Introduction

The knee joint is a complex structure that provides both stability and mobility to the body, which are achieved through the interactions between the anterior cruciate ligament (ACL) and the meniscus1. Acting as shock absorbers and weight distributors, the menisci play a crucial role in enhancing knee stability alongside the ACL. The ACL comprises two bundles: the anteromedial (AM) bundle and the posterolateral (PL) bundle2. Tensile forces exerted on the ACL are influenced by the angle of the knee, resulting in intricate mechanical interactions with the corresponding meniscus, particularly during rotational movements with flexion. Injuries that affect these structures may lead to them rupturing simultaneously3,4,5. Researchers have investigated the roles played by the ACL bundles in determining mechanical tension on the knee6,7,8 and the prevalence of recurrent injuries due to reciprocal failure9. Recent anatomical and biomechanical studies have explored the macroscopic attachments between the ACL and meniscus and their biomechanical interactions10,11. While research on the interconnectivity of the ACL, transverse ligament, and meniscus has contributed to understanding the cause of injury12,13, conflicting results have been documented14,15. Unfortunately, there is still insufficient anatomical information available on the macroscopic properties between each ACL bundle, the systematic connectivity of the menisci, and the tensile strength of each bundle. Therefore, this study aimed to characterize the morphological connectivity between the AM bundle and PL bundle of the ACL and the meniscus using nondestructive three-dimensional micro-CT and histological analyses. Quantitative analyses of histological characteristics of each bundle were also conducted. Our hypothesis was that differences in histological characteristics reflect the distinct biomechanical functions of the AM and PL bundles. Furthermore, it was expected that macroscopic and morphological alterations among bundles would be expected following ACL flexion. Such in-depth laboratory insight into the ACL bundle and meniscal connectivity would not only enhances our understanding of the mechanical causes of injury but also provides fundamental data for characterizing the ACL bundle in clinical ACL reconstruction.

Results

The AM and PL bundles of the ACL (Fig. 1A–D), as well as the meniscal roots to which each ACL is connected were thoroughly analyzed from multiple perspectives using MT, PC, saf-O, modified VG, and micro-CT (Fig. 2A–C).

Fig. 1
figure 1

The overall morphological transverse section of the knee joint complex (A–F). These structures were subsequently compared and analyzed using microcomputed tomography ((micro-CT, (B). Applying modified Masson’s trichrome staining (MT) confirmed the connectivity of the anteromedial (AM) bundle of the anterior cruciate ligament (ACL) (C; blue asterisk), posterolateral (PL) bundle of the ACL (C); red asterisk)), and meniscus (C). The AM bundle tightly connected to the anterior horn of the medial meniscus (aMM) and articular cartilage (F); Ac, blue box)), while the PL bundle loosely connected to the anterior horn of the lateral meniscus (aLM) and connective tissue (E); Ct, red box)). AM anterior cruciate ligament-anteromedial bundle, Ac articular cartilage, aLM anterior horn of lateral meniscus, aMM anterior horn of medial meniscus, Ct connective tissue, ICE intercondylar eminence, LM lateral meniscus, MM medial meniscus, PL anterior cruciate ligament-posterolateral bundle.

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Fig. 2
figure 2

Analysis of immunohistochemical staining (MT, PC, and saf-O) in serial sagittal sections of the knee joint flexed at 15° (A–C). Subsequently, the analysis results were compared using micro-CT. The space between the AM bundle (black asterisk) and the PL bundle (red asterisk) of the ACL was observed to gradually widened from the medial to the lateral side of the knee (indicated by the black arrow). This sequential arrangement of the ACL is illustrated from the red box to the yellow box (Supplemental Video 1). A: anterior, L lateral, M medial, Micro-CT micro-computed tomography, MT Masson’s trichrome staining, Para-M para-medial, PC pentachrome staining, saf-O safranin-O staining, S superior.

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Anatomical connectivity of the AM and PL bundles of the ACL

Cadaveric dissection (Fig. 1A), micro-CT (Fig. 1B) and histological observations (Fig. 1C–F) confirmed that the AM bundle of the ACL is densely connected to the medial meniscus by the articular cartilage (Fig. 1C, F). On the other hand, the PL bundle of the ACL is loosely attached to the transition zone of the lateral meniscus by the connective tissue (Fig. 1C, E).

Applying various histological staining techniques (MT, PC, and saf-O) revealed that in the sagittal view the region between the AM and PL bundles is bridged by connective tissue, and the interbundle space gradually widens from the medial to the lateral side (Fig. 2C and Supplemental Video 1).

