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. 2014 Apr 3;224(6):624–633. doi: 10.1111/joa.12174

A multi-scale structural study of the porcine anterior cruciate ligament tibial enthesis

Lei Zhao 1, Ashvin Thambyah 1,✉, Neil D Broom 1
PMCID: PMC4025890  PMID: 24697495

Abstract

Like the human anterior cruciate ligament (ACL), the porcine ACL also has a double bundle structure and several biomechanical studies using this model have been carried out to show the differential effect of these two bundles on macro-level knee joint function. It is hypothesised that if the different bundles of the porcine ACL are mechanically distinct in function, then a multi-scale anatomical characterisation of their individual enthesis will also reveal significant differences in structure between the bundles. Twenty-two porcine knee joints were cleared of their musculature to expose the intact ACL following which ligament–bone samples were obtained. The samples were fixed in formalin followed by decalcification with formic acid. Thin sections containing the ligament insertion into the tibia were then obtained by cryosectioning and analysed using differential interference contrast (DIC) optical microscopy and scanning electron microscopy (SEM). At the micro-level, the anteromedial (AM) bundle insertion at the tibia displayed a significant deep-rooted interdigitation into bone, while for the posterolateral (PL) bundle the fibre insertions were less distributed and more focal. Three sub-types of enthesis were identified in the ACL and related to (i) bundle type, (ii) positional aspect within the insertion, and (iii) specific bundle function. At the nano-level the fibrils of the AM bundle were significantly larger than those in the PL bundle. The modes by which the AM and PL fibrils merged with the bone matrix fibrils were significantly different. A biomechanical interpretation of the data suggests that the porcine ACL enthesis is a specialized, functionally graded structural continuum, adapted at the micro-to-nano scales to serve joint function at the macro level.

Keywords: anterior cruciate ligament; enthesis; functional adaptation; macro-, micro-, and nano-level structure

Introduction

Ligaments and tendons play a crucial biomechanical role in the musculoskeletal system. The four major ligaments in the knee joint (two cruciate and two collateral ligaments) provide kinematic constraints to the knee both by responding to complex loading conditions and maintaining stability in its 6 degrees of freedom during joint motion.

The microstructure of ligaments is hierarchical and is commonly associated with the classic description of tendon structure originally provided by Kastelic et al. (1977). These investigators identified five hierarchical levels of tendon structure from the micro- to the molecular level, namely fascicle, fibril, sub-fibril, micro-fibril, and tropo-collagen molecule. This complex hierarchy of structure provides the overall mechanical strength of tendons and ligaments.

Apart from the structure and biomechanical properties of ligaments and tendons, of particular interest is their manner of insertion into the bone. Known anatomically as entheses, these insertions represent a functionally graded structural transition between the flexible ligament or tendon and the rigid bone. In effect, the enthesis serves as an anatomic stress reliever that is biomechanically tuned to the mechanical demands of the joint system. There are two broad morphological classifications for an enthesis – direct and indirect. These classifications, based originally on studies of tendinous insertions into bone (Knese, 1957; Knese & Biermann, 1958; Benjamin et al. 1986), have also been used to describe the ligament–bone insertion.

A direct insertion involves a gradual transition of soft tissue to bone over four distinct zones: zone 1 is represented by the tendon or ligament soft tissue; zone 2 is made of fibrocartilage; zone 3 is mineralised fibrocartilage; and zone 4 is bone proper (Schneider, 1956). With these four gradual transitions, the soft tissue generally approaches the bone at large angles (up to 90°) and the fourfold transition occurs in progressing towards the bony substrate. An indirect insertion is when the ligament tissue approaches the bone at an acute angle, merging with the periosteum, and via fibres (Sharpey's) that project deep into the bony substrate. The attachment of the medial collateral ligament to the tibia is an example of this indirect mode of insertion.

