Abstract: BACKGROUND: Knee osteoarthritis is an age-related disease, and aging is closely related to its occurrence and development. Chondrocyte senescence plays a crucial role in the pathological progression of knee osteoarthritis. 
OBJECTIVE: To establish a stable induced knee osteoarthritis model in SD rats. 
METHODS: (1) Animal experiment: Forty Sprague-Dawley rats were randomly divided into four groups: a blank control group (n=10) without modeling; a D-galactose group (n=10) with intra-articular injection of D-galactose solution (once a week for two months), establishing age-related knee osteoarthritis models; an anterior cruciate ligament transection group (n=10) with anterior cruciate ligament transection, establishing knee osteoarthritis models; and a D-galactose + anterior cruciate ligament transection group (n=10) with intra-articular injection of D-galactose solution (once a week for two months) one week after anterior cruciate ligament transection, establishing age-related knee osteoarthritis models. Starting one week after modeling, all four groups of rats underwent running exercise for 30 minutes daily, every other day. At weeks 4 and 8 post-modeling, after behavioral testing (Lequesne MG score), tissue samples were collected for analysis of inflammatory factor levels in knee synovial fluid, pathological morphology of knee cartilage, transmission electron microscopy observation of knee cartilage, and immunohistochemical staining for type II collagen and aggregates in knee cartilage. (2) Cell experiments: At weeks 4 and 8 post-modeling, knee chondrocytes were isolated and extracted from each group of rats. Flow cytometry was used for cell cycle analysis, β-galactosidase staining, and γ-H2AX immunofluorescence staining.
RESULTS AND CONCLUSION: (1) Animal experiments: At week 8 post-modeling, the Lequesne MG scores of rats in all three modeling groups were higher than those in the blank control group (P < 0.05). The Lequesne MG score of rats in the D-galactose + anterior cruciate ligament transection group was higher than that of the D-galactose group and the anterior cruciate ligament transection group (P < 0.05). At weeks 4 and 8 post-modeling, the levels of interleukin-1β, interleukin-6, and tumor necrosis factor-α in the synovial fluid of the knee joints in all three modeling groups were higher than those in the blank control group (P < 0.05). The levels of interleukin-1β, interleukin-6, and tumor necrosis factor-α in the synovial fluid of the knee joints in the D-galactose + anterior cruciate ligament transection group were higher than those in the D-galactose group and the anterior cruciate ligament transection group (P < 0.05). Hematoxylin-eosin and safranin-fast green staining and transmission electron microscopy at weeks 4 and 8 post-modeling showed that cartilage damage and chondrocyte mitochondrial damage were more severe in the D-galactose + anterior cruciate ligament transection group than in the D-galactose group and the anterior cruciate ligament transection group. Immunohistochemical staining results at weeks 4 and 8 after modeling showed that the expression of type II collagen and proteoglycans in the knee cartilage of the blank control group was the highest, while the expression of type II collagen and proteoglycans in the knee cartilage of the D-galactose + anterior cruciate ligament transection group was lower than that of the D-galactose group + anterior cruciate ligament transection group. (2) Cell experiments: At weeks 4 and 8 after modeling, the proportion of chondrocytes in the G0/G1 phase of the D-galactose + anterior cruciate ligament transection group was higher than that of the other three groups (P < 0.05), while the proportion of cells in the S phase and G2/M phase was lower than that of the other three groups (P < 0.05). The positive rate of β-galactosidase staining and the intensity of H2AX immunofluorescence in chondrocytes of the D-galactose + anterior cruciate ligament transection group were both higher than those of the other three groups (P < 0.05). These results indicate that the D-galactose + anterior cruciate ligament transection method can establish an aging knee osteoarthritis model in SD rats, and this model can better simulate the pathological state of aging and degeneration in knee osteoarthritis. 

Key words: knee osteoarthritis, aging, rat, animal model, anterior cruciate ligament transection, D-galactose, tissue construction