- X-ray performance of bilateral TFJ
In 3-month age group, there was no osteophyte formation of bilateral TFJ and K-L grade were all 0. In 6-month age group, small osteophyte was not seen or faintly visible at medial tibial plateau, and K-L grade was 0 in 7 cases, 1 in 3 cases. In 9-month age group, small osteophyte was faintly visible at tibial plateau and intercondylar ridge, and K-L grade was 1 in 3 cases, 2 in 7 cases. In 12-month age group, the tibial plateau showed obvious osteophytes, partial TFJ space stenosis, subchondral bone sclerosis and cystic changes, and K-L grade was 2 in 2 cases, 3 in 8 cases. In 18-month age group, the tibial plateau showed obvious osteophytes, subchondral bone sclerosis and cystic changes, and the bone ends were deformed, and K-L grade were all 4. It can be seen that with the increase of age, the K-L grade gradually increased, suggesting that osteoarthritis gradually worsened (Fig. 2).
- MRI findings of the knee joint
In 3-month and 6-month age group, the medial posterior intercondylar fossa of femur and patellofemoral joint space were both long T1 and long T2 signals, showing synovitis-like changes, but there was no significant difference between the two groups. In 9-month age group, synovitis-like changes were located in the medial posterior side of intercondylar fossa of femur, and the signal was stronger than 3-month and 6-month age group (Fig. 3). In 12-month age group, on the basis of synovitis-like changes, there was a further high-signal area of the subchondral bone in the distal femur and proximal tibia, which suggesting bone marrow lesions (BMLs). In 18-month age group, synovial-like signal intensity was significantly weaker than that of 12-month age group, while BMLs in the distal femur and proximal tibia were significantly enhanced compared with 12-month age group.
The MRI scores of KOA were consistent with 3-month and 6-month age group, and the difference was not statistically significant (P=1.000). Moreover, the score increased gradually after 6 months. The average score was 1.0±0.000 point at 3 and 6 months, 1.8±0.422 points at 9 months, 2.9±0.316 points at 12 months, 4.0±0.000 points at 18-month age group. The difference was statistically significant between each group (F = 303.840, P = 0.000).
- Micro-CT performance of TFJ
In 3-month and 6-month age group, microscopic callus formation was observed in the femoral condyle and proximal tibia. No typical subchondral bone cyst formation was observed, but the density reduction zone was seen between the distal femur and the proximal tibia. In 9, 12 and 18 months age group, the thickness of the subchondral bone plate and the width of the trabecular bone increased significantly. With the increase of age, the density reduction zone between the distal femur and the proximal tibia was gradually surrounded by trabecular bone. In 18-month age group, typical subchondral bone cyst formation can be seen, which was consistent with MRI intramedullary signal characteristics (Fig. 4).
Subchondral bone cysts can be observed at both 12-month and 18-month age group, which were located at the distal femur and proximal tibia. They showed a hardened cancellous bone which was adjacent around the subchondral bone plate and was thickened and not adapted to mechanical conduction. And the typical interior has no trabecular bone structure (Fig. 4).
With the increase of age, the subchondral trabecular bone in the weight-bearing area can collapse and fuse into pieces or clumps, and can merge with the adjacent subchondral bone plate. This phenomenon was found in the distal femur and the proximal tibia weight-bearing area at 9, 12 and 18 months age group (Fig. 4).
Subchondral bone cyst and subchondral trabecular collapse fusion are very similar to human KOA subchondral bone changes.
- Histology results of TFJ
In 6-month age group, articular cartilage had good gloss and transparency, no obvious cartilage defects were found in the distal femoral and proximal tibia articular surface weight-bearing areas, the trochlear crest of femur and proximal tibia appeared to have cartilage-like hyperplasia; In 9-month age group, the articular cartilage had good gloss and transparency, but lower than that of 6-month age group, the trochlear crest of femur was blunt, obvious cartilage-like hyperplasia can be seen at distal femur and proximal tibia; In 12-month age group, articular cartilage was turbid, visible cartilage surface defects were found in the distal femur and the proximal tibia articular surface weight-bearing area, and the defects of medial condyle were particularly obvious. Visible wax-like cartilage or bone-like hyperplasia were observed at the distal femur and proximal tibia. The "U"-shaped cartilage defect in the posterior part of medial condyle of proximal tibia was formed, corresponding to the turbid color of the cartilage in the medial condyle of femur; In 18-month age group, articular cartilage was turbid, except for the distal femur and proximal tibia cartilage surface defect, partial subchondral bone was exposed at the edge of the tibial plateau. The distal femur and the proximal edge of the tibia showed obvious wax-like osteochondral hyperplasia. The "U"-shaped cartilage defect in the posterior part of medial condyle was extended to the middle and front, and the cartilage was significantly thinner (Fig. 5).
4.2 Eosin-hematoxylin staining
In 6-month age group, articular cartilage showed normal or mild degeneration. Normal or reduced cartilage matrix staining was limited to the cartilage surface; the distribution of chondrocytes was normal or slightly increased; the tidal line was intact (Fig. 5).
In 9-month age group, articular cartilage showed mild degeneration. The cartilage surface was slightly irregular; normal or reduced cartilage matrix staining was limited to the cartilage surface; chondrocytes can be locally enlarged, vacuolar-like degeneration, or even partial full-thickness reduction; the tidal line was intact (Fig. 6).
In 12-month age group, articular cartilage can be degenerated at various stages, with moderate degeneration being the most common. The cartilage fissure or loss extended to the middle layer or even the deep layer. The reducing of cartilage matrix staining was limited to the surface layer or extended to the middle layer, and the medial condyle was most obvious; chondrocytes can be locally increased or decreased, while chondrocytes of medial condyle were reduced; intact tidal line, intersecting with blood vessels or multiple tidal lines can be observed (Fig. 6).