Of the two different types of entheses, the direct insertion is much more common in the knee joint and involves a deep interdigitation of the fibrocartilaginous zone into the bone tissue, forming an irregular border between lamellar bone and calcified fibrocartilage (Cooper & Misol, 1970; Hurov, 1986; Gao et al. 1994). The shape of this interdigitation is said to be related to local loading conditions (Schneider, 1956). Gao and Messner (1996), proposed that the frequency and depth of the interdigitations at the bone–soft tissue interface in different entheses is related to the mechanical strength of the associated ligament. Further, it has been suggested that the thickness of the calcified fibrocartilage might be related more to the intensity of loading that takes place at a given insertion (Gao & Messner, 1996; Gao et al. 1996). Such reasoning is based on experiments reporting complex patterns of tensile and compressive strain at the ligament–bone junction when the ligament is loaded (Zantop et al. 2006).

The ligament–bone system is highly complex (Robinson et al. 2005; Giuliani et al. 2009; Kopf et al. 2009) and involves micro- and nano-level structural realities that govern macro-level biomechanical function (Momersteeg et al. 1995; Thornton et al. 2002; Lucas et al. 2009). To date, enthesis reconstruction for ligament repair is limited. Further, tissue engineering efforts to create suitable graft replacements acknowledge the very considerable challenge of effectively ‘mimicking’ the natural structure and function of the ligament–bone system (Laurencin & Freeman, 2005; Mikos et al. 2006; Lu et al. 2010; Ma et al. 2012). A major part of the problem is a limitation in technical capability, but this aspect is increasingly being addressed with emerging tissue engineering technologies that are able to produce ligament constructs at the nano-level and upwards (Laurencin & Freeman, 2005; Freeman et al. 2007; Liu & Goh, 2011). However, there remains a pressing need to achieve a functionally relevant micro- to nanoscale structural elucidation of the ligament–bone anchorage system.

Of interest in the present study is the structure of the anterior cruciate ligament (ACL) and its insertion into bone. The ACL is the most frequently injured knee ligament (Gianotti et al. 2009; Muthuri et al. 2011; Takeda et al. 2011) and there are many published studies relating both to ACL injury prevention and methods of reconstruction employed to restore function following rupture. However, there is continued interest in improving our understanding of ACL structure and function in order to develop new and improved methods that can both duplicate more accurately the anatomy of the full ligament–bone system and achieve reparative outcomes that minimise the likelihood of longer term joint degeneration.

That the ACL consists of at least two separate bundles, the anteromedial (AM) and posterolateral (PL), has been determined by many previous anatomical studies as noted in review articles (Duthon et al. 2006; Kopf et al. 2009; Amis, 2012). This anatomical ‘double-bundle’ feature has provided a rationale for the development of the similarly named ACL reconstruction procedure, one that attempts to restore function by mimicking the natural anatomy of the ACL (Spalazzi et al. 2006; Shen et al. 2007; Tejwani et al. 2007). However, the degree to which replication of the structure and function of the natural ACL is achieved is in part limited by our understanding of the sheer complexity of this tissue system and particularly of the microstructural principles of anchorage at the enthesis.

Previous morphological studies of the ACL have largely been confined to a single structural scale (Amis & Dawkins, 1991a; Duthon et al. 2006; Zantop et al. 2007; Kopf et al. 2009), and the few multi-scale studies incorporating structural relationships between each level (Yahia & Drouin, 1989; Clark & Sidles, 1990) have largely ignored the two-bundle aspect. Further, of the relatively few studies focusing on the ACL enthesis (e.g. Wang et al. 2006; Subit et al. 2008; Moffat et al. 2008) none, to our knowledge, has compared structural differences between the anatomical bundles or provided an adequate description of the structural transition from the macro- to nanoscales.

In this new study we have used novel experimental methods to investigate in detail the macroscale to micro- to nanoscale structure of the ACL and its enthesis using a porcine model. Specifically, the study investigated micro-structurally the biomechanical advantages of the direct type of enthesis in relation to joint function.

