K.). Sites of peroxidase activity were seen as areas of brown or black staining. All experiments were performed with TBS in place of the digoxigenin-conjugated lectin to provide a negative control using a section from the ruptured or control tendons on a random basis.Classification of StainingPer each tendon sample, three slides were randomly selected and examined using with 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 (SWW) 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) existed between the two results, the slides were reassessed with the help of a consultant pathologist (SWBE) 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.The criteria used to score the hematoxylin and eosin–stained slides were adapted from a semiquantitative grading scale (16) validated in our setting (12). 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, and 7) hyalinization. A four-point scoring system was used, where 0 indicates a normal appearance, 1 indicates a slightly abnormal appearance, 2 a moderately abnormal appearance, and 3 a markedly abnormal appearance. Overall, the total score for a given slide could vary between 0 (normal tendon) and 21 (most severely degeneration detectable). As we have already described the histopathological appearance of Achilles tendon rupture (13), we are not reiterating our results in this particular aspect, and we used these finding to make sure that we were actually examining areas of the Achilles tendons with maximum degenerative appearance.For lectin staining, assessment of staining was performed in a blind fashion for comparison with a negative control and a positive control where available. Staining was assessed using the criteria described in Table 2.TABLE 2. Criteria for the classification of lectin staining.StatisticsDescriptive statistics are provided. Data were entered in a commercially available database and analyzed using the SPSS statistical package. Kappa statistics was used to analyze the intraobserver reproducibility of the classification of the tendon appearance. The statistical significance of the differences in staining scores was analyzed using the Mann-Whitney U–Wilcoxon rank sum W test for two independent samples. Statistical significance was set at the 5% level (P < 0.05).RESULTSHistopathological Appearance (Hematoxylin and Eosin)The mean pathology sum score of ruptured tendons was significantly greater than the mean pathology score of the control tendons (18.5 ± 3.2 vs 6.1 ± 2.3). Kappa statistics showed good reproducibility of the mean pathology sum score (0.68, P = 0.04).Lectin StainingAleuria aurantia (AAA).Specimens of ruptured Achilles tendon exhibited significantly stronger staining with Aleuria aurantia than specimens of normal tendon (P < 0.01) (Fig. 1, Table 3). Staining of the normal specimens was mostly located in the connective tissue that surrounded the tendon fascicles. The terminal sugar in the binding sequence of Aleuria aurantia is α-L-fucose.FIGURE 1— Results of staining with Aleuria aurantia.TABLE 3. Results of staining with the various lectins: the median and range are given.AAA: Aleuria aurantia; Con A: Canavalia ensiformis; GNA: Galanthus nivalis; PHA: Phaseolus vulgaris; PNA: Arachis hypogaea; SNA: Sambucus nigra; WGA: Triticum vulgaris.Canavalia ensiformis (Con A).Specimens of ruptured tendon showed significantly stronger staining than specimens of normal tendon (P < 0.01) (Table 3). However, some staining of the normal specimens was apparent (Fig. 2). The binding sequence sugar for which Canavalia ensiformis shows the highest specificity is α-D-mannose.FIGURE 2— Results of staining with Canavalia ensiformis.Galanthus nivalis (GNA).Staining was not observed in any of the specimens that were incubated with Galanthus nivalis (Table 3). The terminal sugar in the binding sequence of Galanthus nivalis is α-D-mannose.Phaseolus vulgaris (PHA).Faint staining was observed in two of ruptured tendon specimens. No staining was observed in any of the other specimens (Table 3). Phaseolus vulgaris is specific for tetra-antennary N-acetyl-lactosamine type glycoproteins.Arachis hypogaea (PNA).There was a significant difference in staining between the specimens of ruptured and normal tendon (P < 0.01), with the ruptured specimens showing consistently stronger staining (Fig. 3, Table 3). Some staining of blood vessel walls was noted in three of the specimens of normal tendon but not in any of the ruptured specimens. The terminal sugar in the binding sequence of Arachis hypogaea is β-D-galactose.FIGURE 3— Results of staining with Arachis hypogaea.Sambucus nigra (SNA).Staining with this lectin showed no real pattern, with the strongest staining classified as patchy in one of the specimens of ruptured tendon (Table 3). There was some faint staining in two of the normal specimens, but no significant difference was observed. Sambucus nigra shows greatest specificity for N-acetylneuraminic acid.Triticum vulgaris (WGA).A significant difference in staining was observed between ruptured and normal tendon (P < 0.01). Strong, or medium to strong, staining was observed in all specimens of ruptured tendon. Only