Micron 37 (2006) 518–525 www.elsevier.com/locate/micron
Comparative ultrastructural analysis of different regions of two digital flexor tendons of pigs V.L.C. Feitosa a, F.P. Reis b, M.A.M. Esquisatto c, P.P. Joazeiro d, B.C. Vidal e, E.R. Pimentel e,* a
Department of Morphology, Federal University of Sergipe, Aracaju, SE, Brazil b University of Tiradentes—UNIT, Aracaju, SE, Brazil c Department of Morphology, Herminio Ometto University Center, Araras, SP, Brazil d Department of Histology, Institute of Biology, State University of Campinas (UNICAMP), Campinas, SP, Brazil e Department of Cell Biology, Institute of Biology, State University of Campinas (UNICAMP), P.O. Box 6109, 13083-863 Campinas, SP, Brazil Received 8 September 2005; accepted 17 January 2006
Abstract Tendons are parallel arrays of collagenous fibers which are specialized in resisting and transmitting tensile forces. In this work we examined the structure of the superficial digital flexor tendon (SDFT) and the deep digital flexor tendon (DDFT) of pigs, which are considered ‘‘wrap around’’ tendons and so receive compression and tension forces. In both tendons, fibrocartilaginous areas were observed in the regions subjected to compression plus frictional loading. Histological and ultrastructural analyses of the tensional region showed an extracellular matrix (ECM) rich in collagen bundles, that were all arranged in the same direction. Fibroblasts were seen closely associated with the collagen bundles. Chondrocytelike cells and high levels of glycosaminoglycans (GAGs) were observed in the compressional regions. The collagen bundles in the compressional region were arranged in several directions and were associated with proteoglycans (PGs). The crimp pattern detected in the tensional region showed that the collagen fibrils were ordered aggregates which formed helical superstructures. # 2006 Elsevier Ltd. All rights reserved. Keywords: Pig; Proteoglycans; Collagen; Fibrocartilage; Tendon
1. Introduction Tendons are specialized structures designed to transmit to bone the tensional forces exerted by muscles (Birk et al., 1989; Cribb and Scott, 1995). Some tendons pass around bony pulleys and receive compressional forces in addition to tensional forces (Merrilees and Flint, 1980; Vogel and Koob, 1989). In the region under compression, these tendons characteristically have a higher amount of proteoglycans (PGs), as well as type II collagen, and a random distribution of type I collagen bundles, which may be associated in heterotypic fibrils with type III and V collagens (van der Rest and Garrone, 1991). Type VI collagen has also been detected in compressional and tensional areas of bullfrog, dog, rabbit, and chicken tendons (Felisbino and Carvalho, 1999).
* Corresponding author. Tel.: +55 19 3788 6117; fax: +55 19 3788 6111. E-mail address:
[email protected] (E.R. Pimentel). 0968-4328/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.micron.2006.01.010
Whereas, collagen accounts for about 90% of the tendon dry mass (Nimni and Harkness, 1988), mainly in the regions under tension, PGs constitute less than 1% of the tendon dry weight, and are represented mostly by small PGs containing dermatan sulfate (Vogel and Heinega˚rd, 1985). Studies using polarized light microscopy indicate that PGs are arranged so that their glycosaminoglycans (GAGs) are helically distributed along the collagen fibres (Vidal and Mello, 1984). In electron micrographs of tendon sections stained with cupromeronic blue under critical electrolyte concentration (CEC) conditions, PGs are seen as filaments regularly attached to the collagen fibrils (Scott, 1985). Other components detected amongst the collagen bundles in tendon include pre-elastic and elastic fibers, which may play a role in the ability of the tendon to bear tensional and compressional forces (Carvalho et al., 1994; Carvalho and Vidal, 1994a, 1995). The cells in tendons and ligaments can detect physicochemical changes in the extracellular matrix (ECM) and coordinate their responses to alter the composition of this matrix. One of the
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most obvious ways in which the ECM of tendons is modified in response to compressional load is by the formation of a fibrocartilaginous matrix at sites where the tendons are under compression. This occurs where the tendon passes around bony pulleys and threads through fibrous retinaculum, and where it attaches to the bone (Benjamin and Ralphs, 1998). Fibrocartilage is a transitional tissue with structural properties intermediate to those of dense fibrous connective tissue and hyaline cartilage (Benjamin and Evans, 1990). In addition to a weave basket-like distribution of collagen fibers and rounded cells, these areas also show an increased amount of large PGs (Vogel et al., 1994). Compressional fibrocartilage, with collagen fibers running in all directions, has been detected in regions of bovine (Vogel et al., 1986; Vogel and Koob, 1989; Robbins et al., 1997) and rabbit (Gillard et al., 1979; Merrilees and Flint, 1980) tendons under compression. In amphibians, a different arrangement of the convoluted and undulated collagen fibers allows the tendon to undergo great