The difference in knee flexion between 15 and 60° was analyzed using micro-CT and identified across various directions (Fig. 3A–F). Micro-CT examination of the coronal view (Fig. 3D), sagittal view (Fig. 3E), and horizontal view (Fig. 3F) at angles exceeding 60 degrees revealed divergences in the spaces occupied by the AM and PL bundles (Fig. 3D and F, and Supplemental Video 2).

Fig. 3
figure 3

Comparative analysis of 15° and 60° of knee flexion using micro-CT (A–F). The coronal view (A,D), sagittal view (B,E), and horizontal view (C,F) were analyzed for each angle to confirm morphological differences between the AM bundle (white arrowhead) and the PL bundle (red arrowhead) of the ACL. At 60 degrees of flexion, the gap between the AM bundle and the PL bundle gradually widened (Supplemental Video 2), becoming distinctly verified (indicated by the yellow arrow; D–F). The transverse ligament is denoted by the pink dotted line (pink asterisk; D,E). A anterior, AM anterior cruciate ligament-anteromedial bundle, LM lateral meniscus, MM medial meniscus, M medial, P posterior, PL anterior cruciate ligament-posterolateral bundle, S superior.

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Ratio of AM and PL bundles of the ACL

The histological characteristics of the AM and PL bundles were analyzed separately into upper and lower regions (Fig. 4A, B).

Among ACL fibers, the AM bundles exhibited a combination of wavy or twisted nonlinear networks, whereas the PL bundles appeared as parallel and regular patterns. Elastic fibers were found to run parallel between collagen bundles. Based on detailed histological findings, the proportions of collagen and elastic fibers were highest in the upper region of the AM bundle, at 84.68 ± 6.49 and 9.59 ± 0.60, respectively; Supplemental Fig. 1 A and 1B). Additionally, the ratio of elastic fibers to collagen was highest in this upper region (11.36 ± 0.90%, p < 0.05) and lowest in its lower region (4.87 ± 0.34%, p < 0.05) (Fig. 5A). The ratio of elastic to collagen fibers within the AM bundle of the ACL is indicative of functional differences in strength and flexibility. The upper AM, with a higher content of elastic fibers, demonstrates clinical responsiveness to movement and enhances flexibility. In contrast, the lower AM, which has a relatively higher collagen content, offers resistance to tensile strength when the upper AM is stretched.

Fig. 4
figure 4

Identification of histomorphological differences between the upper and lower regions of both the AM and PL bundles of the ACL using immunohistochemical methods (VG (a), PC (b), Collagen (c); (A,B). Modified Verhoeff–Van Gieson staining (a) confirmed the characteristics of collagen and elastic fibers in magnified images. Collagen is indicated in red, and black dots represent elastic fibers. In Pentachrome staining (b), elastic fibers appear as brownish collagen clusters with black dots and were further analyzed using collagen immunohistochemical staining. The upper region of the AM bundle (A) displays a dense concentration of elastic fibers, whereas the lower region exhibits a sporadic, multiskewed pattern. Similarly, in the PL bundle (B), the upper region contains more elastic fibers than in the lower region. The collagen arrangement in the AM bundle (A-c) appears to be densely disorganized, while in the PL bundle (B-c), it exhibits an organized pattern with a lower collagen proportion. ACL-AM anterior cruciate ligament-anteromedial bundle, ACL-PL anterior cruciate ligament-posterolateral bundle.

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Quantitative measurements of upper region of ACL

In the upper region of the AM bundle, elastic fibers are abundant and predominantly clustered (9.59 ± 0.60%, p < 0.05), while the collagen fibers of the AM bundle of the ACL are also abundant, dense, and robust, but in a disorganized arrangement in various orientation (84.68 ± 6.49%, p < 0.05, Supplemental Fig. 1A). On the other hand, the collagen fiber in PL bundle of the ACL is in an organized arrangement but constitute a lower proportion that in the AM bundle (69.19 ± 5.55%, p < 0.05, Supplemental Fig. 1A). The elastic fibers exhibit sporadic, multidirectional patterns with a lower proportion (7.13 ± 0.32%, p < 0.05). The ratio of elastic fibers to collagen in the upper region was higher in the AM bundle than in the PL bundle (11.36 ± 0.90%, 10.33 ± 0.37%, respectively; Fig. 5A).

