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The distal tendon of the biceps brachii.
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The distal tendon of the biceps brachii. Structure and clinical correlations Stephan Koch and Bernhard Tillmann Anatomisches Institut der Christian-Albrechts-Universitat zu Kiel, OlshausenstraBe 40, D-24098 Kiel, Germany
Summary. The structure and blood supply of 42 distal biceps tendons were investigated by means of light and electron microscopy as well as by immunohistochemistry. Possible structural causes for the rupture of the tendon are discussed. The distal biceps tendon wraps around the radius during pronation of the forearm. In this area the tendon is exposed to pressure and shearing forces in addition to those caused by tension. Two fibrocartilaginous areas were regularly observed. Large chondrocyte-like cells were found inside the fibrocartilage. As an expression of strain, the extracellular matrix is rich in acidic glycosaminoglycans and stains intensely with toluidine blue at pH 1. Electron microscopy showed a granular pericellular matrix that increases in size towards the gliding surface. Type I collagen is the main component of the distal biceps tendon. Type II collagen is found in tendon fibrocartilage but not in traction tendons. The gliding surface of the tendon is made up of reticular fibres that are equivalent to type III collagen. Monoclonal antibodies revealed the presence of dermatansulfate, keratansulfate and chondroitin-4- as well as chondroitin-6-sulfate. Blood vessels are usually absent in fibrocartilage, as was shown with a polyclonal antibody against the basement membrane component laminine. There are significant differences between the extracellular matrix of traction and gliding tendons, which may be responsible for the location of tendon rupture.
even in younger individuals and athletes by reason of increased sporting and outdoor activities. The majority of patients describe a minor trauma that should not have been strong enough to cause the rupture of a healthy tendon (Bindl et al. 1988; Bindl and Koch 1995; Hegelmaier et al. 1992), so acute trauma does not seem to be the only cause. On reviewing the literature, the underlying causes for the rupture remain unclear. Earlier publications have already demonstrated the correlation between the varying structures of traction and gliding tendons and their functional differences in rabbits (Ploetz 1938). Altmann (1964a, 1964 b) analysed the biomechanical background according to Pauwels' theory of "kausale Histogenese" (1960). The significance of intermittent compression stress for tendon fibrocartilage has also been demonstrated in vitro (Koob et al. 1992). A detailed description of fibrocartilage and its changes with age has been given by Benjamin (1990, 1991). The position within osteofibrous canals (Tillmann and Thomas 1982), as well as the pattern of vascularization (Uthoff et al. 1976; Tillmann and Kolts 1993) could be responsible for the degeneration. The purpose of this study was to gain further knowledge about the structure and blood supply of the distal biceps tendon and the possible structural causes of its rupture.
Key words: Distal biceps tendon - Glycosaminoglycans Biomechanics - Tendon rupture
Material and methods Tissue specimens
Introduction Injuries of the distal biceps tendon used to occur in the fifth or sixth decade of life, but nowadays ruptures are reported Correspondence to: Bernhard Tillmann
Ann Anat (1995) 177: 467 -474 Gustav Fischer Verlag lena
Tendons were obtained from 42 subjects of different ages (22 weeks to 91 years; m: 23 f: 19) during autopsy within 48 hours of death. After macroscopical investigation, each tendon was split longitudinally and the parts prepared for light microscopy, transmission electron microscopy and immunohistochemistry. The intratendinous vasculature was observed in arterial injection samples prepared according to the method of Spalteholz (1914).
Light microscopy For light microscopy, tendons were fixed in Schaffer's solution and embedded in methyl metacrylate. Sections (10 !lm) were mounted on gelatine-coated slides and stained with toluidine blue (pH 1), and by the methods of Gomori, Goldner, von Kossa and with Movat's Pentachrome stain (Romeis 1989).
Electron microscopy For transmission electron microscopy the samples were fixed in 3.5% glutaraldehyde after initial treatment with 0.1 M Sorensen phosphate buffer solution at pH 7.4 (Romeis 1989), and embedded in araldite. Ultrathin sections were cut with a microtome and contrasted with uranyl acetate and lead citrate. Examination was carried out with a Zeiss EM 900 electron microscope.
Immunohistochemistry Samples were snap frozen in liquid nitrogen. Longitudinal frozen sections (10 !lm) were cut with a cryostat at _210 C and mounted on gelatine-coated slides. For immunohistochemistry, frozen sections were pretreated with either testicular hyaluronidase (Boehringer) or chondroitinase ABC (Sigma) in tris-buffered saline (TBS) in a moist chamber at 37 0 C for 30 minutes. The sections were washed three times with TBS and incubated with goat serum for 45 minutes at room temperature. Then incubation with the primary antibody was carried out for 60 minutes at room temperature. Antibodies
used in the investigations (tested and described in detail by Caterson et al. 1983 and Sobue et al. 1988) were: mouse anti collagen (MAC) type I polyclonal antibody (gift from Prof. Dr. P. K. MUller, Lubeck); MAC type II polyclonal antibody (Biodesign) and monoclonal antibody CIID3 (gift from Dr. R. Holmdahl, Uppsala). MAC type III polyclonal antibody (Bio-Science); MAC type IV polyclonal antibody (DAKO); MAC type IX polyclonal antibody (gift from Prof. Dr. P. K. MUller, Lubeck); anti chondroitin-4-sulfate monoclonal antibody (Chemicon International Inc.); Clone BE 123. The antibody is directed against unsaturated uronic-acid residues bound to N-acetyl-galactosamine-4-sulfate; anti chondroitin-6-sulfate monoclonal antibody (Chemicon International Inc.); Clone MK-302. The antibody is directed against uronic-acid residues bound to N-acetyl-galactosamine-6-sulfate. Anti keratansulfate monoclonal antibody (Bio-Science Products AG); Clone 5-D-4. The antibody is directed against hexasaccharides of keratansulfate. Anti dermatansulfate proteoglycan monoclonal antibody (Bio-Science Products AG); Clone 6 - B - 6. The antibody is directed against the core-protein. Mouse anti laminine polyclonal antibody (Medac). Sections were labeled with the respective secondary antibody, fluorescein isothiocyanate (FITC)- conjugated goat anti rabbit or goat anti mouse IgG for 45 minutes. Control sections were incubated only with the FITC-conjugated antibody. Positive controls including tissues with defined antigen sites (human cartilage, skin, kidney, liver, spleen) were used. The slides were examined with a Zeiss-Axiophot microscope equipped for epifluorescence.
