Introduction: The posterior meniscofemoral ligament (pMFL) of knee joint is a ligament that runs posterior to the posterior cruciate ligament and it is known that the height of the pMFL attachment site causes meniscus avulsion. Therefore, understanding the three-dimensional (3D) structure of the pMFL attachment site is essential to better understand the pathogenesis of meniscus disorders. However, the developmental process of pMFL has not been well investigated. The purpose of this study was to analyze pMFL development in rat knee joints using 3D reconstructed images produced from episcopic fluorescence image capture (EFIC) images and examine its relationship with other knee joint components. Methods: Knee joints of Wistar rat embryos between embryonic day (E) 16 and E21 were observed with HE-stained tissues. Serial EFIC images of the hind limbs of E17–E21 were, respectively, captured from which 3D images were reconstructed and the features of pMFL structure: length and angle were measured. Besides, the chronological volume changes and the volume ratio of the knee joint components compared to E17 were calculated to identify the differences in growth by components. Results: pMFL was observed from E17 and was attached to the medial femoral condyle and lateral meniscus at all developmental stages, as in mature rats. 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 developed while maintaining their positional relationship from the onset of development. Conclusion: Current results may support to congenital etiology of meniscus disorder.

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]. 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]. 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]. It is also reported that pMFL acts as a secondary restraint along with PCL, which prevents the posterior drawer of the tibia [8]. 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]. 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.

Animals

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]. 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.

HE Staining

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.

Preparation and Workflow for EFIC

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.

Analysis of 3D Reconstruction Images

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.

Fig. 1.

Measurement methods in the 3D image. a Landmarks for morphometry. Four landmarks were set on the 3D image of the knee joint viewed from the flexed dorsal aspect. aMC, attachment of the pMFL to the medial femoral condyle; aLM, attachment of the pMFL to the posterior horn of lateral meniscus; bMC, bottom of the medial condyle; bLC, bottom of the lateral condyle. Green, pMFL; white, femur; purple, menisci. b Centerline and linear lines of the pMFL. The centerlines of the pMFL were automatically extracted using the Amira software. The linear distance was determined as the distance between the aMC and aLM. c Angle of the pMFL. The angle between the vector comprising aLM and aMC and that comprising bMC and bLC is defined as the pMFL angle.

Fig. 1.

Measurement methods in the 3D image. a Landmarks for morphometry. Four landmarks were set on the 3D image of the knee joint viewed from the flexed dorsal aspect. aMC, attachment of the pMFL to the medial femoral condyle; aLM, attachment of the pMFL to the posterior horn of lateral meniscus; bMC, bottom of the medial condyle; bLC, bottom of the lateral condyle. Green, pMFL; white, femur; purple, menisci. b Centerline and linear lines of the pMFL. The centerlines of the pMFL were automatically extracted using the Amira software. The linear distance was determined as the distance between the aMC and aLM. c Angle of the pMFL. The angle between the vector comprising aLM and aMC and that comprising bMC and bLC is defined as the pMFL angle.

Close modal

Length, Deflection, and Angle of pMFL

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.

Volume of pMFL and Knee Joint Components

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.

Statistical Analysis

Sample size calculations were conducted using G*Power software ver. 3.1.9.7 (Heinrich-Heine-Universität Düsseldorf, Düsseldorf, Germany) [21]. 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.

Hematoxylin Eosin Staining of pMFL and Knee Joint Components

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).

Fig. 2.

Histologic images of the knee joint and attachments of the pMFL in the sagittal section. a-f Knee joints from E16 to E21. Images show the right and left knees at E16–E20 and E21, respectively. Femoral and tibial primordia were identified on E16. The pMFL was observed posterior to the PCL (a one-sided square bracket) from E17, and the boundaries became progressively clearer (arrow). g-k Attachments to the lateral meniscus from E17 to E21. The arrowheads represent the border between the lateral meniscus and pMFL. l-p Attachments to the medial femoral condyle from E17 to E21. Arrowheads represent the border between the medial femoral condyle and pMFL. F, femur; T, tibia. Scale bars = 100 μm.

Fig. 2.