In 18-month age group, articular cartilage can be degenerated at moderate-severe stages, with severe degeneration being the most common. The cartilage fissure or loss located in the middle layer or extended to the deep layer. The reducing of cartilage matrix staining extended to the middle layer or even the deep layer, and the medial condyle was most obvious; chondrocytes showed diffuse increased or focal decreased; incomplete tidal line, intersecting with blood vessels or multiple tidal lines can be observed (Fig. 6).
The Mankin’s score increased with age, and the difference between the groups was statistically significant (F=101.490, P=0.000). Compared with the bilateral scores in each age group, the difference was not statistically significant. (P>0.05, Table 4).
4.3 Toluidine blue staining
The staining results of each age group were consistent with HE staining (Fig. 7). The Mankin’s score increased with age, and the difference between the groups was statistically significant (F=90.405, P=0.000). Compared with the bilateral scores in each age group, the difference was not statistically significant. (P>0.05, Table 5).
4.4 Type 2 collagen immunohistochemical staining
In 6-month age group, the distribution of Col-2a in articular cartilage was clear, and the focal staining of the lateral condyle cartilage weight-bearing area was reduced in the middle layer, and extended to the deep layer in medial condyle. The degree of coloration was coupled with cell proliferation, and the higher the degree of local cell proliferation, the weaker the positive staining of Col-2a (Fig. 8).
In 9-month age group, the reduction of Col-2a staining in articular cartilage was characterized by irregular distribution. The lateral condyle reduction zone was located in the surface layer and middle layer of cartilage, and reached to the deep layer in medial condyle, often accompanied by a decrease in the number of local cells. It was worth noting that the reduction of Col-2a staining may result in a jump change, that was, the deep and middle layer staining were reduced, while the surface layer was still continuous positive (Fig. 8).
In 12-month age group, the degree of reduction and involvement of the articular cartilage Col-2 staining was significantly aggravated compared with 6 and 9 months’ age group. The staining reduction zone was strip or sheet shape. The lateral condyle mainly involved the surface layer, middle layer and even deep layer of cartilage, and the medial condyle obviously involved the deep layer and even the whole layer. There were also cases where the middle layer and deep layer staining reduced, while the surface layer staining enhanced (Fig. 8).
18-month age group: The degree of reduction and involvement of the articular cartilage Col-2 staining was further aggravated compared with 12-month age group.
The staining reduction zone could involve the whole layer of articular cartilage, mainly the middle and deep layers. There was no significant difference in the depth of cartilage involved of internal and external condyle (Fig. 8).
With the increasing of age, the Mankin’s score gradually increased, and the difference between each age group was statistically significant (F=25.399, P=0.000), and the difference between the groups of 6, 9 and 12 months’ age was significant (LSD-t: 6vs9, P=0.006; 9vs12, P=0.006), there were statistical differences between the 12 and 18 months’ age group (P=0.028, Table 6); there was no significant difference in the bilateral scores of the same age group (P>0.05).
4.5 MMP13 immunohistochemical staining
In 6-month age group, the positive MMP13 mainly accumulated in distal femur and proximal tibia, the lateral edge of cartilage and the surface layer of articular cartilage. In the middle and deep layers, MMP13 was scattered in the cells, and distributed in cells and matrix in the surface layer, which was consistent with synovitis (Fig. 9).
In 9-month age group, positive MMP13 was disseminated by the intercondylar and cartilage lateral margins to the central weight-bearing area, showing a map-like distribution, mainly involving the surface layer and intermediate layer of the articular cartilage, and can also affect the whole layer. When the middle layer and the deep layer were involved, there were positive expressions in both cells and matrix (Fig. 9).
In 12-month age group, positive MMP13 showed scattered distribution of articular cartilage, but the expression decreased, which may be related to cell degeneration, necrosis, shedding and cartilage matrix degradation. Positive MMP13 was found in cartilage exfoliation, indicating that cartilage destruction was in progress (Fig. 8).
In 18-month age group, positive MMP13 showed a full-layer distribution of articular cartilage, and MMP13 line-like deposition occurred between the middle layer, deep layer and calcified layer. This phenomenon may reflect the pathological mechanism of cartilage exfoliation (Fig. 9).
With the increase of age, the Mankin’s score increased gradually. There was significant statistical difference between each age group (F=35.120, P=0.000), and the difference between groups was significant between 6, 9 and 12 months (LSD-t: 6vs9, P=0.000; 9vs12, P=0.005), the difference between 12-month and 18-month age group was statistically significant (P=0.032, Table 7); there was no significant difference in the bilateral scores of the same age group (P>0.05).
4.6 TFJ pressure measurement
With the increase of age, the IOP of the distal femur and the proximal tibia increased first and then decreased, which reached its highest in 12-month age group (distal femur IOP= 11.536±2.507 mmHg, proximal tibia IOP= 9.861±3.199 mmHg), the difference was statistically significant (femur side: F=8.261, P=0.000; tibia side: F=8.469, P=0.000). Compared with 6-month age group, the IOP increased slightly in 9-month age group, but the difference was not statistically significant (femur side: P=0.997; tibia side: P=0.973,); Compared with 9-month age group, the IOP was significantly higher in the 12-month age group, and the difference was statistically significant (femur side: P=0.019; tibia side: P=0.019); However, the IOP in 18-month age group was significantly lower than that in 12-month age group, and the difference was statistically significant (femur side: P=0.000; tibia side: P=0.000) (Fig. 10)
In the same age group, the IOP of distal femur was slightly higher than that of proximal tibia, and the difference was not statistically significant (P>0.05).
There was no significant difference in IOP between the distal femur and proximal tibia in the same age group (P>0.05).