distension before exerting resistance (Carvalho and Vidal, 1994a). Some collagenous tissues such as tendons show a morphofunctional characteristic known as crimp, which is generally present at sites where the tissues are subjected to tensile forces (Gathercole and Keller, 1991). The variations in crimp in different regions of the same tendon, as well as the distribution of the fibers and their bundles, could reveal details of the fiber organization in tendons (Vidal, 1995). Observations using polarized light microscopy have shown different crimp patterns and arrangements of the collagen bundles in the compressional and tensional regions of digital flexor tendons (Feitosa et al., 2002a,b). No ultrastructural study has been carried out with ‘‘wrap around’’ tendons of pigs, a mammal with rapid increase of weight during the growth phase. In this work we examined the ultrastructural differences between specific regions of the superficial digital flexor tendon (SDFT) and deep digital flexor tendon (DDFT), taking into account their biomechanical properties. 2. Materials and methods 2.1. Animals Male pigs (large White breed, 45 days old) were obtained from the Center of Medicine and Experimental Surgery at UNICAMP. The pigs were killed with 2.5% thiopental sodium (1 ml/kg body weight).
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region (d), which divides into two branches towards the fingers, and presumably is subject to compressional forces when it passes close to the metatarsophalangeal joint, and the terminal region (t), which extends into the fingers and is also subject to compressional forces. 2.3. Histology Tendon fragments were fixed in 4% paraformaldehyde in Millonig’s buffer at pH 7.4 for 24 h at room temperature. The material was then dehydrated in a graded ethanol series, cleared in xylene and embedded in Paraplast Plus embedding medium. Serial 6 mm thick sections were cut longitudinally and stained with hematoxylin and eosin. After differentiating in 70% ethanol for 1 min, the sections were dehydrated and mounted in Entelan resin. 2.4. Transmission electron microscopy The tendons were fixed with either 2.5% glutaraldeyde and 1% tannic acid in 0.1 M cacodylate buffer at pH 7.3 for 2 h (Cotta-Pereira et al., 1976), or with 2.5% glutaraldeyde containing 0.2% ruthenium red (RR) and 0.2% alcian blue in 0.1 M cacodylate buffer at pH 7.3 for 4 h, for the detection of PGs (Tsuprun and Santi, 1996). After fixation, the tendons were rinsed in cacodylate buffer and post-fixed with 1% osmium tetroxide in the same buffer for 1 h. The sections were impregnated with 1% uranyl acetate in 1.2% NaCl and 7.3% sucrose in water overnight at 4 8C, then dehydrated in a graded ethanol series and embedded in Epon 812 resin. Ultrathin sections were stained with 1% uranyl acetate and lead citrate (Reynolds, 1963), and observed in a Leo 906 transmission electron microscope operated at 40 or 80 kV. 2.5. Scanning electron microscopy After dissection, the tendons were immediately fixed by immersion in 2.5% glutaraldeyde and 1% tannic acid in 0.1 M phosphate buffer at pH 7.4. They were then macerated with 2 M NaOH (Ohtani, 1988; Goranova et al., 1996), post-fixed with osmium tetroxide for 2 h at 4 8C, dehydrated with ethanol and immersed in liquid nitrogen. The material was fractured sagitally with the help of a stainless steel blade, then dried to the critical point and sputter-coated with gold. The samples were observed in a JEOL JSM 5800 LV scanning electron microscope.
2.2. Biological material 3. Results Hind limbs were dissected to obtain the superficial and deep digital flexor tendons. The SDFT was divided into the proximal region (p), which wraps around the tibiotarsal joint and is subject to compressional, frictional and tensional forces, the intermediate region (i), an extended region of the tendon that withstands only tensional forces, and the distal region (d), which passes close to the metatarsophalangeal joint and bears compressional forces. The DDFT was divided into the proximal region (p), which undergoes only tensional forces, the distal
Histological sections of the SDFT and DDFT provided a detailed view of two regions which experience tensional and compression forces (Figs. 1–4). A large number of cells was observed in both tendons. In the tensional areas of both tendons (Figs. 1 and 3), the elongated fibroblasts were parallel to the long axis of the tendon. The unidirectional and undulating aspect of the collagen fibers was obvious. In areas of compression, many cells changed their morphology to round
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Fig. 1–4. Longitudinal sections of the SDFT and DDFT stained by HE. Intermediate region of the SDFT showing elongated fibroblasts (arrowheads) aligned with the collagen bundles (Fig. 1). Proximal region of the SDFT showing tendinocytes (arrowheads) and round cells (arrows) (Fig. 2). Proximal region of the DDFT showing typical fibroblasts with elongated nuclei aligned with the collagen bundles (arrowheads) (Fig. 3). Terminal region of the DDFT showing randomly distributed round cells (arrows) (Fig. 4). Bars = 30 mm; SDFT, superficial digital flexor tendon; DDFT, deep digital flexor tendon.