Histologic images of the knee joint and attachments of the pMFL in the sagittal section. a-f Knee joints from E16 to E21. Images show the right and left knees at E16–E20 and E21, respectively. Femoral and tibial primordia were identified on E16. The pMFL was observed posterior to the PCL (a one-sided square bracket) from E17, and the boundaries became progressively clearer (arrow). g-k Attachments to the lateral meniscus from E17 to E21. The arrowheads represent the border between the lateral meniscus and pMFL. l-p Attachments to the medial femoral condyle from E17 to E21. Arrowheads represent the border between the medial femoral condyle and pMFL. F, femur; T, tibia. Scale bars = 100 μm.

Close modal

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).

3D Reconstructed Images of Knee Joint and the Morphometry

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.

Fig. 3.

Representative 3D reconstruction images of knee joint between E17 and E21 viewed from the flexed dorsal aspect. All knee joint components considerably grew between E19 and E20 and E20 and E21. Between E17 and E21, the pMFL was attached to the medial femoral condyle and lateral meniscus. Gaps were developed between the ACL and PCL with growth. The medial and lateral menisci gradually became thicker. ACL, anterior cruciate ligament; PCL, posterior cruciate ligament. Scale bar = 100 μm.

Fig. 3.

Representative 3D reconstruction images of knee joint between E17 and E21 viewed from the flexed dorsal aspect. All knee joint components considerably grew between E19 and E20 and E20 and E21. Between E17 and E21, the pMFL was attached to the medial femoral condyle and lateral meniscus. Gaps were developed between the ACL and PCL with growth. The medial and lateral menisci gradually became thicker. ACL, anterior cruciate ligament; PCL, posterior cruciate ligament. Scale bar = 100 μm.

Close modal

Length, Deflection, and Angle of pMFL

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.

Fig. 4.

Length and deflection of pMFL. a Centerline length of the pMFL at each developmental stage. b Deflection of pMFL at each developmental stage. The ratio of the centerline length to the linear distance was calculated as the deflection. Each small dot indicates the centerline or deflection value in each sample. *p < 0.05. The Steel-Dwass test was used for statistical analyses.

Fig. 4.

Length and deflection of pMFL. a Centerline length of the pMFL at each developmental stage. b Deflection of pMFL at each developmental stage. The ratio of the centerline length to the linear distance was calculated as the deflection. Each small dot indicates the centerline or deflection value in each sample. *p < 0.05. The Steel-Dwass test was used for statistical analyses.

Close modal
Table 1.

Quantitative measurements of the pMFL from E17 to E21

Embryonic dayAverage (95% CI)SD
Centerline length [µm]  
 17 200.49 (159.29–241.69) 25.89 
 18 302.50 (251.69–353.32) 54.94 
 19 294.77 (245.46–344.08) 46.99 
 20 472.75 (391.20–554.30) 77.71 
 21 550.61 (517.68–583.54) 31.38 
Deflection 
 17 1.045 (1.042–1.049) 0.002 
 18 1.050 (1.038–1.062) 0.013 
 19 1.067 (1.035–1.099) 0.031 
 20 1.093 (1.042–1.145) 0.049 
 21 1.083 (1.036–1.129) 0.045 
Angle [°]  
 17 16.95 (3.62–30.29) 8.38 
 18 21.13 (18.09–24.18) 3.29 
 19 18.87 (14.12–23.62) 4.53 
 20 27.12 (19.98–34.25) 6.80 
 21 25.97 (21.34–30.60) 4.41 
Embryonic dayAverage (95% CI)SD
Centerline length [µm]  
 17 200.49 (159.29–241.69) 25.89 
 18 302.50 (251.69–353.32) 54.94 
 19 294.77 (245.46–344.08) 46.99 
 20 472.75 (391.20–554.30) 77.71 
 21 550.61 (517.68–583.54) 31.38 
Deflection 
 17 1.045 (1.042–1.049) 0.002 
 18 1.050 (1.038–1.062) 0.013 
 19 1.067 (1.035–1.099) 0.031 
 20 1.093 (1.042–1.145) 0.049 
 21 1.083 (1.036–1.129) 0.045 
Angle [°]  
 17 16.95 (3.62–30.29) 8.38 
 18 21.13 (18.09–24.18) 3.29 
 19 18.87 (14.12–23.62) 4.53 
 20 27.12 (19.98–34.25) 6.80 
 21 25.97 (21.34–30.60) 4.41 

CI, confidence interval; SD, standard deviation.

