Three-Dimensional Imaging Analysis of the Developmental Process of Posterior Meniscofemoral Ligaments in Rat Embryos

The posterior meniscofemoral ligament (pMFL) of knee joint, or Wrisberg ligament, is a complex three-dimensional (3D) structure of ligament that runs posterior to the posterior cruciate ligament (PCL) with attachments at the medial femoral condyle and the posterior horn of the lateral meniscus [1, 2]. The anatomy and function of the pMFL has been studied in humans and quadrupeds. Several studies have shown that the pMFL is one of the most important structures for lateral meniscus stability [3]. During knee extension, the pMFL stabilizes the lateral meniscus by drawing the posterior angle of the lateral meniscus anteromedially and ensuring proper femur-tibial load transfer [4, 5]. When the posterior root of the lateral meniscus is injured, the pMFL resists extrusion of the meniscus, provides stability, and prevents the loss of load transfer and lateral contact force [6, 7]. It is also reported that pMFL acts as a secondary restraint along with PCL, which prevents the posterior drawer of the tibia [8, 9]. The presence of which is important in that it plays a compensatory role for damage to the surrounding joint components.

pMFL is related to the discoid lateral meniscus (DLM), a meniscal abnormality [10]. A DLM is a congenital anomaly. DLM is classified into complete, incomplete, and Wrisberg-type DLMs. The Wrisberg-type DLM is unstable because it is attached only to the pMFL (Wrisberg ligament). Besides, whether the Wrisberg-type DLM is a traumatic or congenital abnormality is controversial [11‒13]. Ahn et al. [12] proposed that when pMFL is thick and the attachment is high, tension may cause peripheral avulsion of the meniscus. It means that there is the possibility of attachment site variation of pMFL. Therefore, understanding attachment site structure of pMFL in the fetus using 3D-reconstructed images may lead to a better understanding of the pathogenesis of DLM. For capturing the characteristics of the knee joint during its early developmental stages, rats can be used as appropriate animal model since employing methods involving invasion and tissue destruction is unethical in humans.

Conventional histological methods used to observe tissues have challenges such as artifacts due to thin sections and loss of accurate alignment due to sectioning. To overcome these assignments and capture the 3D developmental process of the pMFL, we used episcopic fluorescence image capture (EFIC) that irradiates excitation light onto the tissue embedded in paraffin blocks and captures the autofluorescence of cells in this study [14‒16]. Imaging the surface of the block can provide continuous images without artifacts or misalignment. Additionally, the higher resolution than other imaging techniques allowed to analyze the development of the knee joint and spatial changes during the development of the cruciate ligament of small samples [17, 18]. In this study, we analyzed the developmental process of the pMFL of the rat knee joint using 3D reconstructed images produced from EFIC images and examined the relationship between the developmental process of the pMFL and other knee joint components.

Fifty-two hind limbs were removed from Wistar rat embryos between embryonic days 16 and 21 (E16 and E21). No distinction was made if the rats were male or female because sex differences are not involved in the appearance of pMFL [2, 19]. Wistar rats were obtained from Shimizu Laboratory Supplies Co. Ltd. (Kyoto, Japan). Hematoxylin and eosin (HE) staining was conducted on the hind limbs between E16 and E21 (E16, 17, and 18, n = 4 for each; E19, n = 3; E20, n = 4; and E21, n = 1) to identify each component and their positional relationship. Additionally, the right hind limbs between E17 and E21 (E17 and E18, n = 7; E19, E20, and E21, n = 6) were used for 3D reconstructed image analysis. The mother rats underwent cesarean section under anesthesia. They were exsanguinated by heart incision after removing the embryos from the uterus. Whole rat embryos were fixed in 4% paraformaldehyde at 4°C overnight before dissecting the hind limbs. All observations and measurements were conducted according to the following procedure to minimize potential confounders.

After the knee joints were embedded in paraffin blocks, 4–6 µm knee joint sections were dehydrated using graded ethanol and xylene and stained with HE.

