3.1 In a rabbit model of impingement knee cartilage injury, we found an innate immune response mechanism at the early stage of injury.
As described above, We applied Borrelli’s method in rabbits to verify the pathological changes of impingement knee cartilage injury. According to the experimental plan, after the surgery of rabbit, we have selected 1, 2 and 4 weeks as the detection time points for research. At 1 week after injury, the articular cartilage was significantly degenerated in rabbits with impingement knee cartilage injury ,as shown by HE and safranin-O & fast green staining(Fig. 1A,B) and the Mankin’s scores were also signifificantly increased compared with those of control rabbits in 0 week (Fig. 1C). All joint specimens obtained were photographed for observation prior to histological examination,We can observe the repair of articular cartilage injury at different time points, the cartilaginous surface of the joint after injury is repaired to varying degrees over time (Fig.S1).As time went on, the degree of cartilage damage gradually decreased, and the cartilage damage was significantly improved four weeks later. We also performed MRI scanning and marked imaging on the articular surface of different groups of rabbit joints, and observed that the normal articular cartilage surface was clear and smooth, with complete morphology and uniform thickness. Form the result,1 week after the injury, the cartilage injury site was obvious and clear, as it is showed by the red arrow marks. Cartilage damage was still visible 2 weeks later, however, after 4 weeks the cartilage basically returned to normal (Fig.1D).
Meanwhile, one week after the injury, the expression of MMP13 and MMP9,a molecule directly involved in cartilage degradation, was significantly increased compared with control rabbits of 0 week ,and the expression of MMPs decreased gradually with the extension of injury time. To further quantitatively detect the expression of genes related to cartilage repair, molecular markers COL1A1, COL2A1, MMP9, MMP13 and ADAMTS-5 were selected to be observed at different time points after cartilage injury. The results indicated that the expression levels of COL1A1, COL2A1 and MMP9 gradually increased 1 week after impaction, and reached a peak at 2 weeks after injury. Meanwhile, the expressions of MMP13 and ADAMTS-5 peaked in the first week after injury, and then decreased gradually (Fig.1E-I). In order to further verify damage, we further tested the protein level of the cartilage, result of western blot showed that the expressions of COL1A1, COL2A1 and MMP9 were the highest in the second week, while the expressions of mmp13 and ADAMTS5 were the highest in the third week (Fig.1H,S2).
Based on these results, we successfully constructed an animal model of cartilage impact injury, and further verified the changes of different injury factors during the injury process through histology and molecular studies.