Materials and methods

A total of 22 porcine knee joints, obtained fresh from a local butchery, formed the basis of this study. Each joint was carefully dissected to reveal the AM and PL bundles, and imaged using macro photography, following which the tibial portion of the anterior cruciate ligament–bone region was harvested. Each ligament–bone sample underwent chemical fixation for 48 h in 10% formalin in its fully relaxed/unloaded state, mildly decalcified, and then cryo-sectioned to produce sagittal slices (∼ 20 μm thick) of the ligament–bone enthesis. The sections were then imaged in their fully hydrated state using both bright field and differential interference contrast (DIC) optical microscopy. In DIC microscopy, a single light source is polarised before being separated into two parts using a Wollaston prism (Slayter & Slayter, 1992). With a very small spatial displacement, the two orthogonally polarised parts then pass through the thin sample slice to generate different optical path lengths. A second Wollaston prism is used to recombine the separated light parts before observation. The specimen structural properties cause variation in the optical path lengths upon final recombination. Thus the recombined beam produces an image of the specimen with contrasts showing the boundaries relating to various microstructural features (Slayter & Slayter, 1992; Thambyah & Broom, 2006).

To study the structure at the nano or fibrillar level, 12 of the samples were prepared for scanning electron microscopy (SEM). The fixed and decalcified samples underwent hexane and graded ethanol treatment, followed by enzymatic digestion for proteoglycan removal. They were then critical-point dried and platinum coated before imaging using SEM. Finally, the average collagen fibril thickness of both AM and PL bundles was obtained from measurements made on high magnification SEM images using the image-processing program imagej (build 1.46r).

Results

The macro-level gross anatomy revealed clearly the AM and PL bundles of the ACL (Fig. 1) and this level of detail has been described previously (Amis & Dawkins, 1991b; Petersen & Zantop, 2007; Kopf et al. 2009). The frontal view of the AM bundle displayed a fanning out at the anterior-most aspect of the tibial plateau relative to the bundle itself (Fig. 2A), which, in turn, tapered to a reduced diameter on approaching the femoral end.

Figure 1.

Figure 1

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Frontal view of the left knee dissected and the femur rotated (flexed). The bundles are teased apart to show their distinct and separate gross morphologies to reveal the anteromedial (AM) bundle, and its insertion into the anterior aspect of the tibia, and the posterolateral (PL) bundle.

Figure 2.

Figure 2

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(A) ACL-on-bone block showing the fanning out of the ligament fibres (black arrows) at the tibial end of the ligament–bone insertion. (B) Cross-sectional view of the ACL showing the ‘crescent’ shaped wrapping (dotted line) of the AM bundle about the PL bundle. The arrowhead in (B) indicates the anterior aspect. AM, anteromedial; PL, posterolateral.

The view in cross-section (shown in Fig. 2B) emphasises a ‘crescent’-shaped wrapping of the AM bundle about the PL bundle. The sagittal section of the AM bundle insertion into the tibia further revealed a differentiated insertion such that a small portion of the anterior-most aspect of bundle appeared to blend into cartilage matrix, while the rest showed an intricate fibre ‘root’ network (Fig. 3).

Figure 3.

Figure 3

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Sagittal view of the AM bundle insertion showing the intense interdigitation of ligament fibres with its rigid substrate (see the boxed region). The vertical white line indicates the depth of fibre rooting. Optical image was obtained by top illumination of the fully hydrated section.

Serial sectioning revealed distinct differences in ligament–bone insertion morphology in traversing from the most medial to the most lateral aspects of the AM and PL bundle entheses (see Fig. 4A,B). Viewed in the most medial section, the bone contours (or cement line profiles) were generally discordant with the approximate plane of the tibial plateau (see dotted line in Fig. 4A), and the general direction of alignment of the compliant ligament fibres was at a relatively high angle to the cement line. Viewed in sections taken nearer the most lateral aspect, the cement line was aligned more generally parallel to the tibial plateau plane, albeit possessing a degree of irregularity in its profile (Fig. 4B).

Figure 4.

Figure 4

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Medial-most sagittal views of (A) the AM bundle and (B) the PL bundle showing the strongly oblique orientation of the cement line (solid line) with respect to the approximate plane of the tibial plateau (see dotted lines). The general direction of the ligament fibre alignment (see arrow) is at a relatively high angle to the cement line.

