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- MATERIALS AND METHODS
- LITERATURE CITED
Hamstring tendons are a commonly used substitute for anterior cruciate ligament (ACL) reconstruction. Ligaments and tendons are similar in composition but the ACL is more complex than hamstring tendons in function and gross morphology, which are highly dependent on its structure and ultrastructure. The purpose of this study was to compare the morphology and ultrastructure of normal human ACL and hamstring tendons, including the cell type and arrangement, expression level of proteoglycans, diameter, and density of collagen fibrils. Twenty semitendinosus or gracilis tendons and 20 ACL specimens were harvested from patients with ACL rupture or osteoarthritis undergoing routine total knee arthroplasty. The specimens were examined histologically and the ultrastructure was observed using scanning and transmission electron microscopy. Semitendinosus and gracilis tendons showed a homogeneous arrangement of collagen fibers and cell type. They had lower fibril density and more widely distributed fibril diameters. In the ACL, there was a more complex arrangement of collagen fibers, distribution of proteoglycans and different cell types. Electronic microscopy demonstrated a combination of parallel, helical and nonlinear networks of ACL fibrils, and fibril diameters were smaller and more nonuniform. This study compared the anatomy of normal human ACL and hamstring tendons, which may provide a standard for evaluating hamstring tendons grafts after ACL reconstruction and may facilitate the application of hamstring tendons in clinical applications. Anat Rec, 2012. © 2012 Wiley Periodicals, Inc.
ACL tears are an increasingly common sports injury. If untreated, these injuries may result in knee instability and secondary damage like meniscal tears and osteoarthritis, as well as an increased risk of reinjury of the knee (Finsterbush et al., 1990). Surgical reconstruction of the ACL is required to stabilize the joint and to help protect against further injuries in young, active populations (Dunn et al., 2004).
Hamstring tendons are the most commonly used autologous grafts in ACL reconstruction because of their similar composition, both of which are composed of closely packed collagen bundles (Kannus, 2000). However, the gross morphology of the ACL is more complex than hamstring tendons. For example, some researchers have divided ACL into two bundles and suggested using double bundle hamstring tendons to anatomically reconstruct the ACL (Girgis et al., 1975). Meanwhile, others pointed out that the ACL is composed of multiple bundles instead of two bundles, and each with their own morphological patterns (Amis and Dawkins, 1991; Hollis et al., 1991; Amiri et al., 2011).
The ACL bundles are comprised of many small collagen fibers, whose arrangement has not been well described and correlated with those of the hamstring tendons. Structural differences in the tendons might be expected since the functions of the ACL and hamstring tendons are obviously different. A tendon, such as the hamstring tendon, is a tough band of fibrous connective tissue that connects muscle to bone and is capable of transmitting forces and withstanding tension during muscle contraction. In contrast, the ACL is an important restraint to the anterior translation and rotation of the tibia relative to the femur (Markolf et al., 1976; Butler et al., 1980; Welsh, 1980). The ACL is required to withstand multiaxial stresses and regionally different tensile strains, whose distribution is not uniform even along the same bundle (Hirokawa et al., 2001). As function is related to structure, it would be expected that the structure and ultrastructure of collagen fiber bundles would be different between the ACL and hamstring tendons according to their different biomechanical properties.
The structure and ultrastructure of tendon grafts and the normal ACL have previously been investigated (Yahia and Drouin, 1989; Strocchi et al., 1992, 1996), but detailed descriptions of collagen fibers, such as their diameter and density in the ACL and hamstring tendons, are inconsistent (Strocchi et al., 1992; Hadjicostas et al., 2007, 2008). We therefore first examined the morphological and ultrastructural features of ACL and semitendinosus and gracilis tendons as these are candidate graft tissues. Then we examined the differences in cell type and morphology of collagen fibers at the proximal, medial, and distal regions from a single specimen of the ACL or hamstring tendons. Lastly in normal ACL and hamstring tendons, the arrangement, diameters, and densities of collagen fibers, which are important properties of collagen structure, were analyzed. Our findings provide fundamental new information about the structure and ultrastructure of the ACL and tendons that are used as surgical substitutes. These observations may provide a standard for evaluating hamstring tendon grafts after ACL reconstruction and could facilitate the application of these grafts in clinical settings.
- Top of page
- MATERIALS AND METHODS
- LITERATURE CITED
Hamstring tendons are commonly used as a surgical substitute for ACL reconstruction. To achieve optimal anatomical reconstruction and help to restore the function of the ACL, it is important fully understand the structure of the whole ACL and hamstring tendons, the arrangement of collagen fibers and differences in fibril diameter and density.
The ACL and hamstring tendons are composed of cells and extracellular matrix. Type I collagen is the major collagen of the extracellular matrix and is responsible for the tensile stresses of the ACL and tendons (Duthon et al., 2005). The ACL has other types of collagen, including Type III, IX, and XI. The fibroblast is the major cell type in both ACL and tendons. Previous studies and our results showed that ACL has two major cell types. One is fusiform or spindle-shaped fibroblasts with negative toluidine blue staining. The other type is round or oval, resembling fibrochondrocytes with lacunae. (Murray and Spector, 1999; Jiang et al., 2001; Wang and Ao, 2004; Duthon et al., 2005). The chondrocyte-like cell mainly located at the insertion sites and middle part of the ACL. These regions showed stronger positive with toluidine blue staining and were more solid and cartilage-like in nature, which indicates a higher expression level of GAG in ACL than hamstring tendons. Aggrecan and versican, two members of large modular PGs, and their partner hyaluronan are likely to provide a higher capacity to resist compressive forces to tendon tissues (Yoon and Halper, 2005). The differences in these components contributes to the ACL having different histological characteristics from tendons or medial collateral ligaments (MCL) (Jiang et al., 2001). It has also been observed that ACL derived cells have the character of mesenchymal stem cells and could differentiate into adipogenic, osteogenic, and chondrogenic lineages (Furumatsu et al., 2010).
