Fig 1.
Imaging of measurement points.
A: A 3D reconstruction of the knee joint was generated from 2D continuous sections with Amira software. Femur and tibia (white), ACL (red) and PCL (blue). Red points indicate the attachment of the ACL to the femur and tibia. Blue points indicate the attachment of the PCL to the femur and tibia. The black point indicates the cross point of both ligaments. B: Measurement points. fACL: femoral attachment of ACL. fPCL: femoral attachment of PCL. tACL: tibial attachment of ACL. tPCL: tibial attachment of PCL. The cross point of ACL and PCL was indicated as intersection of cruciate ligament (iCL). Length of ACL (LACL) was calculated by length of ACL on femoral side (LAf) plus length of ACL on tibial side (LAt). Length of PCL (LPCL) was calculated by length of PCL on femoral side (LPf) plus length of PCL on tibial side (LPt).
Fig 2.
Histological findings of cruciate ligaments.
Histological images of sagittal sections of the knee joint by HE staining. a: Embryonic day (E) 16. The cross section of the knee showed no signs of the ACL and PCL in the interzone. b: E17. The cross section of knee clearly showed the ACL and PCL. c: E18. The cross section showed the PCL. d: E19. The cross section showed the ACL and PCL. e: E20. The border and bundle of the ACL were clearly visible. f: E20. The cross sections of the knee showed buckling of the ACL and PCL. Magnification x100. Bar = 100 μm.
Fig 3.
A: The length of the ACL at E17, E18, E19, and E20. The small red circles indicate the length of the ACL in each sample. *p < 0.05 (Dunnett T3 test). B: The length of the PCL at E17, E18, E19, and E20. The small blue circles indicate the length of the PCL in each sample. ** p < 0.01 (Tukey-Kramer test).
Fig 4.
The distance between the attachment points of the cruciate ligaments on the femur and tibia.
A: The length of the distance between the attachment points of the ACL and PCL on the femur (LfCL). The small black circles indicate the distance in each sample. p < 0.05 (Test of Homogeneity of Variances). B: The length of the distance between the attachment points of the ACL and PCL on the tibia (LtCL). The small black circles indicate the distance in each sample. p < 0.05 (Test of Homogeneity of Variances).
Fig 5.
A: The angle of ACL (αACL) at E17, E18, E19, and E20. The small red circles indicate the angle of ACL in each sample. ** p < 0.01 (Tukey-Kramer test). B: The angle of PCL (αPCL) at E17, E18, E19, and E20. The small blue circles indicate the angle of PCL in each sample. ** p < 0.01(Tukey-Kramer test).
Fig 6.
Cross angle of cruciate ligaments in each dimension.
A: Cross angle of cruciate ligaments in the sagittal plane [β(sagittal)]. Each small circle indicates the cross angle in each sample (purple; E17, green; E18, yellow; E19, red; E20). ** p < 0.01 (Tukey-Kramer test). B: Cross angle of cruciate ligaments in the coronal plane [β(frontal)]. Each small circle indicates the cross angle in each sample (purple; E17, green; E18, yellow; E19, red; E20). ** p < 0.01 (Tukey-Kramer test). C: Cross angle of cruciate ligaments in the horizontal plane [β(horizontal)]. Each small circle indicates the cross angle in each sample (purple; E17, green; E18, yellow; E19, red; E20). ** p < 0.01 (Tukey-Kramer test).
Fig 7.
Spatial changes of the attachment points of the cruciate ligaments in each dimension.
The average coordinates of the attachment points at each stage (purple; E17, green; E18, yellow; E19, red; E20) in the sagittal (A), frontal (B), and horizontal (C) planes. Femoral attachment of ACL (fACL). Femoral attachment of PCL (fPCL). Tibial attachment of ACL (tACL). Tibial attachment of PCL (tPCL). Superior (Sup), interior (Inf), anterior (Ant), posterior (Pos), medial (Med), lateral (Lat).