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Microstructure of the ligament-to-bone attachment complex in the human knee joint
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Research paper
Microstructure of the ligament-to-bone attachment complex in the human knee joint Damien Subit a,b,∗ , Catherine Masson a,1 , Christian Brunet a,2 , Patrick Chabrand c,3 a Laboratoire de Biomécanique Appliquée, Faculté de Médecine Secteur Nord, Boulevard Pierre DRAMARD, 13916 Marseille Cedex 20, France b Laboratoire de Mécanique et d’Acoustique, 31 chemin Joseph-Aiguier, 13402 Marseille Cedex 20, France c Laboratoire d’Aérodynamique et de Biomécanique du Mouvement, Parc Scientifique et Technologique de Luminy, Université de la
Méditerranée et CNRS, Case Postale 918, 13288 Marseille, France
A R T I C L E
I N F O
A B S T R A C T
Article history:
Clinical and experimental studies have shown that injuries in the human knee ligaments
Received 24 July 2007
occur in the ligament midsubstance, at the transition between bone and ligament, and
Received in revised form
in the bone in the vicinity of the ligament-to-bone attachment site. Whereas ligament
21 November 2007
and bone have been thoroughly described, the way they connect to each other remains
Accepted 6 February 2008
unclear. The goal of this study is to provide a description of the microstructure of the
Published online 13 February 2008
ligament-to-bone insertion, with the view of providing a mechanical model capable of
Keywords: Ligament-to-bone attachment Fibrocartilage Microstructure Knee Collagen fibres Mechanical function
predicting the injuries that occur at this insertion. The preparatory literature review showed that there was no description of the insertion microstructure for the human ligaments. The results found for human tendons and animal tendons/ligaments were used to lead the histological and electron — scanning and transmission — microscopy analysis. The posterior cruciate ligament (PCL), and the lateral collateral ligament (LCL) were sampled from one post mortem human subject. Slices were cut along the longitudinal direction of the ligaments, following the fibers direction. The histology analysis showed that the insertion has the same structure as reported in the literature: it is made of a mineralization front between calcified and uncalcified fibrocartilage, which is not crossed by the ligament fibers. The transmission electron microscopy analysis of the calcified fibrocartilage revealed a collagenous structure which has a direction drastically different from the direction of the ligament fibers. The mechanical function of the insertion was discussed and combined with the histological findings to hypothesize the microstructure of the insertion. c 2008 Elsevier Ltd. All rights reserved.
∗ Corresponding author at: University of Virginia, Center for Applied Biomechanics, 1011 Linden avenue, Charlottesville, VA, 22902, USA. Tel: +1 434 296 7288, fax: +1 434 296 3453. E-mail addresses: [email protected] (D. Subit), [email protected] (C. Masson), [email protected] (C. Brunet), [email protected] (P. Chabrand). URLs: http://www.lma.cnrs-mrs.fr, http://www.centerforappliedbiomechanics.org (D. Subit), http://www.inrets.fr (C. Masson), http://www.inrets.fr (C. Brunet), http://www.labm.univ-mrs.fr (P. Chabrand). 1 Tel.: +33 4 91 65 80 15; fax: +33 4 91 65 80 19. 2 Tel.: +33 4 91 65 80 00; fax: +33 4 91 65 80 19. 3 Tel.: +33 4 91 26 62 38; fax: +33 4 91 41 16 91. c 2008 Elsevier Ltd. All rights reserved. 1751-6161/$ - see front matter doi:10.1016/j.jmbbm.2008.02.002
J O U R N A L O F T H E M E C H A N I C A L B E H AV I O R O F B I O M E D I C A L M AT E R I A L S
Nomenclature PCL LCL FOV OM SEM TEM HES MB CFC UFC ECM PMHS
1.
Posterior cruciate ligament Lateral collateral ligament Field of view Optical microscopy Scanning electron microscopy Transmission electron microscopy Haemotoxylin-eosin-saffron Methylene blue Calcified fibrocartilage Uncalcified fibrocartilage Extra cellular matrix Post mortem human subject
Introduction
The structural and material properties of human knee ligaments have been studied for decades, with a focus on injuries caused by impacts, such as sport and road accident related injuries. Experimental and clinical studies of the knee ligament have shown that injuries occur in the ligament itself (ligament midsubstance), in the bone or at the ligament-tobone insertion site (Danto and Woo, 1993; Lee and Hyman, 2002; Noyes et al., 1974). While the ligament and bone structures are well known, the way the ligament inserts into the bone remains obscure. The location of the failure is likely to be explained by the specific structural and material properties of each component (ligament, bone, transition) and how they interact. The dependency of the failure location to the strain rate has been observed experimentally (Arnoux et al., 2005; Lee and Hyman, 2002; Noyes et al., 1974; Subit, 2004), but has not been linked yet to the material properties of the components of the bone-ligament-bone complex. The failure mechanisms in the ligament midsubstance and in the bone have been reported and used to develop the computational models currently utilized to predict injuries (Arnoux et al., 2002; Cardot et al., 2006; Pioletti et al., 1998; Pithioux et al., 2004; Weiss and Gardiner, 2001). However, these models do not account for the mechanical response of the insertion site, which is generally represented as a sudden transition from ligament material to bone material (Puso and Weiss, 1998; Song et al., 2004; Thomopoulos et al., 2006): the mechanical response of the insertion site was described by looking at the stress concentration due to the sudden change in material stiffness (Matyas et al., 1995; Thomopoulos et al., 2006). Indeed, the insertion is the transition between a soft tissue – the ligament – which can stretch of about 10% before failure, and a hard tissue – the bone – which can bear only about 1% of stretch before failure. The insertion is then an astonishing structure from the mechanical point of view. The literature review set about showed that there is no description of the microstructure of the human ligament-tobone insertion. Therefore this review was extended to human tendon,4 because of the similarities between ligament and 4 The term enthesis is widely used to refer to the ligament-tobone junction, even though the word is devoted to the tendon-tobone attachment in anatomy.