Samples for EFIC were prepared as previously described [17]. In brief, the dehydrated samples were permeated and embedded in conditioned paraffin wax. Paraffin blocks were sliced using a Leica SM2500 sliding microtome (Leica Microsystems, Bannockburn, UK) at intervals of 6-7 µm. Autofluorescence on the paraffin block surface was visualized using epifluorescence imaging with mercury illumination and a discosoma red filter. Fluorescence images were captured using a Hamamatsu ORCA-ER low-light charged-coupled device camera (Hamamatsu Photonics K.K., Shizuoka, Japan). The resolution of the camera was 300 pixels/inch. The pixel size was 1,344 × 1,024 pixels. The field of view ranged from 1,897 × 1,444 μm to 4,552 × 3,465 μm. Optical magnification ranged between 25 and 60×, and the digital resolution ranged between 1.41 and 3.39 µm²/pixel.

The stacked 2D images captured by EFIC were reconstructed into 3D images using the Amira software version 5.5.0 (Visage Imaging, Berlin, Germany). The pMFL, anterior cruciate ligament (ACL), PCL, medial and lateral menisci, and femur were manually outlined and reconstructed, without smoothing. The 3D images were observed from various angles to confirm their morphology. Morphometric measurements were conducted using the Amira software. Four landmarks, namely, the attachments of the pMFL to the medial femoral condyle (aMC), posterior horn of the lateral meniscus (aLM), bottom of the medial condyle (bMC), and bottom of the lateral condyle (bLC), were manually set on 3D images viewed from the flexed dorsal aspect [20] (Fig. 1a). These landmarks were used to measure the deflection and angle of the pMFL. The coordinates applied to the 3D images were automatically generated by reconstruction. All measurements were conducted on 3D reconstructed images after E17, the components of which were distinguishable. In E17, four samples whose components could be distinguished were used for measurement.

The centerline of the reconstructed pMFL was extracted and its value was considered as the pMFL length (Fig. 1b). The linear distance between the aMC and aLM was calculated to compare the deflections at each stage. The deflection is defined as the ratio of the centerline length to the linear distance. A vector comprising aMC and aLM and that comprising bMC and bLC was calculated (Fig. 1c). The angle between the two vectors was then determined. The placement of the bMC and bLC and the calculation of angles were repeated thrice considering the possibility of misalignment when determining landmarks. The average value was defined as the angle of the pMFL.

The volumes of pMFL, ACL, PCL, and menisci were calculated by integrating the outlined areas on 2D serial images at each time point to capture the chronological change. The meniscal volume was measured as the combined volume of the medial and lateral menisci. The volume ratio of each knee joint component at each stage from the observation start point, E17, was calculated, respectively, to identify the differences in growth by components.

Sample size calculations were conducted using G*Power software ver. 3.1.9.7 (Heinrich-Heine-Universität Düsseldorf, Düsseldorf, Germany) [21, 22]. A post hoc analysis with a sample size of 32, an effect size of 0.77, and a significance level of 0.05 resulted in a power of 92%, showing that there was sufficient power. After confirmation of normality by Shapiro-Wilk test, comparisons between developmental stages were performed with the Tukey-Kramer test when following a normal distribution and the Steel-Dwass test when not following a normal distribution using JMP Pro version 16 (SAS Institute Inc., Cary, NC, USA). The statistical significance was set at p < 0.05.

The knee joints of E16–E21 were observed using HE-stained tissue sections in the sagittal plane. At E16, femoral and tibial primordia were identified (Fig. 2a). A structure thought to be the cruciate ligament was visible between the bone primordia; however, the pMFL could not be identified, and the boundaries between the tissues were indistinct. A small articular space was observed near the femoral condyle at E17 (Fig. 2b). The margins of the femur and tibia became distinct, and the pMFL was observed as an oval cell aggregate. ACL, PCL, and menisci were also simultaneously identified; however, the boundaries between the tissues were not sharp. From E18 to E19, an articular cavity was clearly observed and morphology of the knee components was clearly defined (Fig. 2c, d). In the sagittal section, pMFL cells were arranged in a granular pattern, and the fibers ran in a direction perpendicular to the observation plane. During E20-E21, cell aggregation of the knee components progressed further, and the pMFL displayed a relatively small circular shape (Fig. 2e, f).