In the lateral-most aspect, the AM bundle exhibited a relatively acute turn into the fibrocartilage-bone substrate and appeared to anchor via a pattern of deep fibre rooting (see Fig. 5A). Conversely, the PL bundle in the same lateral-most aspect displayed a soft tissue insertion approximately orthogonal to the cement line, with a significantly reduced depth of rooting into the fibrocartilage–bone substrate (Fig. 5B).

Figure 5.

Figure 5

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Lateral-most sagittal views of (A) the AM bundle and (B) the PL bundle showing the irregular profile of the cement line (solid line) with respect to the approximate plane of the tibial plateau (see dotted lines). The AM bundle in (A) shows a sharp turn of the ligament fibres (black arrow) into the fibrocartilage–bone substrate, and also deep fibre rooting (vertical line). Conversely the PL bundle in (B) shows a significantly reduced depth of ligament fibre rooting (see vertical line).

With DIC microscopy, the fibrous rooting of the AM bundle insertion into the tibia was shown to involve a significant degree of interdigitation between the soft and hard tissues (Fig. 6A,C), whereas for the PL bundle insertion, the fibers were less deep and transitioned into the bone with a significantly reduced depth of interdigitation (Fig. 6B,D).

Figure 6.

Figure 6

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(A) Sagittal view of the AM bundle of the ACL showing fibrous rooting with a significant degree of deep interdigitation. (B) Sagittal view of the PL bundle of the ACL showing relatively less depth of insertion into the bone. Boxed regions in (A) and (B) are enlarged in (C) and (D), respectively. The cement lines are shown with dotted lines.

The PL insertion further differed from the AM insertion in that the mineralised phase between the soft ligament and hard bone was more complex and appeared to involve differing degrees of mineralisation, especially in the posterior aspect of the bundle (Fig. 7A,B).

Figure 7.

Figure 7

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Sagittal view of posterior aspect of (A) the AM bundle and (B) the PL bundle at the same magnification and showing different degrees of mineralization at the ligament–bone junction. There are three visible zones in the AM bundle, but four in the PL bundle.

Careful tracking of the AM ligament structure into the bone using lower magnification SEM revealed that fibres travelled deep into the bone matrix (Fig. 8A,C) while retaining a relatively unmineralised state compared with the surrounding bone. The PL bundle, on the other hand, appeared to have the ligament tissue end abruptly at the bone junction (Fig. 8B).

Figure 8.

Figure 8

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SEM images of (A) AM bundle and (B) PL bundle insertions into bone. The interdigitation of ligament fibres into the bony substrate (e.g. see boxed region in A enlarged in C) of the AM bundle insertion contrasts with that of the PL bundle. The PL bundle insertion (dotted line in B) shows a distinct and near-linear boundary between ligament and bone.

Analysis of the interface between the AM bundle of the ligament and bone showed that the bone fibrils intertwined and merged with those of the ligament, and at discrete ‘nodal’ points along the length of insertion fibrillar bundles were organised in an approximately transverse orientation with respect to the ligament fibrils (see arrows in Fig. 9). Conversely, the PL bundle fibril-level insertion into bone was characterised by fibril aggregates that tended to end in a tapered arrangement within a relatively shallow bone matrix pocket (Fig. 10).

Figure 9.

Figure 9

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SEM of AM bundle showing fibrillar-level integration with bone. Note also the presence of near-transversely organised collagen fibrils forming well-defined ‘nodal’ clusters along the ligament–bone interface (see arrows).

Figure 10.

Figure 10

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SEM image of PL bundle insertion showing how the ligamentous fibrils end in a shallow bone socket.

Comparing the diameter of the fibrils (n = 10 each), the AM bundle diameter was on average 179.3  ± 17.0 nm and approximately double that of the PL bundle, which was 91.6  ± 14.4 nm (Fig. 11).

Figure 11.

Figure 11

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SEM images taken at the same magnification showing fibril diameter differences in (A) the AM bundle, (B) the PL bundle, and (C) the bone.