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tendon anatomy and function. As a matter of fact, tendon and ligament are often considered identical in the literature (Benjamin et al., 2002; Cooper and Misol, 1970; Evans et al., 1990; Schneider, 1956). Furthermore, animal ligaments studies were as well included. Schneider (1956) and Cooper and Misol (1970) gave the first description of the insertion site (dog patellar tendon and lateral collateral ligament), later confirmed by Benjamin et al. (1986) for human tendon insertions. It is divided in 4 layers: the ligament or tendon, the uncalcified fibrocartilage (UFC), the calcified fibrocartilage (CFC), and the bone. The transition line between uncalcified and calcified fibrocartilage is named tidemark or blue line, this is the mineralization front. The transition line between the calcified fibrocartilage and the bone is named cement line (Fig. 1). The tidemark is a moving mineralization front (human Achilles tendon (Milz et al., 2002), rat Achilles tendon (Rufai et al., 1996)), there is no change in the constituting materials. Besides the mineralization, the difference between UFC and CFC is that the collagen fibres in the latter are not crimped (quadriceps tendon fibers insertion into the patellae of adult rabbits, humans, dogs and sheep (Cooper and Misol, 1970)). The actual frontier between bone and tendon/ligament is the cement line (rabbit knee joint (Gao and Messner, 1996)). The collagen fibres do not cross the cement line (Gao et al., 1996; Milz et al., 2002). Clark and Stechschulte (1998) observed that the tendinous fibres interdigitate with the bone lamellar structure, without merging with them however. A molecular glue could be responsible for binding them together. Some authors attempted to relate the mechanical role of the insertion to its structure. For Schneider (1956), the fibrocartilage prevents the tendon from narrowing when loaded, so that it does not damage the bony part of the insertion. It was suggested similarly that the fibrocartilage would ensure a gradual transition (Knese and Biermann, 1958) from the ligament, which is very soft (E = 20–70 MPa (Arnoux, 2000; Pioletti, 1997)), to the bone (E = 10 000–20 000 MPa (Yamada, 1970)), and even that the insertion has the ability to adapt itself to the load by modifying the amount of calcified tissue (Gao and Messner, 1996) and the layout of the fibres (Rufai et al., 1996). The tendon or ligament fibres can penetrate the UFC at various angles relative to the surface of the UFC (Benjamin et al., 1986): the UFC ensures that the tendon does not bend when it meets the bone, and that the collagen fibres reach the tidemark at an angle approaching a right angle. Therefore, the mechanical function of the UFC would be to offer some protection from wear and tear to the tendon/ligament fibres, by preventing them from being bent, splayed out or compressed during the motion of the joint to which the tendon is attached. Thus the ligament-to-bone attachment would be a way to reduce the mechanical loading transmitted by the ligament to bone. This paper presents the results of the study performed on the knee ligament-to-bone attachment in human to determine its microstructure. The goal was to choose a mechanical model capable of describing its behaviour and its failure (Arnoux et al., 2005; Subit, 2004). Optical microscopy, scanning and transmission electron microscopy devices were used to look at the knee ligament insertion sites in the knee joint of a Post Mortem Human Subject (PMHS). The preparation of the samples is first described, followed by
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Fig. 2 – Overview of the femoral insertion of a LCL (OM, HES staining, FOV width: 21 mm). Fig. 1 – Tendinous and ligamentous insertions (based on Cooper and Misol (1970) and Schneider (1956)).
the results obtained for each device. As a starting point, the microstructure of the insertion site of the ligaments in the human knee was compared to the microstructure reported on in the literature. Combining an in-depth analysis of the histology results and the mechanical function of the insertion, some assumptions are put forward as for its microstructure in the discussion section, to propose a description of the insertion that can be used in computational modeling.
2.
2.2.
The SEM samples were prepared in a similar manner, except that the ligament-bone units were fixed by immersion in a 2.5% glutaraldehyde solution buffered with 0.1 M phosphate mono disodium, pH 7.4. Samples for scanning were collected in two ways. First, the ligament-bone unit was frozen and the samples for scanning were created by manually fracturing the unit in the fibre direction to expose a smooth surface of the ligament-bone interface. Second, samples were created at room temperature by manually tearing the ligamentbone unit in the fibre direction to expose the ligament-bone interface. Finally, the exposed surfaces were coated with gold for scanning.
Materials and methods 2.3.
Ligament samples were harvested from a single human cadaver (age 64) preserved in a Winckler solution (Winckler, 1974). Ligament-bone units were created by taking full-width strips approximately 30 mm long from the tibial and femoral insertion sites of the Posterior Cruciate Ligament (PCL), and the fibular and femoral insertion sites of the Lateral Collateral Ligament (LCL). The strips were then divided to be prepared for optical microscopy, and scanning and transmission electron microscopy. The articular surfaces of the knee joint were confirmed to be devoid of gross pathological change.
2.1.
Scanning electron microscopy
Optical microscopy
The ligament-bone units were fixed at room temperature by immersion for several hours in a 2.5% formaldehyde solution buffered with 0.1 M phosphate, pH 7.4. Following fixation, each unit was washed in the same solution, and dehydrated by dipping in several ethanol solutions of decreasing concentration. After dehydration, the units were dipped in an organic solvent, and embedded in paraffin wax. Full-thickness slices 5 µm wide were cut along the direction of the ligament fibres using a microtome and mounted on glass slides. The slices were then stained either with haematoxylin-eosin-saffron (HES) or with methylene blue (MB).
Transmission electron microscopy
Blocks of the ligament-bone units about 1 mm3 were fixed in a 2.5% glutaraldehyde solution buffered with 0.1 M phosphate mono disodium, pH 7.4. The blocks were then washed in the same solution, and postfixed in buffered 2% osmium tetraoxide. The samples were then dehydrated using graded ethanol solutions and propylene oxide, and soaked in a graded solution of epon/propylene oxide resin to initiate their embedding in this resin, and finally embedded. Once the resin had polymerized, slices of the bone-ligament units 90 nm thick were cut along the fibre direction and placed on a grid. Next, the samples were stained successively with uranyl acetate and lead citrate.
3.
Results
3.1.
Optical microscopy
The overview of a slice (Fig. 2) shows the ligament (L) and the decalcified trabecular bone (B). The box corresponds to the ligament-to-bone insertion site. Fig. 3 is a closer view of the boxed part (higher magnification), for a PCL. The bone is discernable thanks to the osteons (left side). On the right side of the picture are
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Fig. 3 – Tibial insertion of a PCL (OM, MB staining, ×10, FOV width: 0.859 mm).
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Fig. 4 – Femoral insertion of a LCL (OM, HES staining, × 100, FOV width: 2.65 mm).
the collagen fibres. The rows of chondrocytes (Ch) between the collagen fibres show the cartilage-like structure. This part of the ligament can be identified as uncalcified fibrocartilage (UFC). The darkening of the tissue due to the staining used for this slice shows the calcification of the fibrocartilage. The fibre structure is still visible, justifying the name of calcified fibrocartilage (CFC). The CFC zone is 580 ± 163 µm thick (average ± std). The closer view of a LCL shows the same pattern: bone, CFC, UFC and ligament (Fig. 4). The thinner arrow shows the bone/CFC border, the thicker arrow shows the CFC/UFC boarder. A line of chondrocytes (encircled) indicates the cartilage-like structure of the part of ligament adjacent to the ligament-to-bone transition. The CFC zone is about 340 ± 67 µm thick.
3.2.
Scanning electron microscopy
The cortical layer (C) separates the ligament (L) and the bone (B), but is not crossed by ligament fibres (Fig. 5). The box shows the location where the ligament attaches to the bone.
3.3.
Transmission electron microscopy
The mineralization front is scattered with cells which identified as osteoblasts because of their rounded shape and their size. The ECM was not fibrous, and some particles of mineral matter could be identified. The collagen fibres (CgF, Fig. 6a) do not stop at the mineralization front. However their organization changes when they cross the mineralization front (FM, Fig. 6a): the bundles of fibres wrap around another structure which looks orthogonal to them. This structure is referred to as ‘orthogonal fibres’ (OF, Figs. 6 and 7). The orthogonal fibres are in the calcified part of the fibrocartilage. Going deeper in the CFC, the organization of the collagen and orthogonal fibres is more obvious (Fig. 7): they intertwine to create a grid/mesh in the CFC. The observation at higher magnification shows that the orthogonal fibres are collagenous.