chondrocyte-like cells and were randomly distributed (Figs. 2 and 4). Contrary to the tensional regions, the collagen fibers ran in several directions and produced an amorphous aspect. 3.1. Ultrastructural analysis The ultrastructural features of different regions of each tendon, representing areas of tension and compression, were analyzed. In the tensional region of the DDFT (Fig. 5) and SDFT (Fig. 6), the elongated cells were closely associated with collagen fibers. Some cells exhibited well-developed rough endoplasmic reticulum and were surrounded by a large number of collagen fibers (Fig. 6). All cells had condensed chromatin associated with the inner surface of the nuclear envelope, as well as a large amount of non condensed chromatin, indicating a high synthetic activity for the cell. Extracellularly, there was no morphological distinction between the pericellular and the intercellular matrices. The ECM showed a more uniform organization, including closely packed, the longitudinally aligned collagen fibrils. On the other hand, in the p region of the SDFT (Fig. 7) and the d region of the DDFT (Fig. 8), which are pressure-bearing zones, the collagen fibers were arranged in several directions with a wider interfibrillar space than in the tensional region (Figs. 5 and 6). In the d region of the DDFT (Fig. 9), chondrocyte-like cells exhibited an abundant cytoplasm and a large number of intermediate filaments.
Cytoplasmic prolongaments appeared projecting among the collagen fibrils (Fig. 9). Analysis of the RR-alcian blue stained material revealed a pericellular matrix rich in PGs, represented by a fibrilar network with globular precipitates (Fig. 10). These precipitates were linked to each other and to the cell coat (Fig. 11). They also occurred in the wider spaces between bundles of collagen fibrils (Fig. 12) and in association with these fibrils. Strips representing PG were regularly distributed on collagen fibrils in the tensional (Fig. 13) and compressional (Fig. 14) regions of both tendons. SEM showed that the collagen fibers in the p (Fig. 15) and d (Fig. 16) compressional regions of the SDFT and DDFT, respectively, were arranged in several directions in a weave basket-like distribution. These regions contained filamentous material interspersed with thin fibers. These fibrils showed kinks or folds and a large number of surrounded microfibrils. In the tensional region of the SDFT (Fig. 17), a well-defined wavelike pattern known as crimp, was observed, while in the tensional region of the DDFT (Fig. 18), these fibers were more uniform and parallel to the long axis of the tendon. 4. Discussion Tendons are designed to receive and transmit biomechanical loads generated by muscles, but some of them also withstand
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Fig. 5–9. Ultrastructure of cells and surrounded ECM of the DDFT and SDFT. (Fig. 5) Elongated fibroblast of the tensional region of the DDFT associated with collagen bundles which are arranged parallel to the longitudinal axis of the tendon. Note that the collagen fibrils ( ) in the tensional region are arranged in the same direction, and are densely packed in the matrix. Most of the condensed chromatin is associated with the inner surface of the nuclear envelope; nucleus (N). (Fig. 6) Detail of a fibroblast in the tensional region of the SDFT. The cell is very active in protein synthesis, as suggested by the amount of endoplasmic reticulum in the cytoplasm. There is no distinct pericellular matrix and coarse collagen fibers ( ) are in direct contact with the cell. (Fig. 7) Chondrocyte-like cell and ECM of the compression region of the SDFT after treatment with RR-alcian blue. Note the round nucleus (N) and the larger area of cytoplasm compared to cells of the tensional region. Only a few organelles are embedded in the intermediate filaments meshwork. The matrix contains collagen fibrils running in several directions (arrows), RRalcian blue granules corresponding to collapsed PGs (arrowheads) interact with fibrils dispersed in the ECM. (Fig. 8) Aspect of the distal region of the DDFT after RRalcian blue treatment. Note the collagen fibers running in several directions (arrows) and the collapsed PGs associated with collagen fibers. PGs are also randomly distributed in the cytoplasm. (Fig. 9) A typical cell of the compression region of the DDFT after RR-alcian blue treatment, showing the round nucleus (N) with condensed chromatin associated with the inner surface of the nuclear envelope. The cytoplasm has few extensions but contains large quantities of intermediate filaments (arrowheads) and lipid droplets ($). A considerable amount of rough endoplasmic reticulum (arrows) is present. PGs also occurred in the pericellular matrix (short arrow) and many collagen fibers ( ) may be seen spread out for all ECM. Bars = 1 mm; ECM, extracellular matrix; DDFT, deep digital flexor tendon; SDFT, superficial digital flexor tendon; RR, ruthenium red; PGs, proteoglycans.