Fig. 1. Chrondroid cells inside the fibrocartilage of the distal biceps tendon opposite the radius. Toluidine blue, x 90 Fig. 2. The course of collagen fibres follows a rhomboid pattern within the fibrocartilage. It follows the direction of strain. Gomori, x45 Fig. 3. The gliding surface of the distal biceps tendon consists of a dense layer of reticular fibres (arrows) with embedded chondroid cells. Gomori, x 140 Fig. 4. The largest chondroid cells are located beneath the gliding surface. Their size decreases with increasing distance from the surface. Pentachrome, x 140
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Results
type is found that still has features of a fibroblast-like cell, but already shows an oval form (Fig. 6). It is surrounded by a slim granular pericellular matrix. Large chondroid cells are found inside in the fibrocartilage opposite to the radius and show an even larger pericellular matrix (Figs. 7 and 8). These cells contain a marked endoplasmic reticulum and many mitochondria. Cells and pericellular matrix are surrounded by collagen fibres, proteoglycans and other matrix components.
Gross anatomy The distal tendon of the biceps brachii inserts at the radial tuberosity over an area of 3 cm2 • During pronation of the forearm it is wrapped around the radial neck. A thin layer of synovial membrane-like tissue forms the bicipito-radial bursa between the tendon and radius. This bursa is a closed compartement and was present in all cases investigated. Once the bursa is opened, the gliding surface of the distal biceps tendon is visible. It is shiny and appears to be avascular. Blood vessels are visible on the outside of the tendon, reaching the point of insertion via the periosteum of the radius or descending from the muscle belly.
Immunohistochemistry
Light microscopy In all the tendons examined, two different types of cells could be distinguished. Thin fibroblast-like tenocytes are embedded between parallel-orientated collagen fibres on the ulnar aspect of the tendon. Where the tendon faces the pulley, large cells having the appearance of chondrocytes could be observed (Fig. 1). The number and size of the chondroid cells decrease with increasing distance from the gliding surface. This area of the gliding tendon resembles fibrocartilage. The extracellular matrix stains intensely with toluidine blue (pHi). The adult tendon contains two areas that consist of fibrocartilage. One develops where the distal biceps tendon opposes the radius and is not visible until the age of one, the other is part of the tendon-bone interface of the chondro-apophyseal insertion of the tendon. Gomori staining clearly shows the parallel arrangement of the fibres on the ulnar aspect of the tendon. Inside the fibrocartilage the fibres take a swivelled course and are arranged in a rhomboid pattern (Fig. 2). Some fibres run at 90 to the main axis of the tendon and connect the main fibre bundles. Collagen fibres insert at the radial tuberosity at 60 0 • Their course can be followed through the mineralized zone of the attachment site. There seems to be a continuous transition to the bony trabeculae of the radial tuberosity. Small darkly stained reticular fibrils intermingle with the major fibre bundles. A separate layer of reticular fibres was observed at the gliding surface adjacent to the radius (Fig. 3). It is inside this reticular network that the largest chondroid cells are located (Fig. 4). Trichrome staining, according to the method of Masson-Goldner, stained the fibrocartilage inside the biceps tendon an intense red colour. Proximal areas and a small ulnar segment were stained green. Movat's pentachrome staining showed green spotty areas in the fibrocartilage, and von Kossa staining for calcification was negative in all cases. 0
Electron microscopy Transmission electron microscopy reveals three types of tenocytes. Typical fibroblast-like cells are found on the ulnar aspect (Fig. 5). In the middle of the tendon an intermediate
Type I collagen is the main component of the distal biceps tendon. Antibodies against type I collagen show a marked staining which follows the course of the fibres throughout the tendon (Fig. 9). The distal biceps tendon is positive for collagen type II where fibrocartilage is embedded in the tendon (Fig. 10). The area that stains with an antibody against type II collagen is located immediatly under the gliding surface and makes up two thirds of the diameter of the tendon. Between these fibres, a positive reaction with an antibody against collagen type IX is observed and follows the zonal distribution of type II collagen. Antibodies against type III collagen stain small fibrils located between major fibres and a dense layer on the gliding surface of the tendons (Fig. 11). Glycosaminoglycans are an important component of the extracellar matrix of tendons. Monoclonal antibodies against dermatansulfate revealed the presence of this glycosaminoglycan in all tendons and showed a distribution along the course of the collagen fibres (Fig. 12). Keratansulfate was present in all tendons and also distributed along the course of collagen fibres (Fig. 13). There was only faint staining on the ulnar aspect of the tendon and in proximal areas, whereas intense staining was observed in the fibrocartilage. Chondroitin-4-sulfate was present in all gliding tendons, the intensity of the spotty staining decreasing with increasing distance from the gliding surface (Fig. 14). Staining with anti chondroitin-6-sulfate showed only a faint reaction around the fibrils. Occasionly a marked reaction was observed around superficial defects of the tendon. Areas proximal and distal to the fibrocartilage showed a negative reaction for chondroitinsulfates.