Fig. 5 – Tibial insertion of a PCL: L, ligament; C, cortical layer; B, bone (SEM). Scale bar = 2 mm.
4.
Discussion
The microstructure of the PCL and LCL attachment complex is investigated in this study. It is not certain that these results can be extended to the medial collateral ligament and anterior cruciate ligament. Indeed, Benjamin et al. (2002) suggested that the structure of the enthesis depended on the osteogenesic mechanism and the mechanical loading, which is somehow specific to each ligament.
4.1.
The materials at the transition
The results from the optical microscopy showed that there is no cortical bone at the location where the ligament fibres attach to the bone (also confirmed by SEM), and that the ligament-to-bone transitional materials were the UFC and CFC. The structure of the ligament-to-bone transition for the human knee ligaments at the optical microscopy level is identical to the structure described in the literature for the human tendon and animal ligaments (Fig. 1). However, the thicknesses are different. Cooper and Misol (1970) reported a CFC thickness of 100–300 µm for the dog patellar and the lateral collateral ligaments insertion sites, without any
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Fig. 6 – Mineralization front in the femoral insertion of a PCL (TEM). Scale bar= 5 µm. (a) View of the whole mineralization front, (b) Close-up of the the orthogonal fibres.
Fig. 7 – Whorl of collagen fibrils around the orthogonal fibres, femoral insertion of a PCL (TEM). OF, orthogonal fibres; CgF, collagen fibres (TEM). Scale bar = 5 µm.
Fig. 8 – Hypotheses about the attachment of the orthogonal fibres and collagen fibres to the bone. (a) Attachment to the periosteum or cortical bone, (b) Attachment to the trabecular bone. TB: trabecular bone, CgF: collagen fibre, OF: orthogonal fibres, P: periosteum, CB: cortical bone.
details about the location where the measurements were taken. In the current study, the average thicknesses are greater (PCL: 590 µm, LCL: 340 µm). The measurements in this study were performed on one subject at various locations and averaged. They are too few data points in this study
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to perform a statistical analysis of the thickness repartition. The variation of the thickness as a function of the position in the insertion was not further investigated. To the best of our knowledge, it is the first time that the thickness of the PCL and LCL insertions sites are reported, thus no data are available for comparison. It would nevertheless not be surprising to observe different thicknesses for the dog and the human insertion sites. These differences may be due either to the adaptation of the tissue to the loading (different bone remodelling process different for the dog and the human beings), to aging (the specimens used by Cooper and Misol (1970) were likely younger relative to the dog life expectancy, whereas the samples in this study were taken from an elderly), or to the physiological function of the ligament and the loading they bear (quadruped vs. biped). Rufai et al. (1996) reported that the cement line which delimits the CFC and UFC areas (Fig. 1) is a moving line, revealing that this area of the insertion is very active (for the rat). This was confirmed to be the same for the ligaments in this study (presence of osteoblasts in the mineralization front). Evans et al. (1990) mentioned that the CFC was “several” micrometers thick, without providing quantitative results (for human). The thickness of the CFC layer is likely to be function of the location where it is measured: Gao and Messner (1996), (rabbit knee ligaments), Kapandji (1987), Müller (1982), and Evans et al. (1990) (human knee joint) hypothesized that the thickness of the CFC increases to adapt to the increase in the mechanical loading borne by the ligament, in particular the direction of the loading. Benjamin et al. (1991) suggested that the greater thickness of UFC in the lateral meniscus compared to the medial one in the human knee joint was linked to its greater mobility (average values of 0.23 mm for the lateral meniscus, and 0.14 mm for the medial meniscus). The CFC would be thicker for the ligaments peripheral to the knee joint (lateral and medial collateral ligaments), for which the range of motion is greater than for the ligaments closer to the center of rotation of the knee (posterior and anterior cruciate ligaments). The averaged thicknesses measured in this study, which are considered as a first estimate, are larger for the PCL than the LCL, and thus do not follow these assumptions. Meanwhile, Benjamin et al. highlighted the considerable heterogeneity in the different tendons they studied, in particular between direct attachment to the bone and attachment in a synovial joint, and showed that the fibrocartilage has the capability to adapt its structure to the type of motion that it bears, such as the friction with the adjoining bone, or the dynamic behaviour of the joint it is attached to Benjamin et al. (1993, 1995). The PCL is inside the synovial knee joint, whereas the LCL is outside, and the loading patterns during the knee motion are different for each of them, which could explain the differences in fibrocartilage thickness. Finally, it has been shown that embalming could alter material properties (Crandall, 1994), but it is likely that it does not affect the geometry of the microstructure in this structural analysis.
4.2.
The structure of the transition
Gao et al. (1996) reported that the ligamentous collagen fibres attach to the bone at the cement line, without merging
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with the bone (rabbit ligament-to-bone insertion). Likewise, Clark and Stechschulte (1998) observed that the tendon fibres ended against lamella in the lamellar bone or interdigitated between the lamellar structure, without merging with the lamella or splaying into the lamellar structure (rabbit, human, dog, sheep). Therefore, the collagen fibres are not continuous from the ligament/tendon to the bone. However, the way the ligament collagen fibres interweave with the lamella has not been observed yet. Milz et al. (2002) noticed that bone and ligament interlock with each other in a complex manner, and suggested that the degree of interlocking could explain the mechanism by which the Achilles tendon attaches to the bone in human. They envisaged that pieces of CFC could fit into the bone like the pieces of a jigsaw, which would make the bone-CFC coupling stronger as the loading of the insertion increases (when it becomes critical to protect the insertion site). The results of TEM (Fig. 7) showed a grid in the CFC, grid composed of the ligament collagen fibres and the collagen fibres referred to as orthogonal fibres (OF). The darker spots (more numerous on the left-hand side of the Fig. 7) show that the tissue is gradually calcified. This layout was observed in the CFC. To some extent, it supports the hypothesis proposed by Milz et al. (2002): there is a geometrical interaction between the materials composing the ligament-to-bone attachment that makes the transition stronger. Based upon the results of this study, combined with the mechanical role of the attachment site, some realistic patterns are proposed for the geometry insertion site (Fig. 8). It was shown that there is no cortical bone at the insertion site: the orthogonal fibres could ensure the continuity of the cortical bone (Benjamin et al., 2002) (Fig. 8(a)), or attach directly to the underlying bone (Fig. 8(b)) while providing a strong attachment to the ligament fibres. The two concepts have in common that the orthogonal fibres are mechanically loaded mainly in bending, which makes them less sensitive to the direction of loading than a pure tension load. Indeed, if the end of the fibres was directly embedded in the bone (without the orthogonal fibres, Fig. 9), the tension in the fibre would be in the direction of the sealing of the fibre with the bone, loading it along its weakest direction. The crosssection of a loaded collagen fibre shrinks (the ligament at the insertion has an hourglass shape, Fig. 9); consequently the bone-collagen fibres interface has to bear a large load (shear and tension). This kind of attachment is mechanically weak, especially when a soft tissue is involved. Furthermore, it is very sensitive to the direction of the loading: the fibre has to bend at the ligament-to-bone transition which creates a stress concentration and can eventually lead to a premature failure. In the structures illustrated in Fig. 8, the orthogonal fibres, embedded in a cartilaginous matrix, would transmit the load, and consequently share it with the nearby structures (cortical bone, periosteum). As Schneider (1956) suggested it for the CFC, this type of attachment prevents the fibres from narrowing when loaded. Such a structure for the insertion site supports the idea that the collagen fibers do not cross the mineralisation front between the ligament and the bone. It has been shown that the continuity of the collagen fibres through the insertion site was not possible for tissues development reasons (Gao and Messner, 1996; Milz et al., 2002). The results presented
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Fig. 9 – Direct attachment of a collagen fibre (CgF) to the bone, (a) Unloaded and (b) Loaded ligament fibre. The contact interface between the fibre and the bone is damaged because the fibre shrinks when loaded.