The arrangement of collagen fibers is more complex in the ACL than hamstring tendons as observed by both light and electronic microscopy. Fibers in ACL have two types of wave pattern—an undulated wave pattern, known as “crimp,” and a helical wave pattern. These have been reported in canine patellar tendon and ACL (Yahia and Drouin, 1989). During tensile stretch, fibril “crimp” is first straightened out by small loads, after which larger loads are needed to elongate these fibrils (Duthon et al., 2005). Helical fibrils are presented where the tissue must resist multidirectional or unpredictable loading, or where large, reversible changes in size and shape must be accounted for. A typical example is elastic ligaments, where collagen fibrils waving along elastic fibers are progressively straightened and tensioned whenever the ligament is stretched (Ottani et al., 2001). Unlike the uniform pattern of fibers in hamstring tendons, there were more and wider “gaps” with loosely arranged fibers at the two ends of the ACL. Considering the anatomy and function of the insertion site, this may help to buffer the force from ligament to bone and give space for vessels and cells to grow in.
Proteoglycans represent <3% of the dry weight of ligaments and it has been confirmed that the small leucine-rich proteoglycan, decorin, is the most abundant proteoglycan presented in the matrix of ligaments (Ilic et al., 2005). We found that decorin expressed homogeneously in whole semitendinosus and gracilis tendons while inhomogeneously in ACL. It has been reported that Decorin binds to collagen fibrils and actively participates in fibrillogenesis, and it may slow lateral fibril fusion, which could result in uniformly thinner fibrils under certain conditions (Reed and Iozzo, 2002). The structure of collagen fibers supports the toughness of the ACL, and different distributions of decorin may result in the inhomogeneous diameter by affecting fibrillogenesis in different regions of ACL.
Diameter and density are important properties of collagen structure. These properties are known to correlate with fibril functions (Raspanti et al., 1990). Prior studies have shown that the ultimate tensile strength of fibrils is greater in larger fibrils, while the interfibrillar binding, which depends on their surface areas, increases in the smaller fibrils. Each fibril is therefore likely to fulfill a defined functional role (Ottani et al., 2001). Large and nonuniform fibrils are a typical component of tendons and reticular dermis, whose main function is to resist stretching. In contrast, small and uniform fibrils are often found in tissues which need to withstand multidirectional stresses, such as in vascular or intestinal walls (Raspanti et al., 1990). Both large and small fibrils exist in the ACL (Strocchi et al., 1992). Our results showed that ACL has a major proportion of small fibrils, with large fibrils (>150 nm) comprising only about 10%. In contrast, hamstring tendons have a large proportion of large fibrils, and wider distribution of fibril diameters. These results are consistent with different functions of ALC and tendons, similar to those results previously reported (Hadjicostas et al., 2007, 2008).
Collagen fibril densities are changed in response to repetitive compressive and shear stresses in skin tissue, indicating that collagen density should be another indicator for biomechanical properties of connective tissues (Sanders and Goldstein, 2001). In cross-section, the ACL has a higher but more nonuniform density of fibrils than hamstring tendons. These findings suggest that hamstring tendons used as a substitute may provide enough resistance to tensile strength but ACL has better interfibrillar binding and thus creep resistance.
After surgical implantation, the original hamstring tendon graft undergoes remodeling and neo-ligamentization. In this process, the graft regains a blood supply and cells increase and relocate. The collagen fibers synthesized by the cells remodel under the prevailing stresses and therefore the ultrastructure of the grafts change during the process (Arnoczky et al., 1982; Hunt et al., 2005). In consideration of the substantial differences in ultrastructure between these two kinds of tissues, how the tissues remodel and adapt to the new environment is a clinical concern. Several studies have been done on the ultrastructural changes during graft remodeling and differences fibril diameter between the regenerated tissue and the normal semitendinosus tendon graft have been reported (Yoshiya et al., 2004). It has been shown that the hamstring tendons undergo remodeling during the first 2 years after surgery but do not completely resemble the ultrastructure of a normal ACL up to 10 years later (Zaffagnini et al., 2009). So the ultrastructure after surgical replacement might therefore be different from both normal ACL and hamstring tendons, and this could have an effect on ultimate function of the graft. Further studies, however, will be required to define changes in ultrastructure with time in grafted tissues.
The results presented here should to be considered with some caution due to the different ages of ACL and hamstring tendons donors used in this study. Prior studies have documented age-related structural changes in these connective tissues. For example, the diameters of the collagen fibers of human Achilles' tendon were reported to be greater in the 20- to 29-year-old group compared to other groups (Sargon et al., 2005). Additionally in human ACL, fibril diameter was related to the aging process, but the differences were more obvious between young (<20 years) and adult or the elderly (Strocchi et al., 1996). Our previous study indicated that the distribution trend of fibril diameter in adult and elder patients were similar (Jiao et al., 2010).
In conclusion, we compared the ultrastructure and morphology of human hamstring tendons and the anterior cruciate ligament. We demonstrated that the ACL has different cell types, a higher expression level of GAG, a more complex morphology of collagen fibers and distribution of decorin compared to hamstring tendons. The hamstring tendons showed more large fibrils and lower fibril density than the ACL. This work may provide fundamental information on these tissues that may be useful in the application of tendons in the clinical reconstruction of the ACL following injuries.