Vascular supply Arterial injection samples revealed blood vessels that descend from the muscle belly of the biceps brachii. The vessels usually reach the tendon from the outside via a paratenon. From the ulnar side, facing away from the pulley, they run into the depths of the tendon. Intratendinous blood vessels run either within a loose internal peritendineum or directly between collagen fibres. The insertion itself is supplied by small branches of the cubital artery. Macroscopic investigations showed a hypovascular area opposing the radius that was identical with fibrocartilage (Fig. 15). To confirm this finding, immunohistochemical tests with an antibody
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.*
Fig. 5. Electron microscopic view of a fibroblast-like tenocyte from the ulnar aspect of the distal biceps tendon. The cell is embedded between collagen fibres (CO) that are orientated in parallel. Scale bar represents 2.33 Ilm. x 3 000 Fig. 6. Electron microscopic view of a tenocyte of the intermediate layer between fibrocartilage and the ulnar aspect of the distal biceps tendon. The cell is embedded between collagen fibres (CO). The course of the fibres varies and follows the direction of strain. A small territorial matrix is visible (arrow). Scale bar represents 2.33 Ilm. x 3 000 Fig. 7. Electron microscopic view of a chondroid cell inside the fibrocartilage of the distal biceps tendon. Note the marked territorial matrix (star). Scale bar represents 1.59 Ilm. x4400 Fig. 8. Magnification of Fig. 7. The granular territorial matrix that surrounds the cell (star) contains numerous vesicles. The cytoplasm of the chondroid cells is rich in rough endoplasmic reticulum and mitochondria, which is a sign of active synthesis of proteins such as collagen. Scale bar represents 1.00 Ilm. x 7 000
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Fig. 9. Immunohistochemical appearance of type I collagen fibres in the distal biceps tendon. The fibres are orientated in parallel. x180 Fig. to. Immunohistochemical appearance of type II collagen fibres that are present inside fibrocartilage and where the tendon is exposed to pressure. X 180 Fig. 11. Immunohistochemical appearance of type III collagen from the gliding surface of the distal biceps tendon. Chondroid cells (arrows) are embedded in the gliding surface (arrowheads). x 140
Fig. 12. Immunohistochemical appearance of dermatansulfate inside the distal biceps tendon. x 180 Fig. 13. Immunohistochemical appearance of keratansulfate inside the fibrocartilage of the distal biceps tendon. x 180 Fig. 14. Immunohistochemical appearance of chondroitin-4-sulfate in the fibrocartilage of the distal biceps tendon. x 180
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15
Fig. 15. Distal biceps tendon (DBT) of a 72 year-old man after injection of india ink gelatine and preparation according to the method of Spaiteholz. The tendon is inserted at the radial tuberosity (RT). A hypovascular area can be seen where the tendon lies in contact with the radius (arrow). Fig. 16. Immunohistochemical appearance of vessels inside a distal biceps tendon, shown with an antibody against the basement membrane component laminin. An avascular fibrocartilaginous zone (*) is visible in the right upper corner. x 360 against the basement membrane component laminine were used. Blood vessels are clearly visible in all samples. The vessels follow the main course of collagen fibres and are distributed all over the ulnar aspect of the tendons (Fig. 16). Fibrocartilage is avascular in most cases. Only in twelve tendons that showed macroscopical signs of degeneration were blood vessels detected inside fibrocartilage.
Discussion The distal tendon of the biceps brachii is made up of dense, regularly arranged collagen fibrils. It transmits forces from the biceps muscle to the radius and is therefore subject to
great tensile stress. Depending on the position of the forearm during flexion and pronation, the tendon is additionally exposed to pressure and shearing forces. Pressure occurs when the tendon wraps around the radius during pronation of the forearm. Additional shearing forces are brought about during combined movements in the elbow joint. Injuries to the distal biceps tendon nearly always occur close to its insertion, one to two centimetres proximal to the radial tuberosity. Rupture hardly ever occurs before the age of fourty (Agins et al. 1988; Bindl and Holz 1988; Konn and LObbecke 1977; Lang et al. 1988; Morrey et al. 1985). Recent observations suggest that changes inside the distal biceps tendon occur by reason of repeated pronation and supination at the radial tuberosity (Bindl and Koch 1995). Is the location of the rupture really due to the structure of the tendon? The distal biceps tendon is inserted at the radial tuberosity and has the typical structure of a chondro-apophyseal insertion (Knese 1958; Tillmann and Thomas 1982; Benjamin 1992). The structure of the human distal biceps tendon changes at the point where the tendon wraps around the radius. Compared with proximal regions of the tendon, the fibrocartilage opposite to the radius shows a rounded rather than an elongated cellular morphology, and has a marked metachromasia due to the acidic glycosaminoglycans when stained with toluidine blue at pH 1 (Tillmann and Schiinke 1991). These variations are thought to be governed by the prevailing biomechanical conditions: The part of the distal biceps tendon that faces away from the pulley resembles a traction tendon. Tensional stress decreases towards the pulley. The gliding surface and the tissue below it are exposed to compression and shearing forces that decrease with the distance from the pulley. The attachment zone and also an area opposite the radius consist of fibrocartilage and include chondroid cells located between the collagen fibres. They serve to reduce the horizontal shortening of the tendon at sites exposed to multidirectional stress. In accordance with Pauwels' theory of "kausale Histogenese" (Pauwels 1960), chondroid cells increase in size the closer they are to the gliding surface and reflect the load acting on the tendon. Electron microscopy shows that the chondroid cells inside the fibrocartilage are surrounded by a territorial matrix. The closer the cell is located to the pressure-exposed area the wider is the matrix. A territorial matrix around mammalian cells was first described by Clarris and Fraser (1968). A granular territorial matrix around human meniscal chondrocytes, visible in transmission electron microscopy, was described by Ghadially (1983). At present the exact structure of the human territorial matrix is unknown, although the presence of a variety of extracellular glycoproteins and proteoglycans has been demonstrated (Hedmann et al. 1982; Kjellan and Lindahl 1991). According to Lee et al. (1993) the territorial matrix around chondrocytes is composed of hyaluronan-aggrecan complexes. The increased amount of rough endoplasmic reticulum and mitochondria in the chondroid cells could stand for an increased synthesis of extracellular matrix components as are collagens and proteoglycans. Type I collagen fibres are orientated in parallel