here show that the non continuity of the fibres, with a grid of collagen fibres in the CFC, provides actually a stronger attachment from the mechanical point of view. At that point, the next step in the description of the ligament-to-bone attachment is to confirm that the OF are actually fibres, and to characterize (1) how the OF attach to the bone, and (2) how the collagen fibres in the ligament attach to the OF. It was assumed here that the collagen fibres wrap around the orthogonal fibres, but this could involve other kind of attachment such as glue, or interdigitation of fibrils. As for the failure, it was reported a rate sensitivity of the failure location to the loading (Arnoux et al., 2005; Danto and Woo, 1993; Pioletti et al., 1998; Subit, 2004). The grid of collagen fibres, embedded in the fibrocartilage the mechanical response of which is rate sensitive (viscoelasticity), has a complex mechanical response which is as well rate-sensitive. The findings of this study at the microscopic level are in line with the macroscopic level response. As a consequence, the ligament failure is the ‘competition’ between failure in the ligament, in the bone or at the insertion site, depending of the various rate sensitivities of each of these components (Subit, 2004). Besides, the small thickness of the ligamentto-bone insertion and the combined loading that it has to bear (shear, tension) argue for the use of a mechanical model developed for interfaces, known as cohesive zone model (Raous et al., 1999). This model accounts for damage, friction, rate sensitivity and failure. Its capability to describe ligament failure under quasi-static and dynamic loading has been shown in recent works (Arnoux et al., 2005; Subit et al., in revision; Subit, 2004).
5.
Conclusion
This study is the first to report the microstructure of the ligament-to-bone attachments for the LCL and PCL in the human knee joint. Based on the light microscopy results, it was established that the structure is similar to the structure described in the literature for human and animal tendons and ligaments: calcified and uncalcified fibrocartilage are the materials of the insertion. SEM results showed that there is not continuity of the collagen fibres across the insertion. TEM results revealed a change in the structure as well: in the plan of slices (sagittal plan of the ligament), a collagenous structure which has an other orientation than the ligament
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fibres, was observed. Based on mechanical arguments, it was speculated that this structure could be a grid of collagenous fibres (referred to as orthogonal fibres) embedded in the cartilage matrix of the insertion, to which the ligament fibres would attach. The attachment to the orthogonal fibres, rather than to the bone itself, provides the insertion with a better resistance to the load applied through the ligament while being less sensitive to the direction of the loading (shear and tensile loading). This structure needs to be confirmed by repeating the study on other specimens, and observing slices cut in other directions, to assess the presence of the orthogonal fibres in every subject and better describe their geometry and assembly. Besides, only the PCL and LCL were investigated; it is unclear how these results can be extrapolated to the other knee ligaments. Moreover, if the orthogonal fibres happen to be routinely observed, it would be required to investigate how the ligament fibres attach to them, and how the orthogonal fibers attach to the bone. That could be done by a tridimensional reconstruction of the insertion site, to scrutinize if there is any wrapping and interweaving of the fibres, and by histochemical analysis to evaluate the presence of a molecular glue.
Acknowledgements The authors would like to thank their colleagues from Faculté de Médécine de Marseille (Timone): Hubert Lépidi, for his help to perform the study in optical microscopy; Jacques Dejou, Imad About, Claude Alésia, and Hélène Borghi, for their comments and the access to their facilities, and Mireille Rémusat for her support to comment on the results from the histological point of view. Their contribution to this biomechanics study at the border between mechanics and medical science is greatly appreciated.
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Benjamin, M., Ralphs, J.R., Newell, R.L., Evans, E.J., 1993. Loss of the fibrocartilaginous lining of the intertubercular sulcus associated with rupture of the tendon of the long head of biceps brachii. J. Anat. 182 (2), 281–285. Cardot, J., Masson, C., Arnoux, P.J., Brunet, C., 2006. Finite element analysis of cyclist lower limb response in car–bicycle accident. Internat. J. Crashworthiness 11, 115–130. Clark, J., Stechschulte, D.J.J., 1998. The interface between bone and tendon at an insertion site: A study of the quadriceps tendon insertion. J. Anat. 192 (4), 605–616. Cooper, R.R., Misol, S., 1970. Tendon and ligament insertion. A light and electron microscopic study. J. Bone Joint Surg. Am. 52, 1–20. Crandall, J., 1994. The preservation of human surrogates for biomechanical studies. Ph.D. Dissertation, University of Virginia. Danto, M.I., Woo, S.L., 1993. The mechanical properties of skeletally mature rabbit anterior cruciate ligament and patellar tendon over a range of strain rates. J. Orthop. Res. 11, 58–67. Evans, E.J., Benjamin, M., Pemberton, D.J., 1990. Fibrocartilage in the attachment zones of the quadriceps tendon and patellar ligament of man. J. Anat. 171, 155–162. Gao, J., Messner, K., 1996. Quantitative comparison of soft tissuebone interface at chondral ligament insertions in the rabbit knee joint. J. Anat. 188 (2), 367–373. Gao, J., Räsänen, T., Persliden, J., Messner, K., 1996. The morphology of ligament insertions after failure at low strain velocity: An evaluation of ligament entheses in the rabbit knee. J. Anat. 189 (1), 127–133. Kapandji, I.A., 1987. Lower Limb: The Knee. In: The Physiology of Human Joints. Annotated Diagrams of the Mechanics of the Human Joints, vol. 2. Churchill Livingstone, pp. 66–147. Knese, K.H., Biermann, H., 1958. Osteogenesis in tendon and ligament insertions in the area of the original chondral apophyses. Z. Zellforsch. Mikrosk. Anat. 49, 142–187. Lee, M., Hyman, W., 2002. Modeling of failure mode in knee ligaments depending on the strain rate. BMC Musculoskeletal Disorders 3 (3). http://www.biomedcentral.com/1471-2474/3/3. Matyas, J.R., Anton, M.G., Shrive, N.G., Frank, C.B., 1995. Stress governs tissue phenotype at the femoral insertion of the rabbit MCL. J. Biomech. 28, 147–157. Milz, S., Rufai, A., Buettner, A., Putz, R., Ralphs, J.R., Benjamin, M., 2002. Three-dimensional reconstructions of the Achilles tendon insertion in man. J. Anat. 200, 145–152. Müller, W., 1982. Das Knie - Kinematics. Spinger Verlag, Berlin. Noyes, F.R., DeLucas, J.L., Torvik, P.J., 1974. Biomechanics of anterior cruciate ligament failure: an analysis of strain-rate sensitivity and mechanisms of failure in primates. J. Bone Joint Surg. Am. 56, 236–253. Pioletti, D.P., 1997. Viscoelastic properties of soft tissues: Application to knee ligaments and tendons. Ph.D. Dissertation, Ecole polytechnique de Lausanne. Pioletti, D.P., Rakotomanana, L.R., Benvenuti, J.F., Leyvraz, P.F., 1998. Viscoelastic constitutive law in large deformations: Application to human knee ligaments and tendons. J. Biomech. 31, 753–757. Pithioux, M., Subit, D., Chabrand, P., 2004. Comparison of compact bone failure under two different loading rates: Experimental and modelling approaches. Med. Eng. Phys. 26, 647–653. Puso, M.A., Weiss, J.A., 1998. Finite element implementation of anisotropic quasi-linear viscoelasticity using a discrete spectrum approximation. J. Biomech. Eng. 120, 62–70. Raous, M., Cangemi, L., Cocou, M., 1999. A consitent model coupling adhesion, friction and unilateral contact. Comput. Methods Appl. Mech. Eng. 177, 383–399. Rufai, A., Ralphs, J.R., Benjamin, M., 1996. Ultrastructure of fibrocartilages at the insertion of the rat Achilles tendon. J. Anat. 189 (1), 185–191.