The oval shape of pMFL was attached to the lateral meniscus and medial femoral condyle during E17–E21. On the lateral meniscus side, circularly aligned cells separated the pMFL from the lateral meniscus. At E17 and E18, the morphology of the cells of the lateral meniscus and pMFL was similar (Fig. 2g, h). At E19, the cells lined up around the pMFL, and their orientation was clearly different from that of the meniscus cells (Fig. 2i). At E20 and E21, circular clusters of pMFL cells and cells of the meniscus aligned along a triangle (Fig. 2j, k). However, the lining of the pMFL was less distinct than that at E19 and was smooth and continuous with the meniscal tissue. On the femoral side, the boundary between the femur and pMFL was indistinct at E17, and the pMFL lining became visible from E18 (Fig. 2l–p). The pMFL can be distinguished from the femur as an oval-shaped aggregate. At E21, the distinct lining around the pMFL had disappeared and was smoothly connected to the femoral tissue (Fig. 2p).

The pMFL and knee joint constructs grew slightly larger from E17 to E19 as tissue morphogenesis progressed (Fig. 3). At E20, the overall size of the pMFL markedly increased compared with that at E19, and further growth occurred at E21. At E20 and E21, a slight deflection was observed near the center of the pMFL. The ACL and PCL curved toward each other with growth, creating a void between the two ligaments. The medial and lateral menisci gradually thickened from E17 to E21.

The length of the centerline increased between E17 and E18, decreased slightly between E18 and E19, and then began to increase between E19 and E21 (Fig. 4a). The pMFL lengths were significantly longer at E20 and E21than that at E18; those at E20 and E21 were significantly longer than that at E19. The deflection gradually increased from E17 to E20 and decreased between E20 and E21 (Fig. 4b; Table 1). There were no significant differences between stages.

The angle of the pMFL increased between E17 and E18 and between E19 and E20 (Fig. 5; Table 1). However, it decreased between E18 and E19 and between E20 and E21. There were no significant differences between stages.

The volume of pMFL increased moderately between E17 and E18, decreased slightly from E18 to E19, and then increased significantly between E19 and E21 (Fig. 6-(A)-a). The volume at E21 was significantly larger than at E18 and E19.

The ACL, PCL, and meniscal volumes showed similar increasing trends at each developmental stage. They gradually increased from E17 to E19 and rapidly increased between E19 and E21. The ACL had a significantly larger volume at E20 than at E18 (Fig. 6-(A)-b). The volume at E21 was significantly larger than that at E18 and E19. Menisci had same trend (Fig. 6-(A)-d). The PCL had a significantly larger volume at E20 than at E18 (Fig. 6-(A)-c, Table 2). The PCL volume at E21 was significantly larger than that at E18–E20.

The volume ratios of all the components were similar at E18 and E19, and a gradual increasing trend was observed (Fig. 6-(B); Table 2). However, from E20, the increasing trend for each component began to differ. Menisci showed the markedly increase, followed by PCL, which showed a large increase. The ACL and pMFL showed a slower increase in volume than the menisci and PCL.

In the present study, the temporal and spatial development of the pMFL was analyzed using 3D reconstructed images of the knee joints of rat embryos. pMFL was observed from E17 in both the HE-stained and 3D images. The pMFL was attached to the medial femoral condyle and the lateral meniscus at all developmental stages as in mature rats. The length and volume of the pMFL increased significantly between E18 and E21, but the angle did not vary significantly. The volumes of the ACL, PCL, and menisci also significantly increased between E18 and E21. The HE-stained images observed in this study are consistent with the developmental process reported in previous studies, suggesting that the subject followed a normal developmental process [17, 23].

The lack of marked variation in the attachment site and angle of the pMFL with the developmental stage indicates that the pMFL and surrounding knee joint components: lateral meniscus and femur developed while maintaining their positional relationship from the onset of development. Additionally, the pMFL and femur boundary was obscured in the HE-stained image at E21. This suggests that these attachments are firm and that the high tension of the pMFL may have a greater effect on the lateral meniscus, not only for stability but also as a burden. Considering thickness of the pMFL and height of the attachment site were associated with the posterior dissection of the meniscus in patients with a DLM [12] and the patients with a fully DLM have a thicker Wrisberg ligament exhibiting superior attachment to the femur than those in the non-discoid group [24], a thick pMFL and a high attachment site to the femur increase the tension and the range of motion of the lateral meniscus. Consequently, the strain on the meniscus increases, which may contribute to the thickening and detachment of the discoid meniscus. Current results that maintain positional relationship from the onset of development may support to congenital etiology of Wrisberg-type DLM.