Fig. 5
figure 5

Graphs depicting the area ratio of elastic fibers to collagen in four regions of the ACL based on Fig. 4. The ratio for the AM bundle was highest in the upper regions, at 11.36 ± 0.90% (p < 0.05), and lowest in the lower region, at 4.87 ± 0.34% (p < 0.05), (A). Analysis of the regional composition of the PL bundle revealed that the dense area accounted for 10.33 ± 0.37% and the loose area accounted for 5.32 ± 0.78% (p < 0.05), (A). As demonstrated in Figure (B), the figures for each area were presented through the master chart. The boxplots display regional differences in the E/C ratio (elastic fiber/collagen fiber area ratio), with each box indicating median, 25th and 75th percentiles, and range values. Statistical significance (p < 0.05) is indicated by asterisks. E/C ratio, elastic fiber/collagen fiber area ratio; lAM, lower anteromedial bundle; lPL, lower posterolateral bundle; uAM, upper anteromedial bundle; uPL, upper posterolateral bundle.

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Quantitative measurements of lower region of ACL

The ratio of elastic fibers to collagen was lower for both the AM and PL bundles in the lower region than in the upper region of the ACL (4.87 ± 0.34% and 5.32 ± 0.78%, respectively; Fig. 5A). Additionally, the difference in the ratio of elastic fibers to collagen between the dense region (13.08%, Fig. 5B) and the loose region (4.70%) was found to be more pronounced for the PL bundle than the average lower regional ratio (5.32%). In the lower region of the AM bundle, the collagen proportion was 76.84 ± 7.50%, while the PL bundle had the lowest value of 63.08 ± 8.43% (see Supplemental Fig. 1A). Although the collagen content in the lower region was lower for the PL bundle than the AM bundle, the ratio of elastic fibers to collagen was higher in the PL bundle (5.32 ± 0.78%) than in the AM bundle (4.87 ± 0.34%, Fig. 5A). In summary, there appear to be notable differences in AM and PL bundle ratios in the upper and lower regions, which are confirmed by differences in the elastic fiber ratios related to collagen density.

Discussion

Understanding the biomechanical and clinical characterizes of the ACL requires a thorough knowledge of the microscopic properties of its two subparts - AM and PL bundles - which have not been studied in detail histologically. Herein, we have successfully demonstrated that the AM and PL bundles play essential kinetic roles in knee movement, acting as a primary fulcrum and an additional buffering cushion, respectively; (1) the AM bundle is composed of ligamentous segments with a denser, more-disorganized arrangement which are strongly connected to the medial meniscus. (2) the PL bundle is loosely attached to the lateral meniscus and contains ligamentous compartments with an organized arrangement. (3) during knee flexion, the length of the AM bundle extends more elastically with the medial meniscus remaining almost unchanged, whereas the PL bundle deviates laterally with a lower rigidity.

Fibrous blending of ACL and the lateral meniscus has been widely reported based on macroscopic and histological evidence14,15,16,17,18,19,20,21. In our study, two subparts of the ACL were histologically in contact with the adjacent meniscus on their ipsilateral side, not only the lateral but also the medial meniscus (Fig. 1A–F). The AM bundle directly overlapped the medial meniscus and articular cartilage of the medial tibial condyle, implying that its strong attachment to the medial meniscus maximizes joint stability during knee flexion22. Also, the direct connection of the AM bundle to the articular component suggests that responsive collagen production by chondrocytes in this junctional area can maintain the articular surface despite the constant mechanical stress to the knee joint23. Similar to other studies, the PL bundle was found to be inserted anteriorly to the anterior horn of the lateral meniscus10,24. The PL bundle is loosely connected to the lateral meniscus, with a transition zone of loose connective tissue forming the boundary between them.