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Schneider, H., 1956. Zeitschrift für Anatomie und Entwicklungsgeschichte (Structure of tendon attachments). Z. Anat. Entwicklungsgesch 119, 431–456. Song, Y., Debski, R.E., Musahl, V., Thomas, M., Woo, S.L., 2004. A three-dimensional finite element model of the human anterior cruciate ligament: A computational analysis with experimental validation. J. Biomech. 37, 383–390. Subit, D., 2004. Modélisation de la liaison os-ligament dans l’articulation du genou. Ph.D. Dissertation, Université de la Méditerranée. Subit, D., Chabrand, P., Masson, C., A micromechanical model to predict damage and failure in biological tissues. Application
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to the ligament-to-bone attachment in the human knee joint. Journal of Biomechanics (in revision). Thomopoulos, S., Marquez, J.P., Weinberger, B., Birman, V., Genin, G.M., 2006. Collagen fiber orientation at the tendon to bone insertion and its influence on stress concentrations. J. Biomech. 39, 1842–1851. Winckler, G., 1974, Manuel d’anatomie topographique et fonctionnelle. Masson ed. Weiss, J.A., Gardiner, J.C., 2001. Computational modeling of ligament mechanics. Crit. Rev. Biomed. Eng. 29, 303–371. Yamada, H., 1970. Strength of Biological Materials. Williams & Wilkins.
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journal homepage: www.elsevier.com/locate/jmbbm
Research paper
Microstructure of the ligament-to-bone attachment complex in the human knee joint Damien Subit a,b,∗ , Catherine Masson a,1 , Christian Brunet a,2 , Patrick Chabrand c,3 a Laboratoire de Biomécanique Appliquée, Faculté de Médecine Secteur Nord, Boulevard Pierre DRAMARD, 13916 Marseille Cedex 20, France b Laboratoire de Mécanique et d’Acoustique, 31 chemin Joseph-Aiguier, 13402 Marseille Cedex 20, France c Laboratoire d’Aérodynamique et de Biomécanique du Mouvement, Parc Scientifique et Technologique de Luminy, Université de la
Méditerranée et CNRS, Case Postale 918, 13288 Marseille, France
A R T I C L E
I N F O
A B S T R A C T
Article history:
Clinical and experimental studies have shown that injuries in the human knee ligaments
Received 24 July 2007
occur in the ligament midsubstance, at the transition between bone and ligament, and
Received in revised form
in the bone in the vicinity of the ligament-to-bone attachment site. Whereas ligament
21 November 2007
and bone have been thoroughly described, the way they connect to each other remains
Accepted 6 February 2008
unclear. The goal of this study is to provide a description of the microstructure of the
Published online 13 February 2008
ligament-to-bone insertion, with the view of providing a mechanical model capable of
Keywords: Ligament-to-bone attachment Fibrocartilage Microstructure Knee Collagen fibres Mechanical function
predicting the injuries that occur at this insertion. The preparatory literature review showed that there was no description of the insertion microstructure for the human ligaments. The results found for human tendons and animal tendons/ligaments were used to lead the histological and electron — scanning and transmission — microscopy analysis. The posterior cruciate ligament (PCL), and the lateral collateral ligament (LCL) were sampled from one post mortem human subject. Slices were cut along the longitudinal direction of the ligaments, following the fibers direction. The histology analysis showed that the insertion has the same structure as reported in the literature: it is made of a mineralization front between calcified and uncalcified fibrocartilage, which is not crossed by the ligament fibers. The transmission electron microscopy analysis of the calcified fibrocartilage revealed a collagenous structure which has a direction drastically different from the direction of the ligament fibers. The mechanical function of the insertion was discussed and combined with the histological findings to hypothesize the microstructure of the insertion. c 2008 Elsevier Ltd. All rights reserved.
∗ Corresponding author at: University of Virginia, Center for Applied Biomechanics, 1011 Linden avenue, Charlottesville, VA, 22902, USA. Tel: +1 434 296 7288, fax: +1 434 296 3453. E-mail addresses: [email protected] (D. Subit), [email protected] (C. Masson), [email protected] (C. Brunet), [email protected] (P. Chabrand). URLs: http://www.lma.cnrs-mrs.fr, http://www.centerforappliedbiomechanics.org (D. Subit), http://www.inrets.fr (C. Masson), http://www.inrets.fr (C. Brunet), http://www.labm.univ-mrs.fr (P. Chabrand). 1 Tel.: +33 4 91 65 80 15; fax: +33 4 91 65 80 19. 2 Tel.: +33 4 91 65 80 00; fax: +33 4 91 65 80 19. 3 Tel.: +33 4 91 26 62 38; fax: +33 4 91 41 16 91. c 2008 Elsevier Ltd. All rights reserved. 1751-6161/$ - see front matter doi:10.1016/j.jmbbm.2008.02.002
J O U R N A L O F T H E M E C H A N I C A L B E H AV I O R O F B I O M E D I C A L M AT E R I A L S
Nomenclature PCL LCL FOV OM SEM TEM HES MB CFC UFC ECM PMHS
1.