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.

Fig. 5.

Angle of pMFL. The pMFL angle at each developmental stage. Each small dot indicates the angle of the sample. The Tukey-Kramer test was used for statistical analyses.

Fig. 5.

Angle of pMFL. The pMFL angle at each developmental stage. Each small dot indicates the angle of the sample. The Tukey-Kramer test was used for statistical analyses.

Close modal

Volume of pMFL and Knee Joint Components

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.

Table 2.

Quantitative measurements of the pMFL and knee joint components from E17 to E21

VolumeVolume ratio
Embryonic dayAverage (95% CI)SDAverage (95% CI)SD
pMFL [×10−3 mm3   
 17 0.49 (0.20–0.79) 0.19 1.00 (0.40–1.60) 0.38 
 18 1.08 (0.72–1.45) 0.40 2.21 (1.46–2.95) 0.81 
 19 0.90 (0.52–1.28) 0.36 1.83 (1.06–2.61) 0.74 
 20 2.27 (1.48–3.06) 0.75 4.61 (3.01–6.22) 1.53 
 21 3.95 (2.90–4.99) 1.00 8.04 (5.91–10.16) 2.03 
ACL [×10−3 mm3   
 17 1.80 (0.79–2.82) 0.64 1.00 (0.44–1.56) 0.35 
 18 4.09 (2.59–5.59) 1.62 2.27 (1.44–3.10) 0.90 
 19 4.34 (2.11–6.57) 2.12 2.41 (1.17–3.64) 1.18 
 20 10.68 (7.53–13.84) 3.01 5.93 (4.18–7.68) 1.67 
 21 17.13 (13.03–21.23) 3.91 9.50 (7.23–11.78) 2.17 
PCL [×10−3 mm3   
 17 1.22 (0.17–2.26) 0.66 1.00 (0.14–1.86) 0.54 
 18 3.55 (2.46–4.63) 1.17 2.91 (2.02–3.80) 0.96 
 19 4.37 (1.71–7.04) 2.54 3.59 (1.40–5.78) 2.08 
 20 10.25 (8.41–12.09) 1.75 8.41 (6.91–9.92) 1.44 
 21 17.97 (11.97–23.98) 5.72 14.76 (9.82–19.69) 4.70 
Menisci [×10−2 mm3   
 17 0.27 (0.20–0.34) 0.04 1.00 (0.75–1.25) 0.16 
 18 1.05 (0.56–1.54) 0.53 3.87 (2.06–5.69) 1.96 
 19 1.12 (0.59–1.66) 0.51 4.14 (2.18–6.10) 1.87 
 20 3.70 (2.42–4.98) 1.22 13.60 (8.89–18.31) 4.49 
 21 6.66 (4.40–8.92) 2.15 24.52 (16.21–32.83) 7.92 
VolumeVolume ratio
Embryonic dayAverage (95% CI)SDAverage (95% CI)SD
pMFL [×10−3 mm3   
 17 0.49 (0.20–0.79) 0.19 1.00 (0.40–1.60) 0.38 
 18 1.08 (0.72–1.45) 0.40 2.21 (1.46–2.95) 0.81 
 19 0.90 (0.52–1.28) 0.36 1.83 (1.06–2.61) 0.74 
 20 2.27 (1.48–3.06) 0.75 4.61 (3.01–6.22) 1.53 
 21 3.95 (2.90–4.99) 1.00 8.04 (5.91–10.16) 2.03 
ACL [×10−3 mm3   
 17 1.80 (0.79–2.82) 0.64 1.00 (0.44–1.56) 0.35 
 18 4.09 (2.59–5.59) 1.62 2.27 (1.44–3.10) 0.90 
 19 4.34 (2.11–6.57) 2.12 2.41 (1.17–3.64) 1.18 
 20 10.68 (7.53–13.84) 3.01 5.93 (4.18–7.68) 1.67 
 21 17.13 (13.03–21.23) 3.91 9.50 (7.23–11.78) 2.17 
PCL [×10−3 mm3   
 17 1.22 (0.17–2.26) 0.66 1.00 (0.14–1.86) 0.54 
 18 3.55 (2.46–4.63) 1.17 2.91 (2.02–3.80) 0.96 
 19 4.37 (1.71–7.04) 2.54 3.59 (1.40–5.78) 2.08 
 20 10.25 (8.41–12.09) 1.75 8.41 (6.91–9.92) 1.44 
 21 17.97 (11.97–23.98) 5.72 14.76 (9.82–19.69) 4.70 
Menisci [×10−2 mm3   
 17 0.27 (0.20–0.34) 0.04 1.00 (0.75–1.25) 0.16 
 18 1.05 (0.56–1.54) 0.53 3.87 (2.06–5.69) 1.96 
 19 1.12 (0.59–1.66) 0.51 4.14 (2.18–6.10) 1.87 
 20 3.70 (2.42–4.98) 1.22 13.60 (8.89–18.31) 4.49 
 21 6.66 (4.40–8.92) 2.15 24.52 (16.21–32.83) 7.92 

