Osteoarthritis is an important cause of dyskinesia in elderly animals and reduced production performance of in-service animals [17]. At present, late OA, can only be relieved by joint replacement, which is not only expensive, but also painful for patients. Therefore, it is very important to clarify the pathogenesis and early diagnosis of osteoarthritis for the prevention and treatment of OA. At present, the diagnostic methods of OA include X-ray, MRI and arthroscopy. These imaging techniques have different sensitivities for the lesions of cartilage, synovium and subchondral bone [18, 19]. How to make early diagnosis of OA is still a problem to be solved. As a kind of highly sensitive molecules, biomarkers have been widely studied in OA and have broad promising applications [20–23]. However, few scholars have systematically studied the concentration trend of serum molecular markers in different stages of OA. So far, there is no reliable biomarker for veterinary clinical diagnosis. Therefore, in this study, the rat model of OA was induced by ACLT operation, and the pathological changes of OA cartilage and the concentration of CTX-II and C2C were detected longitudinally and jointly, so as to realize the evaluation of condition and early diagnosis of OA.
CTX-II, as a degradation product of type II collagen, reached the highest level of CTX-II in serum at 3 weeks after birth, decreased rapidly at 4 months after birth, and almost could not be detected in serum 7 months later. This study focused on the degradation of type II collagen to explore the biomarkers of OA. When OA occurs, type II collagen is destroyed by cartilage-degrading enzymes, produces CTX-II and then excreted into urine then [24]. Studies did by Reijman and Sowers et al have shown that urinary CTX-II (uCTX-II) is a reliable biomarker for early diagnosis of OA [25, 26]. In our study, two weeks after ACLT surgery, the concentration of serum CTX-II in the model group was significantly higher than that in the control group. And the concentration of CTX-II at each time point was higher than that of the control group. This is also the same result as previous studies [27]. The histological results in our study also have a corresponding experimental trend. This also shows that the articular cartilage has been obviously damaged from the second week in histology, and continued to aggravate with the passage of time, suggesting that CTX-II may also be used as a suitable biomarker for longitudinal study [28]. In addition, we used Mankin scoring system to explore the relationship between the concentration of CTX-II and the degree of articular cartilage degeneration. In our study, we found that the concentration of the model group was significantly higher than that of the control group. And the higher the score was, the more serious the degenerated of articular cartilage and the higher the concentration of CTX-II was. Therefore, the detection of CTX-II may become one of the effective biomarkers for the diagnosis of early OA.
Type Ⅱ collagen is the main component of articular cartilage. In osteoarthritis, type Ⅱ collagen is continuously degraded and cleaved into two length fragments by matrix metalloproteinases. The exposure of a new epitope in a fragment with a length of 3 to 4 is called C2C. Other studies have shown that type II collagen degradation fragment C2C in synovial fluid will also increases after knee injury [29]. Conrozier et al also confirmed that serum C2C was associated with joint space stenosis (JSN) in patients with OA, and the C2C level in OA group was higher than that in normal group [30, 31]. In our study, compared with the control group, the concentration of C2C in the model group also increased significantly. It was higher than that of the control group from the second week, and gradually higher than that of the control group at other time points. We have obtained consistent results from the eye changes and histological results. The relationship between Mankin scoring system and C2C also suggests that in our study, the higher the C2C concentration was, the higher the Mankin score was. It shows that there is a positive correlation between them. Therefore, we think that C2C may help us to detect the early diagnosis of OA.
In this study, both CTX-II and C2C may be used as biomarkers to evaluate OA. Therefore, the combined detection of CTX-II and C2C can more accurately diagnose the degree of degeneration of early osteoarthritis more accurately. Therefore, we carried out Spearman analysis on the change trend of serum CTX-II and C2C concentration and Mankin score in rat OA model. The results showed that there was a significant correlation between CTX-II and C2C and the OA model Mankin score (P < 0.001). It indicates that the change trend of CTX-II and C2C content is closely related to the severity of OA, and the more serious OA is, the stronger the degradation of cartilage matrix and the higher the concentration will be. Some studies have shown that the metabolic cycle of type II collagen in normal cartilage is very long, and the concentration of its metabolic degradation product CTX-II can evaluate the degradation of type II collagen in a certain period of time, and can also reflect the degree of degradation of type II collagen in mineralized tissues [32, 33]. In a follow-up study, Cahue also found that C2C levels were closely related to the severity of the disease [34]. This is similar to our study, but also supports our research. After that, we analyzed the molecular markers C2C and CTX-II and their combined biomarkers by ROC, and evaluated the diagnostic value of C2C and CTX-II in the detection of rat OA. The results showed that the AUC value of combined biomarkers was significantly higher than that of CTX-II and C2C. It shows that the combined detection of serum CTX-II and C2C has a certain diagnostic value for the detection of rat OA.
In this study, the OA model was evaluated from three aspects: gross observation and score, Mankin score and HE staining. The results showed that the degree of articular cartilage injury aggravated with the extension of modeling time, cartilage matrix defect aggravated and chondrocyte loss increased, which was similar to the previous research results [35]. It further indicates that when OA occurs, the matrix synthesis ability of articular cartilage decreases and the degree of degradation increases. In the sham operation group, only incision of the articular capsule without transection of the cruciate ligament would not cause osteoarthritis [36], so the changes of molecular markers in the sham operation group were not significant. We also verified the changes of serum type II collagen degradation products in SD rat OA model, and combined detection can further show that the degradation and metabolism of extracellular matrix of cartilage was enhanced during the occurrence of OA.
During OA, the change trend of CTX-II and C2C in SD rats is similar to that of OA, which has a certain value for the diagnosis of OA. It also shows that it may be used as a more potential combined biomarker for the diagnosis of OA, which is more stable and more sensitive than a single biomarker. However, this study also has some limitations, and the longitudinal research cycle is insufficient. There is no further study on the late changes of CTX-II and C2C. And only the tibial plateau was histologically analyzed. However, this study adopted scientific and effective methods within 10 weeks, so the results are reliable.