Posterior cruciate ligament Lateral collateral ligament Field of view Optical microscopy Scanning electron microscopy Transmission electron microscopy Haemotoxylin-eosin-saffron Methylene blue Calcified fibrocartilage Uncalcified fibrocartilage Extra cellular matrix Post mortem human subject
Introduction
The structural and material properties of human knee ligaments have been studied for decades, with a focus on injuries caused by impacts, such as sport and road accident related injuries. Experimental and clinical studies of the knee ligament have shown that injuries occur in the ligament itself (ligament midsubstance), in the bone or at the ligament-tobone insertion site (Danto and Woo, 1993; Lee and Hyman, 2002; Noyes et al., 1974). While the ligament and bone structures are well known, the way the ligament inserts into the bone remains obscure. The location of the failure is likely to be explained by the specific structural and material properties of each component (ligament, bone, transition) and how they interact. The dependency of the failure location to the strain rate has been observed experimentally (Arnoux et al., 2005; Lee and Hyman, 2002; Noyes et al., 1974; Subit, 2004), but has not been linked yet to the material properties of the components of the bone-ligament-bone complex. The failure mechanisms in the ligament midsubstance and in the bone have been reported and used to develop the computational models currently utilized to predict injuries (Arnoux et al., 2002; Cardot et al., 2006; Pioletti et al., 1998; Pithioux et al., 2004; Weiss and Gardiner, 2001). However, these models do not account for the mechanical response of the insertion site, which is generally represented as a sudden transition from ligament material to bone material (Puso and Weiss, 1998; Song et al., 2004; Thomopoulos et al., 2006): the mechanical response of the insertion site was described by looking at the stress concentration due to the sudden change in material stiffness (Matyas et al., 1995; Thomopoulos et al., 2006). Indeed, the insertion is the transition between a soft tissue – the ligament – which can stretch of about 10% before failure, and a hard tissue – the bone – which can bear only about 1% of stretch before failure. The insertion is then an astonishing structure from the mechanical point of view. The literature review set about showed that there is no description of the microstructure of the human ligament-tobone insertion. Therefore this review was extended to human tendon,4 because of the similarities between ligament and 4 The term enthesis is widely used to refer to the ligament-tobone junction, even though the word is devoted to the tendon-tobone attachment in anatomy.
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tendon anatomy and function. As a matter of fact, tendon and ligament are often considered identical in the literature (Benjamin et al., 2002; Cooper and Misol, 1970; Evans et al., 1990; Schneider, 1956). Furthermore, animal ligaments studies were as well included. Schneider (1956) and Cooper and Misol (1970) gave the first description of the insertion site (dog patellar tendon and lateral collateral ligament), later confirmed by Benjamin et al. (1986) for human tendon insertions. It is divided in 4 layers: the ligament or tendon, the uncalcified fibrocartilage (UFC), the calcified fibrocartilage (CFC), and the bone. The transition line between uncalcified and calcified fibrocartilage is named tidemark or blue line, this is the mineralization front. The transition line between the calcified fibrocartilage and the bone is named cement line (Fig. 1). The tidemark is a moving mineralization front (human Achilles tendon (Milz et al., 2002), rat Achilles tendon (Rufai et al., 1996)), there is no change in the constituting materials. Besides the mineralization, the difference between UFC and CFC is that the collagen fibres in the latter are not crimped (quadriceps tendon fibers insertion into the patellae of adult rabbits, humans, dogs and sheep (Cooper and Misol, 1970)). The actual frontier between bone and tendon/ligament is the cement line (rabbit knee joint (Gao and Messner, 1996)). The collagen fibres do not cross the cement line (Gao et al., 1996; Milz et al., 2002). Clark and Stechschulte (1998) observed that the tendinous fibres interdigitate with the bone lamellar structure, without merging with them however. A molecular glue could be responsible for binding them together. Some authors attempted to relate the mechanical role of the insertion to its structure. For Schneider (1956), the fibrocartilage prevents the tendon from narrowing when loaded, so that it does not damage the bony part of the insertion. It was suggested similarly that the fibrocartilage would ensure a gradual transition (Knese and Biermann, 1958) from the ligament, which is very soft (E = 20–70 MPa (Arnoux, 2000; Pioletti, 1997)), to the bone (E = 10 000–20 000 MPa (Yamada, 1970)), and even that the insertion has the ability to adapt itself to the load by modifying the amount of calcified tissue (Gao and Messner, 1996) and the layout of the fibres (Rufai et al., 1996). The tendon or ligament fibres can penetrate the UFC at various angles relative to the surface of the UFC (Benjamin et al., 1986): the UFC ensures that the tendon does not bend when it meets the bone, and that the collagen fibres reach the tidemark at an angle approaching a right angle. Therefore, the mechanical function of the UFC would be to offer some protection from wear and tear to the tendon/ligament fibres, by preventing them from being bent, splayed out or compressed during the motion of the joint to which the tendon is attached. Thus the ligament-to-bone attachment would be a way to reduce the mechanical loading transmitted by the ligament to bone. This paper presents the results of the study performed on the knee ligament-to-bone attachment in human to determine its microstructure. The goal was to choose a mechanical model capable of describing its behaviour and its failure (Arnoux et al., 2005; Subit, 2004). Optical microscopy, scanning and transmission electron microscopy devices were used to look at the knee ligament insertion sites in the knee joint of a Post Mortem Human Subject (PMHS). The preparation of the samples is first described, followed by
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Fig. 2 – Overview of the femoral insertion of a LCL (OM, HES staining, FOV width: 21 mm). Fig. 1 – Tendinous and ligamentous insertions (based on Cooper and Misol (1970) and Schneider (1956)).
the results obtained for each device. As a starting point, the microstructure of the insertion site of the ligaments in the human knee was compared to the microstructure reported on in the literature. Combining an in-depth analysis of the histology results and the mechanical function of the insertion, some assumptions are put forward as for its microstructure in the discussion section, to propose a description of the insertion that can be used in computational modeling.
2.
2.2.
The SEM samples were prepared in a similar manner, except that the ligament-bone units were fixed by immersion in a 2.5% glutaraldehyde solution buffered with 0.1 M phosphate mono disodium, pH 7.4. Samples for scanning were collected in two ways. First, the ligament-bone unit was frozen and the samples for scanning were created by manually fracturing the unit in the fibre direction to expose a smooth surface of the ligament-bone interface. Second, samples were created at room temperature by manually tearing the ligamentbone unit in the fibre direction to expose the ligament-bone interface. Finally, the exposed surfaces were coated with gold for scanning.
Materials and methods 2.3.
Ligament samples were harvested from a single human cadaver (age 64) preserved in a Winckler solution (Winckler, 1974). Ligament-bone units were created by taking full-width strips approximately 30 mm long from the tibial and femoral insertion sites of the Posterior Cruciate Ligament (PCL), and the fibular and femoral insertion sites of the Lateral Collateral Ligament (LCL). The strips were then divided to be prepared for optical microscopy, and scanning and transmission electron microscopy. The articular surfaces of the knee joint were confirmed to be devoid of gross pathological change.
2.1.
Scanning electron microscopy
Optical microscopy
The ligament-bone units were fixed at room temperature by immersion for several hours in a 2.5% formaldehyde solution buffered with 0.1 M phosphate, pH 7.4. Following fixation, each unit was washed in the same solution, and dehydrated by dipping in several ethanol solutions of decreasing concentration. After dehydration, the units were dipped in an organic solvent, and embedded in paraffin wax. Full-thickness slices 5 µm wide were cut along the direction of the ligament fibres using a microtome and mounted on glass slides. The slices were then stained either with haematoxylin-eosin-saffron (HES) or with methylene blue (MB).