CI, confidence interval; SD, standard deviation.

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.

Fig. 6.

Volumetric measurement. (A) Volume of knee joint components in each developmental stage. a pMFL volume. b ACL volume. c PCL volume. d Menisci volume. Each small dot indicates the volume value in each sample. *p < 0.05. The Steel-Dwass test was used for statistical analyses. (B) Volume ratio of each component. The ratio of the volume at each stage to the mean value at E17 was calculated. The values are shown as mean with a 95% confidence interval.

Fig. 6.

Volumetric measurement. (A) Volume of knee joint components in each developmental stage. a pMFL volume. b ACL volume. c PCL volume. d Menisci volume. Each small dot indicates the volume value in each sample. *p < 0.05. The Steel-Dwass test was used for statistical analyses. (B) Volume ratio of each component. The ratio of the volume at each stage to the mean value at E17 was calculated. The values are shown as mean with a 95% confidence interval.

Close modal

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].

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]. 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]. 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.

Additional Information

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.

1.
Deckey
DG
,
Tummala
S
,
Verhey
JT
,
Hassebrock
JD
,
Dulle
D
,
Miller
MD
,
.
Prevalence, biomechanics, and pathologies of the meniscofemoral ligaments: a systematic review
.
Arthrosc Sports Med Rehabil
.
2021
;
3
(
6
):
e2093
101
. .
2.
Pękala
PA
,
Łazarz
DP
,
Rosa
MA
,
Pękala
JR
,
Baginski
A
,
Gobbi
A
,
.
Clinical anatomy of the posterior meniscofemoral ligament of Wrisberg: an original MRI study, meta-analysis, and systematic review
.
Orthop J Sports Med
.
2021
;
9
(
2
):
232596712097319
. .
3.
Fox
AJS
,
Wanivenhaus
F
,
Burge
AJ
,
Warren
RF
,
Rodeo
SA
.
The human meniscus: a review of anatomy, function, injury, and advances in treatment
.
Clin Anat
.
2015
;
28
(
2
):
269
87
. .
4.
Moran
CJ
,
Poynton
AR
,
Moran
R
,
Brien
MO
.
Analysis of meniscofemoral ligament tension during knee motion
.
Arthroscopy
.
2006
;
22
(
4
):
362
6
. .
5.
Ohori
T
,
Mae
T
,
Shino
K
,
Tachibana
Y
,
Fujie
H
,
Yoshikawa
H
,
.
Complementary function of the meniscofemoral ligament and lateral meniscus posterior root to stabilize the lateral meniscus posterior horn: a biomechanical study in a porcine knee model
.
Orthopaedic J Sports Med
.
2019
;
7
(
1
):
232596711882160
. .
6.
Forkel
P
,
Herbort
M
,
Schulze
M
,
Rosenbaum
D
,
Kirstein
L
,
Raschke
M
,
.
Biomechanical consequences of a posterior root tear of the lateral meniscus: stabilizing effect of the meniscofemoral ligament
.
Arch Orthop Trauma Surg
.
2013
;
133
(
5
):
621