The mechanical relationship between the subparts of the ACL and two menisci is important in distributing the axial load on the knee to ensure joint stability, and therefore meniscal tear are also strongly associated with ACL injuries25. Posterior displacement of the menisci during tibial rotation by knee flexion results in ACL retraction. The medial meniscus is more tightly attached to the bone, making it relatively less mobile than the lateral meniscus. In our study, the AM bundle may be a more solid fulcrum (Figs. 1 and 2). Namely, the AM bundle–medial meniscus–medial tibial condyle complex is stronger, more rigid, and more resistant than its counterpart8. In this ‘medial’ complex, injuries to the joints, ligaments, and meniscus are more commonly result mechanical damage or degenerative changes26,27,28,29. The fragile histological characteristics of the PL bundle and its connections to surrounding structures explain why the lateral meniscus is more susceptible to damage from ligament instability than from direct mechanical stress21,30,31,32. It corresponds that excessive stress on the knee joint would be concentrated on its lateral side, whereas it would be evenly distributed on the medial side, suggesting that the lateral meniscus is more susceptible to acute injury than the medial meniscus33,34,35,36. Arner et al.37 also argued that ACL injury increases the likelihood of lateral meniscus displacement, alters the contact position of the articular cartilage, and elevates chronic tension in the medial meniscus. In other words, our finding implies that temporary acute displacement due to injury is more common in the PL bundle, whereas sustained extrinsic forces causing injurious compression are more common in the AM bundle (Figs. 1 and 1).

At 60 and 90 degrees of knee flexion, tibial anterior shear force increases, placing greater stress on the ACL. Our findings suggest that the two bundles diverge laterally from the medial side of the knee due to the stretching of the connective tissue that occurs during knee flexion (Fig. 3 and Supplemental Video 2). The meniscus contributes to widening the space between these bundles and the likelihood of injury increases when the knee is flexed beyond 60 degrees. Stress in front of the ACL is localized at the anterior site of the intercondylar fossa, altering the orientation of the ligament. The transverse ligament, anchored to the front of bilateral menisci, is critical for knee rotation and restricts early flexion of the medial meniscus, thereby influencing ACL alignment. Up to 60 degrees of flexion, the position of the menisci remains stable, but flexion beyond this point can lead to meniscal damage. Extreme torque can lead to the unfortunate triad syndrome in which injuries to the ACL and adjacent structures occur simultaneously.

Differences between the bundles in collagen density and mechanical behavior have implications for shear force capacity and flexibility, as they may indicate injury susceptibility and kinematic differences within the ACL. In our study, the AM and PL bundles differ in collagen density and mechanical behavior, which affects shear force capacity and flexibility, and may indicate injury susceptibility and kinematic differences within the ACL (Figs. 4 and 5). The eccentric tensile strength of the AM bundle exceeds that of the PL bundle at knee flexions greater than 60 degrees. Previous arthroscopic studies have associated ACL hypermobility and damage to the anterior horn of the medial meniscus with anterior knee pain and an increased risk of meniscal tears38. The tensile force is higher in the AM bundle attached to the anterior horn of the medial meniscus than in the PL bundle attached to the lateral meniscus, which is due to the difference in size of the attachment sites on the horn39. The stiffness of these sites and the different tissue characteristics may play a role in the mechanism of injury.

The study faced several challenges: (1) The sample size was small, yet we established data significance and used a combination of macroscopic histology and 3D micro-CT to compensate for the sample number. (2) Our focus was on the histology and proportions of the AM and PL bundles, not including the kinematic analysis of translation and torsion relating to the transverse ligament. (3) The age of the cadaver specimens might have affected the outcomes, as collagen content and tensile strength decrease with age40,41. Hence, more comprehensive biomechanical studies on torque values are recommended for future research.

Materials and methods

Specimen preparation

This descriptive cadaveric study was conducted in the translatory laboratory for clinical anatomy at our medical research facility. Ten unembalmed human cadavers (5 males and 5 females; 15 sides) with an age at death of 69.1 ± 8.3 years (mean ± standard deviation) were included. Body donors had previously provided an informed consent statement to participate in the body donation program of Surgical Anatomy Education Centre at Yonsei University College of Medicine. Ethical approval for this study was obtained from the Institutional Review Board of Yonsei University College of Medicine (Yonsei IRB Approval number: 4-2023-0823). This study was conducted in accordance with the principles of the Declaration of Helsinki.

Manual dissection

The specimens were dissected while taking particular care to remove all soft tissues surrounding the knee joint, with special attention given to preserving the two bundles of the ACL. Cadavers with a history of traumatic events, clinically visible deformities, meniscal tears, or arthroscopic partial meniscectomy around the knee were excluded. An anatomical saw was used for dissection in order to prevent damage to the ACL and neighboring tissues, except for the ACL, meniscus, collateral ligaments, surrounding soft tissues, and vessels. The medial parapatellar incision was performed meticulously, the patella was flipped over, and the ACL at the bone–ligamentous junction of the femur and tibia was incised and harvested. The surrounding fibrous membrane and fatty tissue were methodically excised and separated along the marginal border between the AM and PL bundles of the ACL.