Transmission electron microscopy
Blocks of the ligament-bone units about 1 mm3 were fixed in a 2.5% glutaraldehyde solution buffered with 0.1 M phosphate mono disodium, pH 7.4. The blocks were then washed in the same solution, and postfixed in buffered 2% osmium tetraoxide. The samples were then dehydrated using graded ethanol solutions and propylene oxide, and soaked in a graded solution of epon/propylene oxide resin to initiate their embedding in this resin, and finally embedded. Once the resin had polymerized, slices of the bone-ligament units 90 nm thick were cut along the fibre direction and placed on a grid. Next, the samples were stained successively with uranyl acetate and lead citrate.
3.
Results
3.1.
Optical microscopy
The overview of a slice (Fig. 2) shows the ligament (L) and the decalcified trabecular bone (B). The box corresponds to the ligament-to-bone insertion site. Fig. 3 is a closer view of the boxed part (higher magnification), for a PCL. The bone is discernable thanks to the osteons (left side). On the right side of the picture are
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Fig. 3 – Tibial insertion of a PCL (OM, MB staining, ×10, FOV width: 0.859 mm).
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Fig. 4 – Femoral insertion of a LCL (OM, HES staining, × 100, FOV width: 2.65 mm).
the collagen fibres. The rows of chondrocytes (Ch) between the collagen fibres show the cartilage-like structure. This part of the ligament can be identified as uncalcified fibrocartilage (UFC). The darkening of the tissue due to the staining used for this slice shows the calcification of the fibrocartilage. The fibre structure is still visible, justifying the name of calcified fibrocartilage (CFC). The CFC zone is 580 ± 163 µm thick (average ± std). The closer view of a LCL shows the same pattern: bone, CFC, UFC and ligament (Fig. 4). The thinner arrow shows the bone/CFC border, the thicker arrow shows the CFC/UFC boarder. A line of chondrocytes (encircled) indicates the cartilage-like structure of the part of ligament adjacent to the ligament-to-bone transition. The CFC zone is about 340 ± 67 µm thick.
3.2.
Scanning electron microscopy
The cortical layer (C) separates the ligament (L) and the bone (B), but is not crossed by ligament fibres (Fig. 5). The box shows the location where the ligament attaches to the bone.
3.3.
Transmission electron microscopy
The mineralization front is scattered with cells which identified as osteoblasts because of their rounded shape and their size. The ECM was not fibrous, and some particles of mineral matter could be identified. The collagen fibres (CgF, Fig. 6a) do not stop at the mineralization front. However their organization changes when they cross the mineralization front (FM, Fig. 6a): the bundles of fibres wrap around another structure which looks orthogonal to them. This structure is referred to as ‘orthogonal fibres’ (OF, Figs. 6 and 7). The orthogonal fibres are in the calcified part of the fibrocartilage. Going deeper in the CFC, the organization of the collagen and orthogonal fibres is more obvious (Fig. 7): they intertwine to create a grid/mesh in the CFC. The observation at higher magnification shows that the orthogonal fibres are collagenous.
Fig. 5 – Tibial insertion of a PCL: L, ligament; C, cortical layer; B, bone (SEM). Scale bar = 2 mm.
4.
Discussion
The microstructure of the PCL and LCL attachment complex is investigated in this study. It is not certain that these results can be extended to the medial collateral ligament and anterior cruciate ligament. Indeed, Benjamin et al. (2002) suggested that the structure of the enthesis depended on the osteogenesic mechanism and the mechanical loading, which is somehow specific to each ligament.
4.1.
The materials at the transition
The results from the optical microscopy showed that there is no cortical bone at the location where the ligament fibres attach to the bone (also confirmed by SEM), and that the ligament-to-bone transitional materials were the UFC and CFC. The structure of the ligament-to-bone transition for the human knee ligaments at the optical microscopy level is identical to the structure described in the literature for the human tendon and animal ligaments (Fig. 1). However, the thicknesses are different. Cooper and Misol (1970) reported a CFC thickness of 100–300 µm for the dog patellar and the lateral collateral ligaments insertion sites, without any
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Fig. 6 – Mineralization front in the femoral insertion of a PCL (TEM). Scale bar= 5 µm. (a) View of the whole mineralization front, (b) Close-up of the the orthogonal fibres.
Fig. 7 – Whorl of collagen fibrils around the orthogonal fibres, femoral insertion of a PCL (TEM). OF, orthogonal fibres; CgF, collagen fibres (TEM). Scale bar = 5 µm.
Fig. 8 – Hypotheses about the attachment of the orthogonal fibres and collagen fibres to the bone. (a) Attachment to the periosteum or cortical bone, (b) Attachment to the trabecular bone. TB: trabecular bone, CgF: collagen fibre, OF: orthogonal fibres, P: periosteum, CB: cortical bone.
details about the location where the measurements were taken. In the current study, the average thicknesses are greater (PCL: 590 µm, LCL: 340 µm). The measurements in this study were performed on one subject at various locations and averaged. They are too few data points in this study
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to perform a statistical analysis of the thickness repartition. The variation of the thickness as a function of the position in the insertion was not further investigated. To the best of our knowledge, it is the first time that the thickness of the PCL and LCL insertions sites are reported, thus no data are available for comparison. It would nevertheless not be surprising to observe different thicknesses for the dog and the human insertion sites. These differences may be due either to the adaptation of the tissue to the loading (different bone remodelling process different for the dog and the human beings), to aging (the specimens used by Cooper and Misol (1970) were likely younger relative to the dog life expectancy, whereas the samples in this study were taken from an elderly), or to the physiological function of the ligament and the loading they bear (quadruped vs. biped). Rufai et al. (1996) reported that the cement line which delimits the CFC and UFC areas (Fig. 1) is a moving line, revealing that this area of the insertion is very active (for the rat). This was confirmed to be the same for the ligaments in this study (presence of osteoblasts in the mineralization front). Evans et al. (1990) mentioned that the CFC was “several” micrometers thick, without providing quantitative results (for human). The thickness of the CFC layer is likely to be function of the location where it is measured: Gao and Messner (1996), (rabbit knee ligaments), Kapandji (1987), Müller (1982), and Evans et al. (1990) (human knee joint) hypothesized that the thickness of the CFC increases to adapt to the increase in the mechanical loading borne by the ligament, in particular the direction of the loading. Benjamin et al. (1991) suggested that the greater thickness of UFC in the lateral meniscus compared to the medial one in the human knee joint was linked to its greater mobility (average values of 0.23 mm for the lateral meniscus, and 0.14 mm for the medial meniscus). The CFC would be thicker for the ligaments peripheral to the knee joint (lateral and medial collateral ligaments), for which the range of motion is greater than for the ligaments closer to the center of rotation of the knee (posterior and anterior cruciate ligaments). The averaged thicknesses measured in this study, which are considered as a first estimate, are larger for the PCL than the LCL, and thus do not follow these assumptions. Meanwhile, Benjamin et al. highlighted the considerable heterogeneity in the different tendons they studied, in particular between direct attachment to the bone and attachment in a synovial joint, and showed that the fibrocartilage has the capability to adapt its structure to the type of motion that it bears, such as the friction with the adjoining bone, or the dynamic behaviour of the joint it is attached to Benjamin et al. (1993, 1995). The PCL is inside the synovial knee joint, whereas the LCL is outside, and the loading patterns during the knee motion are different for each of them, which could explain the differences in fibrocartilage thickness. Finally, it has been shown that embalming could alter material properties (Crandall, 1994), but it is likely that it does not affect the geometry of the microstructure in this structural analysis.