6
. .
7.
Knapik
DM
,
Salata
MJ
,
Voos
JE
,
Greis
PE
,
Karns
MR
.
Role of the meniscofemoral ligaments in the stability of the posterior lateral meniscus root after injury in the ACL-deficient knee
.
JBJS Rev
.
2020
;
8
(
1
):
e0071
. .
8.
Gupte
CM
,
Bull
AMJ
,
Thomas
RD
,
Amis
AA
.
A review of the function and biomechanics of the meniscofemoral ligaments
.
Arthroscopy
.
2003
;
19
(
2
):
161
71
. .
9.
Lertwanich
P
,
Martins
CAQ
,
Kato
Y
,
Ingham
SJM
,
Kramer
S
,
Linde-Rosen
M
,
.
Contribution of the meniscofemoral ligament as a restraint to the posterior tibial translation in a porcine knee
.
Knee Surg Sports Traumatol Arthrosc
.
2010
;
18
(
9
):
1277
81
. .
10.
Kim
JH
,
Ahn
JH
,
Kim
JH
,
Wang
JH
.
Discoid lateral meniscus: importance, diagnosis, and treatment
.
J Exp Orthop
.
2020
;
7
(
1
):
81
. .
11.
Ahn
JH
,
Lee
SH
,
Yoo
JC
,
Lee
YS
,
Ha
HC
.
Arthroscopic partial meniscectomy with repair of the peripheral tear for symptomatic discoid lateral meniscus in children: results of minimum 2 years of follow-up
.
Arthroscopy
.
2008
;
24
(
8
):
888
98
. .
12.
Ahn
JH
,
Wang
JH
,
Kim
DU
,
Lee
DK
,
Kim
JH
.
Does high location and thickness of the Wrisberg ligament affect discoid lateral meniscus tear type based on peripheral detachment
.
Knee
.
2017
;
24
(
6
):
1350
8
. .
13.
Lee
DH
,
Kim
TH
,
Kim
JM
,
Bin
SI
.
Results of subtotal/total or partial meniscectomy for discoid lateral meniscus in children
.
Arthrosc J Arthrosc Relat Surg
.
2009
;
25
(
5
):
496
503
. .
14.
Weninger
WJ
,
Mohun
TJ
.
Phenotyping transgenic embryos: a rapid 3-D screening method based on episcopic fluorescence image capturing
.
Nat Genet
.
2002
;
30
(
1
):
59
65
. .
15.
Rosenthal
J
,
Mangal
V
,
Walker
D
,
Bennett
M
,
Mohun
TJ
,
Lo
CW
.
Rapid high resolution three dimensional reconstruction of embryos with episcopic fluorescence image capture
.
Birth Defects Res C Embryo Today
.
2004
;
72
(
3
):
213
23
. .
16.
Tsuchiya
M
,
Yamada
S
.
High-resolution histological 3D-imaging: episcopic fluorescence image capture is widely applied for experimental animals
.
Congenital Anom
.
2014
;
54
(
4
):
250
1
. .
17.
Takaishi
R
,
Aoyama
T
,
Zhang
X
,
Higuchi
S
,
Yamada
S
,
Takakuwa
TT
.
Three-dimensional reconstruction of rat knee joint using episcopic fluorescence image capture
.
Osteoarthritis Cartilage
.
2014
;
22
(
10
):
1401
9
. .
18.
Zhang
X
,
Aoyama
T
,
Takaishi
R
,
Higuchi
S
,
Yamada
S
,
Kuroki
H
,
.
Spatial change of cruciate ligaments in rat embryo knee joint by three-dimensional reconstruction
.
PLoS One
.
2015
;
10
(
6
):
e0131092
. .
19.
Gupte
CM
,
Bull
AMJ
,
Murray
R
,
Amis
AA
.
Comparative anatomy of the meniscofemoral ligament in humans and some domestic mammals
.
Anat Histol Embryol
.
2007
;
36
(
1
):
47
52
. .
20.
van der Merwe
J
,
van den Heever
DJ
,