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to the main axis of traction where the distal biceps tendon is exposed to pull. Tendon fibrocartilage also consists of typ I collagen, but the fibres follow a swivelled course due to the different directions of strain. Immunohistochemistry shows a spotty distribution of type II collagen ' between type I collagen fibres. The presence of type II collagen is an indicator for the exposure to pressure in this part of the tendon, as the extracellular matrix of normal traction tendons does not include type II collagen. Where collagen type II is present, collagen type IX can be shown as a small connecting collagen. Reticular fibrils consist of type III collagen. They are located between major collagen fibre bundles and form a neat reticular layer on the gliding surface itself. Glycosaminoglycans as part of extracellular matrix proteoglycans of tendons have been investigated over the past years in bovine, rat and rabbit tendons (Daniel and Mills 1988; Honda et al. 1987; Vogel 1986, 1987, 1988; Thonar 1988). Gel-electrophoretic investigations show, that there are significant differences between the composition of proteoglycans of traction and gliding rat and bovine tendons (Vogel 1986 and 1987). Investigations of the human posterior tibial tendon proved these results (Vogel 1993). The present study showed the presence of dermatansulfate, keratansulfate and chondroitinsulfates in the pressure-exposed fibrocartilage of all human biceps tendons, and also showed regional differences in the distribution of glycosaminoglycans throughout the tendon. Those parts that are mainly exposed to traction did not react with antibodies against chondroitinsulfates or keratansulfate, although they were positive for dermatansulfate. Dermatansulfate is supposed to influence tissue repair (Rosenberg 1992) and may play an important role in the development of degeneration within the distal biceps tendon. In some tendons only a weak staining for chondroitinsulfates was observed in the pressurized area. This result may be explained by possible immobilization of the extremity, shortly before death, since a loss of the chondroitinsulfate content of tendon has been described after the reduction of a compressive load (Koob 1992). Injection samples and immunohistochemical experiments showed a continuous vascular network extending from the biceps muscle to the bony insertion of the distal biceps tendon. Fibrocartilage inside the tendon is usually devoid of blood vessels. Occasionally vessels were found within fibrocartilage, but only in tendons that had shown macroscopic signs of degeneration. These results go along well with previous findings in other gliding tendons (Tillmann and Kolts 1993; Kolts et al. 1994). Vascular architecture represents a functional adaption to the load acting on a gliding tendon. The absence of blood vessels inside the fibrocartilage has led to the assumption that this avascular area would be prone to degeneration (Uthoff et al. 1976). It seems more likely that healthy fibrocartilage does not need vessels. Like avascular articular cartilage, or the menisci of the adult human knee, fibrocartilage seems to be nourished by convection (Tillmann 1987), which is much more efficient if one considers the intermittent multidirectional stress to which the tendon is exposed. The bicipito-radial bursa that lies be-
tween tendon and bone as a closed compartment is reminiscent of a joint cavity that nourishes the adjacent structures. In cases of overuse injuries, a traumatized bursa or a chronic bursitis might cause an inflammatory response and secondary ingrowth of vessels into malnourished fibrocartilage. Our own experiments showed that ruptures of the tendon occur either proximal to the fibrocartilage opposite the radius, or immediatly proximal to the insertion. Two fibrocartilaginous areas are located in the adult tendon. In the distal 3 cm of the tendon, three changes from a normal traction tendon to a fibrocartilaginous gliding tendon were observed. As the composition of the extracellular matrix changes between these areas, so do the mechanical features. With regard to tensile strength, it seems logical that weak spots do exist where two mechanically different structures mix. Acknowledgements: Our thanks are due to Mrs. K. Stengel, Mrs. E. Schongarth, Mrs. A. Haupt, Mrs. R. Worm, Mr. H. Mrohs and Mr. R. G. Klaws for technical assistance, and particularly to Dr. Holmdahl and Dr. Muller for the donation of antibodies.
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normal and torn menisci of the human knee joint. J Anat 136: 773-791 Grafe H (1969) Aspekte zur Atiologie der subkutanen Achillessehnenruptur. Zentralbl Chir 94: 1073 -1082 Hedman K, Johansson S, Vartio T, Kjellen L, Vaheri A, Hook M (1982) Structure of the pericellular matrix: association of heparan and chondroitin sulfates with fibronectin-procollagen fibres. Cell 28: 663 -671 Hegelmaier C, Schramm W, Lange P (1992) Die distale Bicepssehnenruptur. Unfallchirurg 95: 9 -16 Holz U (1980) Achillessehnenruptur und Achillodynie. Zur Bedeutung der Gewebsreaktion. Fortschr Med 98: 1517 -1520 Honda T, Katagiri K, Kuroda A, Matsunaga E, Shinkai H (1987) Age-related changes of the dermatan sulfate containing small proteoglycans in bovine tendon. Coli Rei Res 7: 171 - 184 Kjellan L, Lindahl U (1991) Proteoglycans: structures and interaction. Ann Rev Biochem 60: 443 -475 Knese KH, Biermann H (1958) Die Knochenbildung an Sehnenund Bandansatzen im Bereich ursprtinglich chondraler Apophysen. Z Zellforsch 49: 142 -187 Konn G, LObbecke F (1977) Bizepssehnenrisse aus der Sicht des Pathologen. Schriftenreihe Unfallmed Tagung, Heft 32: 137 -144 Kolts I, Tillmann B, Ltillmann-Rauch R (1994) The structure and vascularization of the biceps brachii long head tendon. Ann Anat 176: 75 - 80 Koob TJ, Clark PE, Hernandez DJ, Thurmond FA, Vogel KG (1992) Compression loading in vitro regulates proteoglycan synthesis by tendon fibrocartilage. Arch Biochem Biophys 298: 303 - 312 Lang E, Meeder P, Hontsch D (1988) Die distale BizepssehnenruptUT. Klinik-Therapie-Ergebnisse. Akt Traumatol18: 209 - 214 Lee GM, Johnstone B, Jacobson K, Caterson B (1993) The dynamic structure of the pericellular matrix on living cells. J Cell Bioi 123: 1899-1907 Morrey BF, Askew LJ, An KN, Dobyns JH (1985) Rupture of the distal tendon of biceps brachii. J Bone Joint Surg 67-A: 418-421 Pauwels F (1960) Eine neue Theorie tiber den EinfluB mechanischer Reize auf die Differenzierung der Stfitzgewebe. Zehnter Beitrag zur funktionellen Anatomie und kausalen Morphologie des Stfitzapparates. Z Anat Entwickl-Gesch 121: 478 - 515 Ploetz E (1938) Funktioneller Bau und funktionelle Anpassung der Gleitsehnen. Z Orth 67: 212-234 Riley GP, Harrall RL, Constant CR, Chard MD, Cawston TE, Hazleman BL (1994) Glycosaminoglycans of human rotator cuff tendons: changes with age and in chronic rotator cuff tendinitis. Ann Rheum Dis 53: 367 - 376 Romeis B (1989) Mikroskopische Technik. 17. Aufl Urban & Schwarzenberg, Miinchen Wi en Baltimore, pp 494 - 565