4.2.
The structure of the transition
Gao et al. (1996) reported that the ligamentous collagen fibres attach to the bone at the cement line, without merging
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with the bone (rabbit ligament-to-bone insertion). Likewise, Clark and Stechschulte (1998) observed that the tendon fibres ended against lamella in the lamellar bone or interdigitated between the lamellar structure, without merging with the lamella or splaying into the lamellar structure (rabbit, human, dog, sheep). Therefore, the collagen fibres are not continuous from the ligament/tendon to the bone. However, the way the ligament collagen fibres interweave with the lamella has not been observed yet. Milz et al. (2002) noticed that bone and ligament interlock with each other in a complex manner, and suggested that the degree of interlocking could explain the mechanism by which the Achilles tendon attaches to the bone in human. They envisaged that pieces of CFC could fit into the bone like the pieces of a jigsaw, which would make the bone-CFC coupling stronger as the loading of the insertion increases (when it becomes critical to protect the insertion site). The results of TEM (Fig. 7) showed a grid in the CFC, grid composed of the ligament collagen fibres and the collagen fibres referred to as orthogonal fibres (OF). The darker spots (more numerous on the left-hand side of the Fig. 7) show that the tissue is gradually calcified. This layout was observed in the CFC. To some extent, it supports the hypothesis proposed by Milz et al. (2002): there is a geometrical interaction between the materials composing the ligament-to-bone attachment that makes the transition stronger. Based upon the results of this study, combined with the mechanical role of the attachment site, some realistic patterns are proposed for the geometry insertion site (Fig. 8). It was shown that there is no cortical bone at the insertion site: the orthogonal fibres could ensure the continuity of the cortical bone (Benjamin et al., 2002) (Fig. 8(a)), or attach directly to the underlying bone (Fig. 8(b)) while providing a strong attachment to the ligament fibres. The two concepts have in common that the orthogonal fibres are mechanically loaded mainly in bending, which makes them less sensitive to the direction of loading than a pure tension load. Indeed, if the end of the fibres was directly embedded in the bone (without the orthogonal fibres, Fig. 9), the tension in the fibre would be in the direction of the sealing of the fibre with the bone, loading it along its weakest direction. The crosssection of a loaded collagen fibre shrinks (the ligament at the insertion has an hourglass shape, Fig. 9); consequently the bone-collagen fibres interface has to bear a large load (shear and tension). This kind of attachment is mechanically weak, especially when a soft tissue is involved. Furthermore, it is very sensitive to the direction of the loading: the fibre has to bend at the ligament-to-bone transition which creates a stress concentration and can eventually lead to a premature failure. In the structures illustrated in Fig. 8, the orthogonal fibres, embedded in a cartilaginous matrix, would transmit the load, and consequently share it with the nearby structures (cortical bone, periosteum). As Schneider (1956) suggested it for the CFC, this type of attachment prevents the fibres from narrowing when loaded. Such a structure for the insertion site supports the idea that the collagen fibers do not cross the mineralisation front between the ligament and the bone. It has been shown that the continuity of the collagen fibres through the insertion site was not possible for tissues development reasons (Gao and Messner, 1996; Milz et al., 2002). The results presented
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Fig. 9 – Direct attachment of a collagen fibre (CgF) to the bone, (a) Unloaded and (b) Loaded ligament fibre. The contact interface between the fibre and the bone is damaged because the fibre shrinks when loaded.
here show that the non continuity of the fibres, with a grid of collagen fibres in the CFC, provides actually a stronger attachment from the mechanical point of view. At that point, the next step in the description of the ligament-to-bone attachment is to confirm that the OF are actually fibres, and to characterize (1) how the OF attach to the bone, and (2) how the collagen fibres in the ligament attach to the OF. It was assumed here that the collagen fibres wrap around the orthogonal fibres, but this could involve other kind of attachment such as glue, or interdigitation of fibrils. As for the failure, it was reported a rate sensitivity of the failure location to the loading (Arnoux et al., 2005; Danto and Woo, 1993; Pioletti et al., 1998; Subit, 2004). The grid of collagen fibres, embedded in the fibrocartilage the mechanical response of which is rate sensitive (viscoelasticity), has a complex mechanical response which is as well rate-sensitive. The findings of this study at the microscopic level are in line with the macroscopic level response. As a consequence, the ligament failure is the ‘competition’ between failure in the ligament, in the bone or at the insertion site, depending of the various rate sensitivities of each of these components (Subit, 2004). Besides, the small thickness of the ligamentto-bone insertion and the combined loading that it has to bear (shear, tension) argue for the use of a mechanical model developed for interfaces, known as cohesive zone model (Raous et al., 1999). This model accounts for damage, friction, rate sensitivity and failure. Its capability to describe ligament failure under quasi-static and dynamic loading has been shown in recent works (Arnoux et al., 2005; Subit et al., in revision; Subit, 2004).
5.
Conclusion
This study is the first to report the microstructure of the ligament-to-bone attachments for the LCL and PCL in the human knee joint. Based on the light microscopy results, it was established that the structure is similar to the structure described in the literature for human and animal tendons and ligaments: calcified and uncalcified fibrocartilage are the materials of the insertion. SEM results showed that there is not continuity of the collagen fibres across the insertion. TEM results revealed a change in the structure as well: in the plan of slices (sagittal plan of the ligament), a collagenous structure which has an other orientation than the ligament
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fibres, was observed. Based on mechanical arguments, it was speculated that this structure could be a grid of collagenous fibres (referred to as orthogonal fibres) embedded in the cartilage matrix of the insertion, to which the ligament fibres would attach. The attachment to the orthogonal fibres, rather than to the bone itself, provides the insertion with a better resistance to the load applied through the ligament while being less sensitive to the direction of the loading (shear and tensile loading). This structure needs to be confirmed by repeating the study on other specimens, and observing slices cut in other directions, to assess the presence of the orthogonal fibres in every subject and better describe their geometry and assembly. Besides, only the PCL and LCL were investigated; it is unclear how these results can be extrapolated to the other knee ligaments. Moreover, if the orthogonal fibres happen to be routinely observed, it would be required to investigate how the ligament fibres attach to them, and how the orthogonal fibers attach to the bone. That could be done by a tridimensional reconstruction of the insertion site, to scrutinize if there is any wrapping and interweaving of the fibres, and by histochemical analysis to evaluate the presence of a molecular glue.
Acknowledgements The authors would like to thank their colleagues from Faculté de Médécine de Marseille (Timone): Hubert Lépidi, for his help to perform the study in optical microscopy; Jacques Dejou, Imad About, Claude Alésia, and Hélène Borghi, for their comments and the access to their facilities, and Mireille Rémusat for her support to comment on the results from the histological point of view. Their contribution to this biomechanics study at the border between mechanics and medical science is greatly appreciated.
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