The deflection and angle of pMFL did not change markedly during development. In human knees, multiple patterns of pMFL attachment to the medial femoral condyle have been identified [25]. Studies have also suggested that the shape of the femoral intercondylar fossa influences the pMFL angle [26]. Although neither the relative position of the pMFL attachment site nor the classification based on the femoral intercondylar notch shape was not examined in this study, the lack of consistency in angles and deflections of pMFL indicates that different attachment patterns may be mixed at each developmental stage, affecting those of angle. The deflection was maximum at E20 and then decreased at E21. The pMFL runs posterior to the PCL and assists its function by running along and near the attachment site of the PCL to the tibia [27‒29]. Considering that the meniscus and PCL showed significant growth while their positional relationship was constant during development, the deflection of the pMFL that occurred at E20 was likely due to extrusion induced by the increased volume of the adjacent PCL. The slight decrease in the deflection at E21 could also be attributed to the increase in volume of the lateral meniscus that exceeded the volume of the PCL, thereby stretching the pMFL.

The body weight of rat fetuses continues to increase almost exponentially from E16 to E21, whereas the volume of amniotic fluid surrounding the fetus peaks at E18 or E19 and decreases dramatically thereafter. Simultaneously, the space in which the fetus can move freely also decreases dramatically from E19 to E21 [30, 31]. Except for pMFL, the volume of the knee joint component continued to increase throughout E17–E21, which is consistent with fetal weight gain. The significant increase in volume from E18 to E21 occurs at about the same time as the decrease in free space for the fetus and gradually replacing the development of hind limb activity. This suggests that the development of the knee joint in rats does not necessarily proceed simultaneously with the formation and growth of each joint component but is divided into two stages: a period wherein the movement is initiated when free space is available for movement and the stimulus facilitates the smooth progression of morphogenesis and a period wherein the volume and other aspects of growth are prioritized after a certain degree of morphogenesis. This switchover occurs between E18 and E19, which is likely to be an important turning point for the maturation of the knee joint. The decrease in some results of pMFL measurements at E19 may be related to this turning point. However, the influence of individual differences in fetuses cannot be dismissed, and further investigation is needed to clarify whether this is related to the aforementioned switch.

In the human knee, the load is applied in extension, whereas in quadrupeds, it is applied in flexion [32, 33]. Therefore, the pMFL is thicker in quadrupeds than in humans [19]. In the present study, the pMFL was attached to and developed on the lateral meniscus and the femoral medial condyle throughout E17–E21. However, the occurrence of a similar process in the human knee joint remains unclear. Comparative studies are required to determine the similarities or differences in knee joint development between humans and quadrupeds. On the other hand, this is the first report on the 3D structure of pMFL development, and understanding the structure of the pMFL attachment site during the embryonic period in this study will help to elucidate the pathogenesis of postnatal meniscus avulsion.

This study has several limitations. First, there may have been individual differences. To overcome the limitation, verification using more samples is needed. Second, artifacts may be present in 3D reconstruction as it was conducted manually. The regions of interest in the EFIC images were extracted by referring to the positional relationships in the HE-stained images at all developmental stages. For structures with similar brightness, some deviation has occurred between the original tissue contour and the segmentation site during the 3D reconstruction.

The authors would like to thank Ryota Takaishi for his skilled technical assistance and advice.

All animal experiments were approved by the Institutional Animal Research Committee and conducted according to the Guidelines for Animal Experiments of Kyoto University (Permit Number: Med Kyo 22536).

The authors declare no conflicts of interest.

This work was supported by the Asahi Glass Foundation and JSPS KAKENHI (Grant No. JP21K17472).

M.T., T.T., and T.A. designed and supervised the study. K.I. performed the experiments, analyzed the data, and drafted the manuscript. K.I., M.T., A.I., T.T., and T.A. interpreted the data. MT and SY provided technical guidance. T.A. and M.T. provided financial support. M.T., A.I., S.Y., T.T., and T.A. gave their comment on the first version of the manuscript and approval of the final manuscript.

Momoko Nagai-Tanima and Kanon Ishida contributed equally to this work.

All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author.