Discussion

The present study describes in detail the multiscale structural make-up of the porcine ACL tibial enthesis and is to our knowledge the first study to reveal the detailed micro- to nanoscale insertion of the AM and PL bundles of the ACL into the tibia. It is therefore important to acknowledge here the limitation of applying the current findings from the porcine ACL to other species, given that variations exist in the anatomy of the knee between species (Proffen et al. 2012). However, we do suggest that any structure–function insights derived from the present porcine-based study can provide a strong motivation for similar investigations into other species.

It has been previously assumed that the ACL insertion into bone is only of the direct type involving the gradual change from ligament proper to bone via a transition of uncalcified or calcified fibrocartilage, or both (Benjamin et al. 2002). The additional sub-types revealed by the present study are shown to depend on location and include either a deep-rooted and strongly interdigitated insertion of soft tissue with hard bone or an insertion involving significantly less interdigitation but more gradual blending of the soft and hard tissues (see Figs 4 and 5, comparing AM and PL).

The PL bundle insertion with its different zonal changes in structure is typical of a direct enthesis. The AM bundle with its deep-rooted fibres may be mistakenly identified as an indirect type enthesis containing ‘Sharpey's fibers’. However, Sharpey's fibers are the deeply embedded extensions from the periosteum and thus cannot accurately describe the deep ligamentous fibres buried in bone and seen continuous with the AM bundle, as demonstrated in this study. Further, the deep-rooted fibrils of the AM bundle are shown to interweave with those of the bone matrix collagen (Fig. 9) and this highlights the degree of mechanical complexity involved in attempting to relate the ligament–bone attachment's nanoscale structure to the macro-level joint mechanics.

The interdigitation of the AM bundle towards the lateral aspect and the PL bundle to a lesser degree (see Fig. 5), together with the bone cement line adaptation on the medial aspect (see Fig. 4), suggest that within each bundle these structural variations are ‘tuned’ to reflect region-specific mechanical requirements in the enthesis. The question therefore arises as to the extent to which the observed differences in interdigitation between the porcine AM and PL bundle entheses are of relevance to or influenced by the requirement for specific modes of porcine knee stability during joint function.

While very little is known about porcine knee biomechanics, previous studies on the human knee have shown that in passive extension the PL bundle is taut, whereas in relative flexion the AM bundle becomes taut and the PL bundle relaxes (Amis & Dawkins, 1991a; Petersen & Zantop, 2007). It has also been suggested that the PL bundle's primary role is to provide rotational stability, whereas the AM bundle is designed to resist anterior tibial translation (Gabriel et al. 2004; Steckel et al. 2007; Amis, 2012). Importantly, the few published biomechanical studies of the porcine ACL show that it, too, is subject to differential levels of AM and PL recruitment with respect to flexion angle. With the ligament being required to provide stability for both anterior tibial translation and rotation, the PL bundle has been shown to be more important in restraining the latter (Li et al. 1998; Kato et al. 2010).

Thus, if in the porcine knee there exists a structure–function relationship between the double bundle morphology and joint-level mechanical function, it is then not unreasonable to assume that in the human knee, the joint level mechanical function of the ACL (Amis & Dawkins, 1991a; Petersen & Zantop, 2007) may reflect a similar structure–function relationship. This assumption forms the basis of any clinical relevance that the present study might have. In any case, from an anatomical perspective, too, the structure–function relationship of the porcine knee is still of interest and will require further elaboration, as discussed in the next section.

We propose that the relatively shallow interdigitation of the PL bundle into bone compared with that of the AM bundle (see Fig. 6) may be a microstructural reflection of macro-level differences in biomechanical function, with the three sub-types of enthesis contributing to distinctly different functions –illustrated schematically in Fig. 12. First, the medial-most sections showing the irregular bone contours may indicate an adaptive response designed to reduce shear such that fibres are aligned more perpendicular to the tidemark (Figs 4 and 12B). Secondly, the PL bundle with its highly graded structural continuum (Figs 6B, 7B and 12C) may indicate a design that is more resistant to torsional or distorsional/twisting strains. Finally, the deeply rooted and interdigitated fibres of the AM bundle (Figs 5A, 6A and 12D) may be optimally suited to resisting more multi-directional pull-out forces, arising from different knee motions including rotation.