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Fig. 10–14. Aspects of the extracellular matrix after treatment with RR-alcian blue. (Fig. 10) Distal region of the DDFT showing interconnected PGs (arrowheads) and PGs interacting with collagen fibrils (arrows); bar = 1 mm. (Fig. 11) Detail of the compression region of the SDFT showing granules of PGs (arrowheads) interconnected to each other, and to microfibrils (mfl) in the pericellular matrix; bar = 0.5 mm. (Fig. 12) PGs (arrowheads) in the proximal region of the SDFT detected among the collagen fibrils (arrows). Note that the collagen fibrils of this pressure region are arranged in several directions (arrows). A cellular process ( ) with some vesicles and dilated endoplasmic reticulum can be seen. Bar = 0.3 mm; RR, ruthenium red; DDFT, deep digital flexor tendon; PGs, proteoglycans; SDFT, superficial digital flexor tendon. Aspects of the fibrous portions of the tensional (Fig. 13) and compression (Fig. 14) regions of the SDFT stained with RR-alcian blue. PGs are associated with collagen fibrils and bridge them (arrows). Note the kinking of the collagen fibrils (Fig. 14) in the region under compression. Fig. 13; bar = 0.5 mm. Fig. 14; bar = 0.3 mm.
perpendicular compressional forces as they pass under a joint. The SDFT and DDFT of pigs show two well-defined organizations: an arrangement of classic tendons designed to resist tensional forces, and a fibrocartilaginous structure that appears in areas under compression. Sections from regions of
tension in both tendons stained by HE showed elongated fibroblasts parallel to the long axis of the collagen bundles, whereas regions under compression contained chondrocytelike cells and a collagen distribution similar to that of corresponding tendons from other mammals (Merrilees and
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Fig. 15–18. Scanning electron microscopy of the proximal and terminal regions of the SDFT (15) and DDFT (16), respectively, both under compression. The fibers of the two regions run in several directions and are arranged in a basket-weave pattern. Large spaces are present between the fibers. (15) bar = 10 mm. (16) bar = 10 mm; (Fig. 17) Scanning electron microscopy of the intermediate (tensional) region of the SDFT showing the crimp structure. Note the fibrous structures which follow a zag path. Bar = 21 mm. SDFT, superficial digital flexor tendon. (Fig. 18) Scanning electron microscopy of the proximal (tensional) region of the DDFT showing regularly distributed, uniform collagen bundles arranged in the same direction; bar = 5 mm. DDFT, deep digital flexor tendon.