Histology

Tissue blocks were decalcified and embedded in paraffin wax, followed by serial sectioning into 5-µm-thick slices. The histological analysis was performed using modified Masson’s trichrome staining (MT), Pentachrome staining (PC), and Safranin-O staining (saf-O), modified Verhoeff–Van Gieson staining (VG) techniques.

Modified Masson’s trichrome staining

Specimens were incubated in a decalcification solution for 3 days and neutralized by soaking in a 0.25% lithium-carbonate solution for 4 h. After embedding in paraffin, 5-µm thick sections were obtained. Deparaffinization and rehydration were performed, followed by preservation in Bouin’s solution at 60 ℃ for 1 h. Weigert’s iron hematoxylin and Biebrich scarlet/acid fuchsin solutions were used, and differentiation was achieved in a 2.5% phosphomolybdic acid–phosphotungstic acid solution. Aniline blue solution was applied, followed by 1% acetic acid solution.

Pentachrome staining

After dehydration, the elastic fibers were stained using Verhoeff’s iron hematoxylin stain, followed by staining of glycosaminoglycan (GAG) with Alcian blue (pH 2.5) solution. The muscles were subsequently stained with Biebrich scarlet–acid fuchsin solution and differentiated with 5% phosphotungstic acid solution. Finally, collagen was stained with a yellow solution. Through this process, PC staining intricately delineates the tissue architecture by displaying the collagen, elastic fibers, muscle fibers, fibrin, and GAG in distinct colors.

Safranin-O staining

To stain the nuclei, 5 μm-thick tissue sections were immersed in Weigert’s iron hematoxylin solution for 10 min. Subsequently, a 0.001% solution of Fast Green (FCF) was applied for 5 min, followed by 1% Acetic acid solution and 0.1% saf-O solution for 5 min each. This process results in the cartilage matrix is being stained red while the surrounding background appears green.

Modified verhoeff–Van Gieson staining

Sections underwent a deparaffinization process using xylene, followed by sequential rehydration in 100%, 95%, and 70% ethanol solutions for 1 min each. After a 5-minute immersion in distilled water, the sections were incubated in a working Elastic Stain Solution (Abcam, Cambridge, United Kingdom) for 30 min and subsequently washed in running tap water. Differentiation was achieved using 2% ferric chloride, followed by treatment with 5% sodium thiosulfate for 1 min. The sections were then incubated in modified VG solution for 3 min, dehydrated in ethanol, and cleared with xylene. Following staining, the samples underwent further ethanol dehydration and were cleared in xylene.

Analysis of the AM and PL bundle of the ACL

An optical microscope (BX51, Olympus, Tokyo, Japan) were used to examine all sections. Using ImageJ software (Java 1.8.0, National Institutes of Health, Bethesda, MD, United States) was used to measure the two-dimensional area of signals indicating the presence of collagen type I and elastic fibers at identical positions. The densities of collagen and elastic fibers were compared and analyzed by delineating the upper and lower regions of the AM and PL bundles of the ACL. The ratio of elastic fibers to collagen was calculated relative to the cross-sectional area.

The statistical significance of the ratio of elastic fibers to collagen was standardized using R software (version 4.2.2 in a MS Windows). Morphometric data are presented as a mean ± standard deviation. A paired t-test was utilized due to the normal distribution of the data, with a p-value < 0.05 considered significant.

PTA preparation and micro-CT analysis

The knee joints used in the study was kept intact, without any removal of skin or fat, and free from degenerative soft tissue changes. In order to ensure that the two bundles of the ACL and meniscus could be clearly visualized, the intercondylar portion of the femur was carefully sectioned while preserving the attachment sites. It has been reported that the micro-CT preparation technique can increase the contrast enhancement of soft tissues42.

The knee specimens from the seven cadavers were subjected to a sequential immersion process in 30%, 50%, and 70% ethanol solutions overnight, which was followed by a 2% PTA solution with 70% ethanol for a period of 1 month. The specimens were then scanned using a micro-CT scanner (Skyscan1173, Bruker, Kontich, Belgium) with the following conditions: source voltage of 110 kV, source current of 72 µA, image pixel grid of 940 × 759; and image pixel size of 72 inches. For 3D visualization and analysis, all scanned images were reconstructed using NRecon and CTVox software (version 2.7 software, Bruker, Kontich, Belgium) and analyzed using Mimics software (version 20.0 software, Materialise, Leuven, Belgium).