patchy or faint staining was observed in the specimens of normal tendon (Fig. 4, Table 3). Strong staining of blood vessel walls was again noted in the specimens of normal tendon. The terminal sugar of the recognized carbohydrate binding sequence of Triticum vulgaris is β-D-N-acetyl-glucosamine.FIGURE 4— Results of staining with Triticum vulgaris.DISCUSSIONWe have previously shown that ruptured tendons show profound histopathological changes, whereas nonruptured tendon samples from aged individuals have little evidence of histopathology at hematoxylin and eosin staining (8,12). The samples from ruptured Achilles tendons showed marked collagen degeneration and disorganization, increased cellularity and rounding of nuclei, and, in some specimens, hypervascularity. The increase in extracellular matrix, coupled with the decrease in collagen fibers, shows an imbalance between the two structural components of the tendon tissue, and it is not known which process precedes the other. The increase in GAG content may be a result of mechanical overloading, and this, in 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 (15).Lectin StainingFour of the seven lectins used in this study exhibited significantly positive results. Lectins are generally not specific for a single sugar residue but for a carbohydrate sequence. Glycoproteins are proteins with covalently attached oligosaccharides. Most proteins secreted from eukaryotic cells are glycoproteins (11). A key feature of many glycoproteins is their ability to interact with cells and with other macromolecules in the extracellular matrix. The location of glycoprotein can play a role in pathological process in the tendon, and it is therefore of interest to be able to locate them in specific areas of the musculoskeletal tissues (10).β-D-galactose is a major component of the GAG keratan sulfate (11). GAGs form a large proportion of the extracellular matrix. They are composed of repeating disaccharide units, and one of the monosaccharides is always either N-acetylglucosamine or N-acetylgalactosamine. They form solutions with high viscosity and elasticity. Keratan sulfate is the most heterogeneous of the major GAGs, and it contains small amounts of fucose, mannose, and N-acetylglucosamine (23). GAGs are attached to extracellular proteins to form proteoglycans, and proteoglycans and collagen form a mesh-like structure in the extracellular matrix of connective tissue. In normal tendons, two forms of proteoglycans have been demonstrated, small and large. The small ones have a short core protein to which one or two dermatan sulfate chains attach, whereas the large proteoglycans are rich in chondroitin sulfate (7). The proteoglycan component of connective tissue confers rigidity to the extracellular matrix and allows the diffusion of molecules. Proteoglycans can also influence the formation of collagen fibrils in vitro (22). Tendons contain only a small amount of proteoglycans, which can be visualized at electron microscopy as thin filaments, arranged orthogonally to the collagen fibers (22).Aleuria aurantiaThis lectin is derived from orange peel fungus, and its binding specificity is greatest for α-L-fucose, a deoxy sugar found in the complex oligosaccharide components of glycoproteins and glycolipids. Fibronectin, a high molecular weight “adhesive” glycoprotein (1) detected on the torn surface of ruptured Achilles tendons (9), has a greater affinity for denatured than for normal collagen (5). Therefore, its presence in ruptured Achilles tendons may indicate prior tissue degeneration (9). The fact that Aleuria aurantia has detected an increase in the concentration of terminal α-L-fucose residues does not necessarily represent an increased concentration of fibronectin. However, as increased concentrations of fibronectin have been previously described in ruptured tendons, its presence has to be considered a strong possibility. The presence of fibronectin in ruptured Achilles tendons confirms a degenerative etiology behind the tendon rupture.Canavalia ensiformisCanavalia ensiformis derives from the Jack bean. It shows specificity for terminal α-D-mannose, α-D-glucose, and N-acetylglucosamine residues. All three of these residues are involved in the oligosaccharide component of glycoproteins. Together, they form a 14 residue, core oligosaccharide molecule involved in targeting of glycoproteins secreted by cells (11).Arachis hypogaeaThis lectin is derived from the peanut. It shows greatest specificity for terminal residues of β-D-galactose. The significance of the positive staining result with Arachis hypogaea may be that it indicates an increased concentration of proteoglycans. Because proteoglycans contribute to the rigidity of the tissue, a pathological increase in their concentration may make the tissue excessively rigid, thus predisposing it to damage.Triticum vulgarisTriticum vulgaris derives from wheat germ. It shows greatest binding specificity for β-D-N-acetylglucosamine. N-acetylglucosamine is one of the integral components of GAGs and is present in hyaluronate, dermatan sulfate, keratan sulfate, and heparin sulfate. Dermatan sulfate is