Rosenberg LC (1992) Structure and function of dermatan sulfate proteoglycans in articular cartilage. In: Kuettner KE, Schleyerbach R, Peyron JG, Hascall VC (eds) Articular cartilage and osteoarthritis. Raven Press, New York, pp 45 - 63 Sobue M, Nakashima N, Fukatsu T, Nagasaka T, Katoh T, Ogura T, Takeuchi J (1988) Production and characterization of monoclonal antibody to dermatan sulfate proteoglycan. J Histochem Cytochem 36: 479-485 SpalteholzKW (1914) Ober das Durchsichtigmachen von menschlichen und tierischen Praparaten. 2. Aufl. Hirzel, Leipzig Sweet MBE, Maldonado BA, Thonar EJMA, Williams J, Lenz ME, Schnitzer T, Kuettner KE (1989) Studies on the metabolism of keratansulphate-bearing proteoglycans of cartilage. In: Greiling H, Scott JE (eds) Keratan sulphate. The Biochemical Society London, pp 184 - 190 Tillmann B, Thomas W (1982) Anatomie typischer Sehnenansatze, -urspriinge und Engpasse. Orthop Praxis 12: 910- 917 Tillmann B (1987) Binde- und Stiitzgewebe des Bewegungsapparates. In: Tillmann B, Tondury G (Hrsg) Rauber/ Kopsch Anatomie des Menschen Bd. I, Bewegungsapparat. Thieme, Stuttgart, pp 13 - 50 Tillmann B, Schiinke M (1991) Struktur und Funktion extrazellularer Matrix. Verh Anat Ges 84 (Anat Anz Suppl 168): 23-26 Tillmann B, Kolts I (1993) Ruptur der Ursprungssehne des Caput longum musculi bicipitis brachii. Struktur und Blutversorgung der Bizepssehne. Operat Orthop Traumatol2: 107 - 111 Uthoff HK, Sarkar K, Maynard JA (1976) Calcifying tendinitis. A new concept of its pathogenesis. Clinical Orthop Rei Res 118: 164-168 Vogel KG (1994) Glycosaminoglycans and proteoglycans. In: Yurchenko PD, Birk DE, Mecham RP (eds) Extracellular matrix assembly and structure. Academic Press, San Diego, pp 243-280 Vogel KG, Keller EJ, Lenhoff RJ, Campbell K, Koob TJ (1986) Proteoglycan synthesis by fibroblast cultures initiated from regions of adult bovine tendon subjected to different mechanical forces. Europ J Cell Bio141: 102 -112 Vogel KG, Evanko SP (1987) Proteoglycans of fetal bovine tendon. J Bioi Chern 262: 13607 -13613 Vogel KG, Thonar EJMA (1988) Keratan sulfate is a component of proteoglycans in the compressed region of adult bovine flexor tendon. J Orthop Res 6: 434-442 Vogel KG, Ordog A, Pogany G, Olah J (1993) Proteoglycans in the compressed region of human tibialis posterior tendon and in ligaments. J Orthop Res 11: 68 - 77
Accepted February 9, 1995
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The distal tendon of the biceps brachii. Structure and clinical correlations Stephan Koch and Bernhard Tillmann Anatomisches Institut der Christian-Albrechts-Universitat zu Kiel, OlshausenstraBe 40, D-24098 Kiel, Germany
Summary. The structure and blood supply of 42 distal biceps tendons were investigated by means of light and electron microscopy as well as by immunohistochemistry. Possible structural causes for the rupture of the tendon are discussed. The distal biceps tendon wraps around the radius during pronation of the forearm. In this area the tendon is exposed to pressure and shearing forces in addition to those caused by tension. Two fibrocartilaginous areas were regularly observed. Large chondrocyte-like cells were found inside the fibrocartilage. As an expression of strain, the extracellular matrix is rich in acidic glycosaminoglycans and stains intensely with toluidine blue at pH 1. Electron microscopy showed a granular pericellular matrix that increases in size towards the gliding surface. Type I collagen is the main component of the distal biceps tendon. Type II collagen is found in tendon fibrocartilage but not in traction tendons. The gliding surface of the tendon is made up of reticular fibres that are equivalent to type III collagen. Monoclonal antibodies revealed the presence of dermatansulfate, keratansulfate and chondroitin-4- as well as chondroitin-6-sulfate. Blood vessels are usually absent in fibrocartilage, as was shown with a polyclonal antibody against the basement membrane component laminine. There are significant differences between the extracellular matrix of traction and gliding tendons, which may be responsible for the location of tendon rupture.
even in younger individuals and athletes by reason of increased sporting and outdoor activities. The majority of patients describe a minor trauma that should not have been strong enough to cause the rupture of a healthy tendon (Bindl et al. 1988; Bindl and Koch 1995; Hegelmaier et al. 1992), so acute trauma does not seem to be the only cause. On reviewing the literature, the underlying causes for the rupture remain unclear. Earlier publications have already demonstrated the correlation between the varying structures of traction and gliding tendons and their functional differences in rabbits (Ploetz 1938). Altmann (1964a, 1964 b) analysed the biomechanical background according to Pauwels' theory of "kausale Histogenese" (1960). The significance of intermittent compression stress for tendon fibrocartilage has also been demonstrated in vitro (Koob et al. 1992). A detailed description of fibrocartilage and its changes with age has been given by Benjamin (1990, 1991). The position within osteofibrous canals (Tillmann and Thomas 1982), as well as the pattern of vascularization (Uthoff et al. 1976; Tillmann and Kolts 1993) could be responsible for the degeneration. The purpose of this study was to gain further knowledge about the structure and blood supply of the distal biceps tendon and the possible structural causes of its rupture.
Key words: Distal biceps tendon - Glycosaminoglycans Biomechanics - Tendon rupture
Material and methods Tissue specimens
Introduction Injuries of the distal biceps tendon used to occur in the fifth or sixth decade of life, but nowadays ruptures are reported Correspondence to: Bernhard Tillmann
Ann Anat (1995) 177: 467 -474 Gustav Fischer Verlag lena
Tendons were obtained from 42 subjects of different ages (22 weeks to 91 years; m: 23 f: 19) during autopsy within 48 hours of death. After macroscopical investigation, each tendon was split longitudinally and the parts prepared for light microscopy, transmission electron microscopy and immunohistochemistry. The intratendinous vasculature was observed in arterial injection samples prepared according to the method of Spalteholz (1914).
