Table 1.
Sequences of the used primers and their references.
Fig 1.
A) The femoral condyles of the ACLR knee joint of animal #8952 showed no visible change/deterioration of the cartilage. B) The femoral condyles of the ACLR knee joint of animal #8955 showed different characteristics in form of a reduced cartilage coating (1) in the lateral condyle and even erosion of the cartilage (2) in the medial condyle. C) The lateral tibia plateau surface of the sham-operated knee joint of animal #8958 showed a slightly rough cartilage surface (arrow). A-C: The scale bars were determined with the corresponding x-ray images using the program RadiAnt DICOM viewer (build 3.4.1.13367, retrieved from http://www.radiantviewer.com; October 12th 2016; Medixant (Poznan, Poland)).
Fig 2.
Results of the histological scoring after consensus.
The score for each area of interest–the medial/lateral condyle and the medial/lateral tibiaplateau of the ACLR and sham-operated knee joints- of eight Göttingen Minipigs are shown. The used score [35] ranges from 0 points with no changes up to 25 points. As the results of the scoring showed, the sham knee joints were also affected with no significant differences between the ACLR and sham operated knee joints.
Fig 3.
Comparison of MRI (A and B) with histological findings (C). The medial condyle region of the ACLR knee joint of animal #8958 showed no salience neither in the sagittal scan through the medial compartment of the knee (A) nor in the coronal MRI-scan (B). The scale bars for A and B were determined with the program RadiAnt DICOM viewer (see caption Fig 1). The histological safranin-o staining (C) showed a normal cartilage tissue (Little-score: 0 points). The histological and MRI results did match.
Fig 4.
Comparison of MRI (A and B) with histological findings (C). The medial tibia plateau region of the ACLR knee joint of animal #8952 showed in the sagittal MRI-scan (A) through the medial compartment no salience but in the coronal MRI-scan (B) a signal irregularity (1) of the normally bright appearing cartilage was present and was therefore rated as pathological; no cyst or osteophytes were present. The scale bars for A and B were determined with the program RadiAnt DICOM viewer (see caption Fig 1). C) The histological safranin-o staining showed a mild degeneration (Little-score: 4.0 points) with only a slight reduction in staining (1). The darker formations in the cartilage (2) were interpreted as creases of the slice and were not included in the scoring. Thus, the histological and MRI findings did not match.
Fig 5.
Comparison of MRI (A and B) with histological findings (C). The lateral tibia plateau region of the sham knee joint of animal #8958 showed no salience neither in the sagittal MRI-scan (A) through the lateral compartment of the knee nor in the coronal MRI-scan (B) and was therefore rated as unchanged. The scale bars for A and B were determined with the program RadiAnt DICOM viewer (see caption Fig 1). In contrast to the MRI results, the histological safranin-o staining (C) showed a moderate degeneration (Little-score: 10.5 points) with a fissure (1) in the cartilage and detachment of the topmost layer of the cartilage tissue. The histological finding and the MRI result did not concur.
Fig 6.
Comparison of MRI (A and B) with histological findings (C). The medial condyle region of the ACLR knee joint of animal #8955 showed in the sagittal MRI-scan (A) through the medial compartment no salience but the coronal MRI-scan (B) showed a signal irregularity (1) in the cartilage layer and was therefore rated as pathologically changed. The scale bars for A and B were determined with the program RadiAnt DICOM viewer (see caption Fig 1). The histological safranin-o staining (C) showed a severe degeneration (Little-score: 19.25 points) with a fissure (1) reaching to the subchondral lamella and severe erosion (2) of the cartilage. Reduced safranin-o staining of the remaining cartilage tissue indicated a reduced GAG content and a severe loss of chondrocytes could be seen. The histological and MRI results did match.
Fig 7.
MRI scans of eight Göttingen Minipigs were scored according to an adapted WORMS-score. The score ranges from 0–22 points. The results showed that in the samples, either from ACLR or sham knee joints, no or only minor changes were visible in the MRI images.
Fig 8.
The ACLR knee joint A) of animal #4085 showed only the ligamentum cruciatum posterius (arrow) whereas the sham knee joint B) of the same animal showed both cruciate ligaments were present (arrows). This showed that the operation of resecting the ligamentum cruciatum anterius was successful (A). A, B: The scale bars were determined with the program RadiAnt DICOM viewer (see caption Fig 1).
Fig 9.
Radiological grading (from 0 to 4) according to the Kellgren and Lawrence Score [32]. The examined areas of the ACLR and sham sites showed similar findings in terms of degeneration. This also supported our assumption that the sham-operated knee joints may not qualify as a reliable control group. Another aspect of the x-ray analysis was that only the medial condyles of the ACLR and sham sites showed changes of any kind. This would indicate that changes would be initially starting in the medial condyle.
Fig 10.
Normalized (ß-actin) gene expression for the evaluated genes from the examined regions of ACLR and sham knee joints. The results showed no evident differences between the ACLR and the sham operated side for the assessed genes A) col1A1, D) mmp1, E) mmp3, F) mmp13, H) adamts5, I) il-1ß and J) vegf. This would concur with the histological findings, where the sham operated group was as degenerated as the ACLR group and therefore not usable as a serious control group. The gene expressions of B) col2, C) acan and G) adamts4 at first sight appeared to be differently expressed but as standard deviation was high, significant differences were not found. So no significant differences between the ACLR and sham sites were found in terms of gene expression in the end and therefore, no discrimination between an induced (ACLR) and spontaneous (sham) OA could be made.
Table 2.
Correlation gene expression vs histology.
Correlation (r-values) of the gene expression findings with the scored histological findings.
Fig 11.
Results of the immunohistochemical staining of the examined regions of ACLR and sham operated knee joints with A) collagen II and B) collagen I. The score ranges from 0 (no staining) to 3 points. The results indicated no evident differences between the ACLR side and the sham side for neither Col II (A) nor Col I (B). This was in concurrence with our histological results (Fig 2). Sample of an immunhistological staining of collagen II (C) and collagen I (D) from the moderate degenerated (histological score: 9 points) sham operated side of the lateral tibia plateau of animal #8952 with an intense staining for collagen I (3 points) and a weak staining for collagen II (1 point).