Erasmus
P
.
Variability, agreement and reliability of MRI knee landmarks
.
J Biomech
.
2019
;
95
:
109309
. .
21.
Faul
F
,
Erdfelder
E
,
Lang
AG
,
Buchner
AG
.
G*Power 3: a flexible statistical power analysis program for the social, behavioral, and biomedical sciences
.
Behav Res Methods
.
2007
;
39
(
2
):
175
91
. .
22.
Faul
F
,
Erdfelder
E
,
Buchner
A
,
Lang
AG
.
Statistical power analyses using G*Power 3.1: tests for correlation and regression analyses
.
Behav Res Methods
.
2009
;
41
(
4
):
1149
60
. .
23.
Ito
M
,
Kida
MY
.
Morphological and biochemical re-evaluation of the process of cavitation in the rat knee joint: cellular and cell strata alterations in the interzone
.
J Anat
.
2000
;
197 Pt 4
(
Pt 4
):
659
79
. .
24.
Kim
EY
,
Choi
SH
,
Ahn
JH
,
Kwon
JW
.
Atypically thick and high location of the Wrisberg ligament in patients with a complete lateral discoid meniscus
.
Skeletal Radiol
.
2008
;
37
(
9
):
827
33
. .
25.
Kim
MS
,
Park
HJ
,
Kim
SJ
,
Kim
JN
.
Attachment type, thickness, and volume of the posterior meniscofemoral ligament and meniscal pathology
.
J Digit Imaging
.
2022
;
35
(
6
):
1590
8
. .
26.
Minic
M
,
Zivanovic-Macuzic
I
,
Jakovcevski
M
,
Kovacevic
M
,
Minic
S
,
Jeremic
D
.
The influence of the morphometric parameters of the intercondylar notch on occurrence of meniscofemoral ligaments
.
Folia Morphol
.
2022
;
81
(
1
):
190
5
. .
27.
Amis
AA
,
Bull
AMJ
,
Gupte
CM
,
Hijazi
I
,
Race
A
,
Robinson
JR
.
Biomechanics of the PCL and related structures: posterolateral, posteromedial and meniscofemoral ligaments
.
Knee Surg Sports Traumatol Arthrosc
.
2003
;
11
(
5
):
271
81
. .
28.
Amis
AA
,
Gupte
CM
,
Bull
AMJ
,
Edwards
A
.
Anatomy of the posterior cruciate ligament and the meniscofemoral ligaments
.
Knee Surg Sports Traumatol Arthrosc
.
2006
;
14
(
3
):
257
63
. .
29.
Nagasaki
S
,
Ohkoshi
Y
,
Yamamoto
K
,
Ebata
W
,
Imabuchi
R
,
Nishiike
J
.
The incidence and cross-sectional area of the meniscofemoral ligament
.
Am J Sports Med
.
2006
;
34
(
8
):
1345
50
. .
30.
Robinson
SR
,
Smotherman
WP
.
Behavioral response of altricial and precocial rodent fetuses to acute umbilical cord compression
.
Behav Neural Biol
.
1992
;
57
(
2
):
93
102
. .
31.
Robinson
SR
,
Smotherman
WP
.
The emergence of behavioral regulation during fetal development
.
Ann N Y Acad Sci
.
1992
;
662
:
53
83
. .
32.
Oláh
T
,
Cai
X
,
Michaelis
JC
,
Madry
H
.
Comparative anatomy and morphology of the knee in translational models for articular cartilage disorders. Part I: large animals
.
Ann Anat
.
2021
;
235
:
151680
. .
33.
Oláh
T
,
Michaelis
JC
,
Cai
X
,
Cucchiarini
M
,
Madry
H
.
Comparative anatomy and morphology of the knee in translational models for articular cartilage disorders. Part II: small animals
.
Ann Anat
.
2021
;
234
:
151630
. .