Light microscopy For light microscopy, tendons were fixed in Schaffer's solution and embedded in methyl metacrylate. Sections (10 !lm) were mounted on gelatine-coated slides and stained with toluidine blue (pH 1), and by the methods of Gomori, Goldner, von Kossa and with Movat's Pentachrome stain (Romeis 1989).
Electron microscopy For transmission electron microscopy the samples were fixed in 3.5% glutaraldehyde after initial treatment with 0.1 M Sorensen phosphate buffer solution at pH 7.4 (Romeis 1989), and embedded in araldite. Ultrathin sections were cut with a microtome and contrasted with uranyl acetate and lead citrate. Examination was carried out with a Zeiss EM 900 electron microscope.
Immunohistochemistry Samples were snap frozen in liquid nitrogen. Longitudinal frozen sections (10 !lm) were cut with a cryostat at _210 C and mounted on gelatine-coated slides. For immunohistochemistry, frozen sections were pretreated with either testicular hyaluronidase (Boehringer) or chondroitinase ABC (Sigma) in tris-buffered saline (TBS) in a moist chamber at 37 0 C for 30 minutes. The sections were washed three times with TBS and incubated with goat serum for 45 minutes at room temperature. Then incubation with the primary antibody was carried out for 60 minutes at room temperature. Antibodies
used in the investigations (tested and described in detail by Caterson et al. 1983 and Sobue et al. 1988) were: mouse anti collagen (MAC) type I polyclonal antibody (gift from Prof. Dr. P. K. MUller, Lubeck); MAC type II polyclonal antibody (Biodesign) and monoclonal antibody CIID3 (gift from Dr. R. Holmdahl, Uppsala). MAC type III polyclonal antibody (Bio-Science); MAC type IV polyclonal antibody (DAKO); MAC type IX polyclonal antibody (gift from Prof. Dr. P. K. MUller, Lubeck); anti chondroitin-4-sulfate monoclonal antibody (Chemicon International Inc.); Clone BE 123. The antibody is directed against unsaturated uronic-acid residues bound to N-acetyl-galactosamine-4-sulfate; anti chondroitin-6-sulfate monoclonal antibody (Chemicon International Inc.); Clone MK-302. The antibody is directed against uronic-acid residues bound to N-acetyl-galactosamine-6-sulfate. Anti keratansulfate monoclonal antibody (Bio-Science Products AG); Clone 5-D-4. The antibody is directed against hexasaccharides of keratansulfate. Anti dermatansulfate proteoglycan monoclonal antibody (Bio-Science Products AG); Clone 6 - B - 6. The antibody is directed against the core-protein. Mouse anti laminine polyclonal antibody (Medac). Sections were labeled with the respective secondary antibody, fluorescein isothiocyanate (FITC)- conjugated goat anti rabbit or goat anti mouse IgG for 45 minutes. Control sections were incubated only with the FITC-conjugated antibody. Positive controls including tissues with defined antigen sites (human cartilage, skin, kidney, liver, spleen) were used. The slides were examined with a Zeiss-Axiophot microscope equipped for epifluorescence.
Fig. 1. Chrondroid cells inside the fibrocartilage of the distal biceps tendon opposite the radius. Toluidine blue, x 90 Fig. 2. The course of collagen fibres follows a rhomboid pattern within the fibrocartilage. It follows the direction of strain. Gomori, x45 Fig. 3. The gliding surface of the distal biceps tendon consists of a dense layer of reticular fibres (arrows) with embedded chondroid cells. Gomori, x 140 Fig. 4. The largest chondroid cells are located beneath the gliding surface. Their size decreases with increasing distance from the surface. Pentachrome, x 140
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Results
type is found that still has features of a fibroblast-like cell, but already shows an oval form (Fig. 6). It is surrounded by a slim granular pericellular matrix. Large chondroid cells are found inside in the fibrocartilage opposite to the radius and show an even larger pericellular matrix (Figs. 7 and 8). These cells contain a marked endoplasmic reticulum and many mitochondria. Cells and pericellular matrix are surrounded by collagen fibres, proteoglycans and other matrix components.
Gross anatomy The distal tendon of the biceps brachii inserts at the radial tuberosity over an area of 3 cm2 • During pronation of the forearm it is wrapped around the radial neck. A thin layer of synovial membrane-like tissue forms the bicipito-radial bursa between the tendon and radius. This bursa is a closed compartement and was present in all cases investigated. Once the bursa is opened, the gliding surface of the distal biceps tendon is visible. It is shiny and appears to be avascular. Blood vessels are visible on the outside of the tendon, reaching the point of insertion via the periosteum of the radius or descending from the muscle belly.