Figure 12.

Figure 12

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Schematics summarising the important structure–function features of the ACL enthesis as shown in the present study. (A) Schematic illustrating how the ACL is designed to resist both anterior tibial translation and internal tibial rotation. The crescent-like shape of the AM bundle about the PL may help to distribute shear forces between these two functionally different ligament bundles. (B) One of the insertion types seen in the medial-most section is that of an adaptive bone contour that results in an orthogonal ligament alignment with regard to the cement line. (C). Another type of insertion, more common in the PL bundle, is the functionally graded material change from soft-to-hard tissue, which would appear to be designed to resist torsional or distortional/twisting strains. (D) The deep-rooted fibres, seen mainly in the lateral aspect of the ACL and most significantly in the AM bundle insertion, would appear to be designed to resist multiply directed pull-out forces.

However, the role of the AM bundle in providing rotational stability may be unique to the porcine knee, where its attachment to the tibia is found to be further from the knee central axis than that in the human (Kato et al. 2010). It is therefore suggested that the micro- to nanoscale ligament insertion into bone in human knees will be different to that in the porcine knee to reflect the difference in the position of the AM bundle tibial footprint between these two species. Such biomechanical aspects will be important considerations for future studies by our group into the human knee ACL and its insertion into bone.

A more universal application of the findings from the present study can be seen in the micromechanics of annulus-endplate insertion in the intervertebral disc. A recent study demonstrated a significant degree of sub-bundle interdigitation of the annular fibres into the calcified cartilage of the endplate (Rodrigues et al. 2012). This similarity in insertion morphology suggests a common functional rationale in the design of different entheses. The obliquely directed lines of action of the force-bearing fibres in these two quite different soft/hard tissue systems would appear to share a design principle that is based on a similar functional requirement of an interdigitated-type integration into the rigid substrate. Such interdigitation would act to maximise the area over which interface shear forces are distributed and thus increase the strength of anchorage of the fiber bundles in tension (Rodrigues et al. 2012).

The present study has shown that the porcine enthesis can be viewed as a specialised, functionally graded structural continuum adapted to serve both the local micromechanical environment and joint function at the macro- or gross level. Thus, if in the human knee a similar relationship exists between the micro- to nanoscale structure of the ACL with its corresponding macro-level joint function, then this study has a twofold significance. First, we propose that it is important to understand the micro- to nanoscale structural detail as this serves as a ‘blue-print’ for the design of ligament replacement systems. Current attempts to use artificial materials (e.g. Dacron and Gortex) to replace damaged ligament have not proven to be effective, with failure occurring in artificial replacements despite their being several times stronger that the natural ligament (Laurencin & Freeman, 2005; Legnani et al. 2010). Secondly, although tissue engineered solutions are a promising new option (Petrigliano et al. 2006) there are many issues relating to the use of auto/allografts and engineered ligaments. One of the fundamental limitations in this whole area concerns the question of just what are the multiscale structural requirements (and related mechanical properties) for effective ligament design (Vieira et al. 2009). In addressing these limitations the question of how to reproduce the critical soft-to-hard tissue integration in the healthy native tissue (Phillips et al. 2008; Place et al. 2009; Yang & Temenoff, 2009) is clearly of considerable importance, given that failure to establish this critical interface has been reported as compromising graft stability and long-term clinical outcomes (Moffat et al. 2009).

Conclusions

The porcine ACL tibial enthesis is shown to be a specialised, functionally graded, micro- to nanoscale structural continuum consisting of at least three distinct insertion morphologies that appear to be adapted to serve joint function at the macro level.

Acknowledgments

The authors are grateful to the Wishbone Trust NZ and The University of Auckland (Faculty Research Development Fund) for their grants in support of this work.

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