the glycoprotein found in the greatest concentrations in adult tendons (22). It consists of repeating disaccharide units of L-iduronate and sulfated N-acetylglucosamine.Hyaluronate is a central component of the extracellular matrix of tendon, contributing to the strength and elasticity of the extracellular matrix (11), forming the backbone of the proteoglycan molecules. Therefore, an increase in the amount of hyaluronate within tendon may also contribute to an increase in the rigidity of the tissue, thus predisposing it to damage.Summary of Information Obtained from Lectin HistochemistryThe information obtained from lectin histochemistry appears to highlight two important areas that may be important in the etiology of Achilles tendon rupture. Glycoproteins have previously been detected in specimens of ruptured Achilles tendon and may indicate collagen degradation. The results of staining with Aleuria aurantia and Canavalia ensiformis may indicate that an increase in the amount of glycoproteins has been detected in the specimens of ruptured Achilles tendon in this study.Staining with Arachis hypogaea and Triticum vulgaris indicated an increase in some of the component sugar residues of important GAGs and proteoglycans. These substances are important components of the extracellular matrix of tendon, and contribute to the rigidity and elasticity of the tissue. Therefore, an increase in their concentration may result in the tissue being excessively stiff and thus more prone to damage under tensile loads.Limitations of the StudyThere are several limitations to this study. For example, our study population of ruptured 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 (3). 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 (17) 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 were strong enough to justify our conclusions. Also, as aging causes at least some morphological changes in the tendons, and, as our control tendons were harvested from donors some 15 yr older than patients with a ruptured Achilles tendon, the use of an age-matched control population would have been likely to have further highlighted the histopathological differences that we have reported.CONCLUSIONSWe cannot offer an explanation of what causes the alteration in glycosylation recognized by the lectins used in the present study. At present, we can only hypothesize that posttranslational changes in the extracellular matrix produce alterations in the biochemistry of the tendon that might interfere with the interaction with the lateral sugar residues of the collagen molecules or cause steric blockade (19). These, coupled with the collagen abnormalities that are a feature of tendon degeneration (13), may result in foci of lesser resistance and of inferior biomechanical properties that may eventually result in a tear.Recent work on rotator cuff tears has identified the presence of amyloid in the torn supraspinatus tendon (4). Deposits of amyloid have three major components: amyloid itself, amyloid P protein, and GAGs. GAGs are thought to exert an important role in the amyloidogenic pathways, accelerating the formation of fibrils and protecting against proteolysis (14). Also, there is evidence that tendon amyloidosis can occur in other tendons, including the Achilles tendon (7, pp. 406–407). The results of the present study offer indirect evidence that amyloid deposition could be involved in the etiopathogenesis of Achilles tendon rupture and support the contention that Achilles tendon tears are due to intrinsic degenerative changes within the tendon.Dr. Waterston was partially financed through a Summer Studentship of the Wellcome Trust.No benefit in any form has been received or will be received from a commercial party related directly or indirectly to the subject of this article.Address for correspondence: N. Maffulli, Department of Trauma and Orthopaedic Surgery, Keele University School of Medicine, North Staffordshire Hospital, Thornburrow Drive, Hartshill, Stoke on Trent, Staffordshire, ST4 7QB, UNITED KINGDOM; E-mail: n.maffulli@keele.ac.uk.REFERENCES1. Ayad, S., R. P. Boot-Handford, M. J. Humphries, K. E. Kadler, and C. A. Shutlleworth. The Extracellular Matrix Facts Book. London: Academic Press, 1984, pp. 1–356. [Context Link]2. Brooks, S. A., A. J. C. 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[CrossRef] [Medline Link] [Context Link] ACHILLES TENDON RUPTURE; HISTOPATHOLOGY; GLYCOSAMINOGLYCANSovid.com:/bib/ovftdb/00005768-200207000-0000100004624_1989_71_100_carr_calcaneal_|00005768-200207000-00001#xpointer(id(R3-1))|11065405||ovftdb|SL0000462419897110011065405P83[Medline Link]2914976ovid.com:/bib/ovftdb/00005768-200207000-0000100004780_1978_147_1584_engvall_fibronectin_|00005768-200207000-00001#xpointer(id(R5-1))|11065213||ovftdb|SL000047801978147158411065213P85[CrossRef]10.1084%2Fjem.147.6.1584ovid.com:/bib/ovftdb/00005768-200207000-0000100004780_1978_147_1584_engvall_fibronectin_|00005768-200207000-00001#xpointer(id(R5-1))|11065405||ovftdb|SL000047801978147158411065405P85[Medline 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B.CLINICAL SCIENCES: Clinical Investigations734