Flint, 1980; Vogel et al., 1986; Okuda et al., 1987; Evanko and Vogel, 1990; Ralphs et al., 1992; Carvalho and Felisbino, 1999; Felisbino and Carvalho, 1999). Ultrastructural analysis showed marked differences in cell morphology, matrix organization, collagen bundle arrangement, and the distribution of PGs in the different regions of the two tendons. The regions under pressure and frictional forces had fibrocartilaginous features, with collagen bundles arranged in several directions and an increased content of PGs, as well as the presence of chondrocyte-like cells. Similar aspects have been found in other wrap around tendons which receive perpendicular compressional forces (Vogel and Koob, 1989; Benjamin and Evans, 1990; Carvalho, 1995). On the other hand, in the tensional regions of these same tendons, the fibrous matrix was closely associated with fibroblasts which were delimited by the surrounding closely packed collagen fibrils. The RR-alcian blue stained granules, representing PGs, were interconnected by thin filaments, as in rat tail tendon (Vidal and Mello, 1984). The PGs also joined collagen fibrils to each other, as occurs in rat and mouse tendons stained with cupromeronic blue (Cribb and Scott, 1995). The interactions of PGs with collagen fibers and other PGs, are non-covalent and therefore reversible (Scott, 1988). According to Hascall and Sajdera (1970), PGs appear as granules because of the preparative procedures, mainly dehydration. Vidal and Mello (1984) demonstrated that in Achilles tendon of newborn rats, PGs
occurred as dense interconnected globules attached to the cell coat through fibrillar formations. These authors also observed an intimate association of collagen fibrils with the ruthenium red stained cell coat. The cell-matrix interactions observed here, especially in the tensional regions in both tendons, indicated that the cell morphology and metabolism were closely related to the distribution of biomechanical forces. The structural and ultrastructural findings confirmed the organizational differences between the tensional and compression regions of both tendons. The appearance of the compressional region of both tendons was typical of fibrocartilages, which are described as an intermediate structure between dense fibrous connective tissue and hyaline cartilage (Benjamin and Evans, 1990). The greater occurrence of PGs in compressional areas compared to the tensional regions was expected as based on quantitative analyses and swelling tests done for the SDFT (Feitosa et al., 2002a,b), as well as on results obtained for other tendons (Koob and Vogel, 1987; Evanko and Vogel, 1990; Carvalho and Vidal, 1994a,b; Covizi et al., 2001). The fibrillar arrangement seen in the pericellular matrix of the compressional regions of both tendons may correspond to the accumulation of type VI collagen microfibrils, which can associate with PG to form the structure seen in the pericellular environment of cells of regions under compression (Felisbino and Carvalho, 1999).
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Structural and biochemical studies done with these two tendons (Feitosa et al., 2002a,b), have shown the compressional regions have a greater amount of PGs, as demonstrated by the intense metachromatic basophilia after staining with toluidine blue; such staining is not seen in the regions under tensional forces. Similar results were reported for the bovine flexor tendon (Koob and Vogel, 1987) and the digital flexor tendon of rats (Covizi et al., 2001). Some of the GAGs found in the compressional regions are chondroitin sulfate and probably correspond to the large PGs. In bovine tendons under compression, 40% of PGs can aggregate with hyaluronan (Vogel and Heinega˚rd, 1985; Evanko and Vogel, 1990), thereby allowing the tendon to withstand compressional forces (Vogel and Heinega˚rd, 1985; Vogel et al., 1986; Benjamin and Evans, 1990). The pattern of crimp in the regions of tension observed in this work by SEM reflect the three-dimensional helical superstructure of the collagen bundles (Vidal, 1995, 2003) in response to the biomechanical demands of the region. Recent work on the optical anisotropy of the SDFT of pigs (Feitosa et al., 2002a) has shown that the crimp patterns were quite different for the tensional and compression regions. This difference reflects the importance of the arrangement and organization of the collagen fibers to meet the biomechanical requirements of the tissue. The ability of fibrocartilage to resist compression probably reflects the presence of large aggregating PGs in the tissue as well as the specific arrangement of the collagen fibers (Carvalho, 1995). Specific models for studying the macromolecular interactions and biomechanics of tendon fibrocartilages are needed in order to understand better the function of each component. Our results once again indicate that mechanical forces can influence the composition and structure of fibrous connective tissues. Acknowledgments The authors thank to Dr. Mary Anne Heidi Dolder and Dr. Jose´ Lino Neto for use of the ultramicrotome and for help during preparation of the specimens, and to Ana Cristina de Moraes for the facilities to obtain the animals. V.L.C. Feitosa was the recipient of a CAPES-PICDT fellowship. References Benjamin, M., Evans, E.J., 1990. Fibrocartilage. J. Anat. 171, 1–15. Benjamin, M., Ralphs, J.R., 1998. Fibrocartilage in tendons and ligaments—an adaptation to compressive load. J. Anat. 193, 481–494. Birk, D.E., Southern, J.F., Zycband, E.I., Fallon, J.T., Trelstad, R.L., 1989. Collagen fibril bundles: a branching assembly unit in tendon morphogenesis. Development 107, 437–443. Carvalho, H.F., Vidal, B.C., 1994a. The unique arrangement of the bullfrog pressure-bearing tendon as an indicative of great deformability. Biol. Cell 82 (1), 59–65. Carvalho, H.F., Vidal, B.C., 1994b. Structure and histochemistry of a pressurebearing tendon of the frog. Ann. Anat. 176, 161–170. Carvalho, H.F., Neto, J.L., Taboga, S.R., 1994. Microfibrils: neglected components of pressure-bearing tendons. Ann. Anat. 176, 155–159. Carvalho, H.F., 1995. Understanding the biomechanics of tendon fibrocartilages. J. Theor. Biol. 172, 293–297.
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