Immunohistochemistry
Light microscopy In all the tendons examined, two different types of cells could be distinguished. Thin fibroblast-like tenocytes are embedded between parallel-orientated collagen fibres on the ulnar aspect of the tendon. Where the tendon faces the pulley, large cells having the appearance of chondrocytes could be observed (Fig. 1). The number and size of the chondroid cells decrease with increasing distance from the gliding surface. This area of the gliding tendon resembles fibrocartilage. The extracellular matrix stains intensely with toluidine blue (pHi). The adult tendon contains two areas that consist of fibrocartilage. One develops where the distal biceps tendon opposes the radius and is not visible until the age of one, the other is part of the tendon-bone interface of the chondro-apophyseal insertion of the tendon. Gomori staining clearly shows the parallel arrangement of the fibres on the ulnar aspect of the tendon. Inside the fibrocartilage the fibres take a swivelled course and are arranged in a rhomboid pattern (Fig. 2). Some fibres run at 90 to the main axis of the tendon and connect the main fibre bundles. Collagen fibres insert at the radial tuberosity at 60 0 • Their course can be followed through the mineralized zone of the attachment site. There seems to be a continuous transition to the bony trabeculae of the radial tuberosity. Small darkly stained reticular fibrils intermingle with the major fibre bundles. A separate layer of reticular fibres was observed at the gliding surface adjacent to the radius (Fig. 3). It is inside this reticular network that the largest chondroid cells are located (Fig. 4). Trichrome staining, according to the method of Masson-Goldner, stained the fibrocartilage inside the biceps tendon an intense red colour. Proximal areas and a small ulnar segment were stained green. Movat's pentachrome staining showed green spotty areas in the fibrocartilage, and von Kossa staining for calcification was negative in all cases. 0
Electron microscopy Transmission electron microscopy reveals three types of tenocytes. Typical fibroblast-like cells are found on the ulnar aspect (Fig. 5). In the middle of the tendon an intermediate
Type I collagen is the main component of the distal biceps tendon. Antibodies against type I collagen show a marked staining which follows the course of the fibres throughout the tendon (Fig. 9). The distal biceps tendon is positive for collagen type II where fibrocartilage is embedded in the tendon (Fig. 10). The area that stains with an antibody against type II collagen is located immediatly under the gliding surface and makes up two thirds of the diameter of the tendon. Between these fibres, a positive reaction with an antibody against collagen type IX is observed and follows the zonal distribution of type II collagen. Antibodies against type III collagen stain small fibrils located between major fibres and a dense layer on the gliding surface of the tendons (Fig. 11). Glycosaminoglycans are an important component of the extracellar matrix of tendons. Monoclonal antibodies against dermatansulfate revealed the presence of this glycosaminoglycan in all tendons and showed a distribution along the course of the collagen fibres (Fig. 12). Keratansulfate was present in all tendons and also distributed along the course of collagen fibres (Fig. 13). There was only faint staining on the ulnar aspect of the tendon and in proximal areas, whereas intense staining was observed in the fibrocartilage. Chondroitin-4-sulfate was present in all gliding tendons, the intensity of the spotty staining decreasing with increasing distance from the gliding surface (Fig. 14). Staining with anti chondroitin-6-sulfate showed only a faint reaction around the fibrils. Occasionly a marked reaction was observed around superficial defects of the tendon. Areas proximal and distal to the fibrocartilage showed a negative reaction for chondroitinsulfates.
Vascular supply Arterial injection samples revealed blood vessels that descend from the muscle belly of the biceps brachii. The vessels usually reach the tendon from the outside via a paratenon. From the ulnar side, facing away from the pulley, they run into the depths of the tendon. Intratendinous blood vessels run either within a loose internal peritendineum or directly between collagen fibres. The insertion itself is supplied by small branches of the cubital artery. Macroscopic investigations showed a hypovascular area opposing the radius that was identical with fibrocartilage (Fig. 15). To confirm this finding, immunohistochemical tests with an antibody
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.*
Fig. 5. Electron microscopic view of a fibroblast-like tenocyte from the ulnar aspect of the distal biceps tendon. The cell is embedded between collagen fibres (CO) that are orientated in parallel. Scale bar represents 2.33 Ilm. x 3 000 Fig. 6. Electron microscopic view of a tenocyte of the intermediate layer between fibrocartilage and the ulnar aspect of the distal biceps tendon. The cell is embedded between collagen fibres (CO). The course of the fibres varies and follows the direction of strain. A small territorial matrix is visible (arrow). Scale bar represents 2.33 Ilm. x 3 000 Fig. 7. Electron microscopic view of a chondroid cell inside the fibrocartilage of the distal biceps tendon. Note the marked territorial matrix (star). Scale bar represents 1.59 Ilm. x4400 Fig. 8. Magnification of Fig. 7. The granular territorial matrix that surrounds the cell (star) contains numerous vesicles. The cytoplasm of the chondroid cells is rich in rough endoplasmic reticulum and mitochondria, which is a sign of active synthesis of proteins such as collagen. Scale bar represents 1.00 Ilm. x 7 000
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Fig. 9. Immunohistochemical appearance of type I collagen fibres in the distal biceps tendon. The fibres are orientated in parallel. x180 Fig. to. Immunohistochemical appearance of type II collagen fibres that are present inside fibrocartilage and where the tendon is exposed to pressure. X 180 Fig. 11. Immunohistochemical appearance of type III collagen from the gliding surface of the distal biceps tendon. Chondroid cells (arrows) are embedded in the gliding surface (arrowheads). x 140
Fig. 12. Immunohistochemical appearance of dermatansulfate inside the distal biceps tendon. x 180 Fig. 13. Immunohistochemical appearance of keratansulfate inside the fibrocartilage of the distal biceps tendon. x 180 Fig. 14. Immunohistochemical appearance of chondroitin-4-sulfate in the fibrocartilage of the distal biceps tendon. x 180
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15
Fig. 15. Distal biceps tendon (DBT) of a 72 year-old man after injection of india ink gelatine and preparation according to the method of Spaiteholz. The tendon is inserted at the radial tuberosity (RT). A hypovascular area can be seen where the tendon lies in contact with the radius (arrow). Fig. 16. Immunohistochemical appearance of vessels inside a distal biceps tendon, shown with an antibody against the basement membrane component laminin. An avascular fibrocartilaginous zone (*) is visible in the right upper corner. x 360 against the basement membrane component laminine were used. Blood vessels are clearly visible in all samples. The vessels follow the main course of collagen fibres and are distributed all over the ulnar aspect of the tendons (Fig. 16). Fibrocartilage is avascular in most cases. Only in twelve tendons that showed macroscopical signs of degeneration were blood vessels detected inside fibrocartilage.
Discussion The distal tendon of the biceps brachii is made up of dense, regularly arranged collagen fibrils. It transmits forces from the biceps muscle to the radius and is therefore subject to
great tensile stress. Depending on the position of the forearm during flexion and pronation, the tendon is additionally exposed to pressure and shearing forces. Pressure occurs when the tendon wraps around the radius during pronation of the forearm. Additional shearing forces are brought about during combined movements in the elbow joint. Injuries to the distal biceps tendon nearly always occur close to its insertion, one to two centimetres proximal to the radial tuberosity. Rupture hardly ever occurs before the age of fourty (Agins et al. 1988; Bindl and Holz 1988; Konn and LObbecke 1977; Lang et al. 1988; Morrey et al. 1985). Recent observations suggest that changes inside the distal biceps tendon occur by reason of repeated pronation and supination at the radial tuberosity (Bindl and Koch 1995). Is the location of the rupture really due to the structure of the tendon? The distal biceps tendon is inserted at the radial tuberosity and has the typical structure of a chondro-apophyseal insertion (Knese 1958; Tillmann and Thomas 1982; Benjamin 1992). The structure of the human distal biceps tendon changes at the point where the tendon wraps around the radius. Compared with proximal regions of the tendon, the fibrocartilage opposite to the radius shows a rounded rather than an elongated cellular morphology, and has a marked metachromasia due to the acidic glycosaminoglycans when stained with toluidine blue at pH 1 (Tillmann and Schiinke 1991). These variations are thought to be governed by the prevailing biomechanical conditions: The part of the distal biceps tendon that faces away from the pulley resembles a traction tendon. Tensional stress decreases towards the pulley. The gliding surface and the tissue below it are exposed to compression and shearing forces that decrease with the distance from the pulley. The attachment zone and also an area opposite the radius consist of fibrocartilage and include chondroid cells located between the collagen fibres. They serve to reduce the horizontal shortening of the tendon at sites exposed to multidirectional stress. In accordance with Pauwels' theory of "kausale Histogenese" (Pauwels 1960), chondroid cells increase in size the closer they are to the gliding surface and reflect the load acting on the tendon. Electron microscopy shows that the chondroid cells inside the fibrocartilage are surrounded by a territorial matrix. The closer the cell is located to the pressure-exposed area the wider is the matrix. A territorial matrix around mammalian cells was first described by Clarris and Fraser (1968). A granular territorial matrix around human meniscal chondrocytes, visible in transmission electron microscopy, was described by Ghadially (1983). At present the exact structure of the human territorial matrix is unknown, although the presence of a variety of extracellular glycoproteins and proteoglycans has been demonstrated (Hedmann et al. 1982; Kjellan and Lindahl 1991). According to Lee et al. (1993) the territorial matrix around chondrocytes is composed of hyaluronan-aggrecan complexes. The increased amount of rough endoplasmic reticulum and mitochondria in the chondroid cells could stand for an increased synthesis of extracellular matrix components as are collagens and proteoglycans. Type I collagen fibres are orientated in parallel
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to the main axis of traction where the distal biceps tendon is exposed to pull. Tendon fibrocartilage also consists of typ I collagen, but the fibres follow a swivelled course due to the different directions of strain. Immunohistochemistry shows a spotty distribution of type II collagen ' between type I collagen fibres. The presence of type II collagen is an indicator for the exposure to pressure in this part of the tendon, as the extracellular matrix of normal traction tendons does not include type II collagen. Where collagen type II is present, collagen type IX can be shown as a small connecting collagen. Reticular fibrils consist of type III collagen. They are located between major collagen fibre bundles and form a neat reticular layer on the gliding surface itself. Glycosaminoglycans as part of extracellular matrix proteoglycans of tendons have been investigated over the past years in bovine, rat and rabbit tendons (Daniel and Mills 1988; Honda et al. 1987; Vogel 1986, 1987, 1988; Thonar 1988). Gel-electrophoretic investigations show, that there are significant differences between the composition of proteoglycans of traction and gliding rat and bovine tendons (Vogel 1986 and 1987). Investigations of the human posterior tibial tendon proved these results (Vogel 1993). The present study showed the presence of dermatansulfate, keratansulfate and chondroitinsulfates in the pressure-exposed fibrocartilage of all human biceps tendons, and also showed regional differences in the distribution of glycosaminoglycans throughout the tendon. Those parts that are mainly exposed to traction did not react with antibodies against chondroitinsulfates or keratansulfate, although they were positive for dermatansulfate. Dermatansulfate is supposed to influence tissue repair (Rosenberg 1992) and may play an important role in the development of degeneration within the distal biceps tendon. In some tendons only a weak staining for chondroitinsulfates was observed in the pressurized area. This result may be explained by possible immobilization of the extremity, shortly before death, since a loss of the chondroitinsulfate content of tendon has been described after the reduction of a compressive load (Koob 1992). Injection samples and immunohistochemical experiments showed a continuous vascular network extending from the biceps muscle to the bony insertion of the distal biceps tendon. Fibrocartilage inside the tendon is usually devoid of blood vessels. Occasionally vessels were found within fibrocartilage, but only in tendons that had shown macroscopic signs of degeneration. These results go along well with previous findings in other gliding tendons (Tillmann and Kolts 1993; Kolts et al. 1994). Vascular architecture represents a functional adaption to the load acting on a gliding tendon. The absence of blood vessels inside the fibrocartilage has led to the assumption that this avascular area would be prone to degeneration (Uthoff et al. 1976). It seems more likely that healthy fibrocartilage does not need vessels. Like avascular articular cartilage, or the menisci of the adult human knee, fibrocartilage seems to be nourished by convection (Tillmann 1987), which is much more efficient if one considers the intermittent multidirectional stress to which the tendon is exposed. The bicipito-radial bursa that lies be-
tween tendon and bone as a closed compartment is reminiscent of a joint cavity that nourishes the adjacent structures. In cases of overuse injuries, a traumatized bursa or a chronic bursitis might cause an inflammatory response and secondary ingrowth of vessels into malnourished fibrocartilage. Our own experiments showed that ruptures of the tendon occur either proximal to the fibrocartilage opposite the radius, or immediatly proximal to the insertion. Two fibrocartilaginous areas are located in the adult tendon. In the distal 3 cm of the tendon, three changes from a normal traction tendon to a fibrocartilaginous gliding tendon were observed. As the composition of the extracellular matrix changes between these areas, so do the mechanical features. With regard to tensile strength, it seems logical that weak spots do exist where two mechanically different structures mix. Acknowledgements: Our thanks are due to Mrs. K. Stengel, Mrs. E. Schongarth, Mrs. A. Haupt, Mrs. R. Worm, Mr. H. Mrohs and Mr. R. G. Klaws for technical assistance, and particularly to Dr. Holmdahl and Dr. Muller for the donation of antibodies.
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Accepted February 9, 1995
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