Abstract: BACKGROUND: The current prevalence of osteoporosis and osteoarthritis comorbidity is extremely high in the elderly population, which is a major cause of pain, disability, fracture risk and socio-economic costs globally, but its pathogenesis remains unclear.
OBJECTIVE: To explore a suitable animal model of osteoporosis and osteoarthritis comorbidity, thereby providing a research basis for animal experiments of osteoporosis and osteoarthritis comorbidity.
METHODS: Sixty female Sprague-Dawley rats were randomly divided into sham-operated group 1, sham-operated group 2, osteoporosis-osteoarthritis model group and osteoarthritis-osteoporosis model group. In the osteoporosis-osteoarthritis model group, the osteoporosis model was first established by bilateral ovariectomy in rats, and 4 weeks later, the osteoarthritis model was constructed by the modified Hulth method. In the osteoarthritis-osteoporosis model group, the osteoarthritis model was first established by the modified Hulth method, and 4 weeks later, the osteoporosis model was constructed by bilateral ovariectomy. In the sham-operated group 1, the ovaries were not removed, and only the joint cavity was opened without damaging the intra-articular tissues, with the rest procedures being the same as those in the osteoporosis-osteoarthritis model group. In the sham-operated group 2, only the joint cavity was opened without damaging the intra-articular tissues, and the ovaries were not removed, with the rest procedures being the same as those in the osteoarthritis-osteoporosis model group. At 4, 8, and 12 weeks after modeling, Micro CT was used to detect the imaging changes of the knee joint and analyze the changes in bone microstructure. The expressions of osteoprotegerin, receptor activator of nuclear factor kappa B ligand, tumor necrosis factor α, and interleukin 1β in serum were detected by ELISA. Hematoxylin-eosin staining and safranin O-fast green staining were used to observe the pathological changes in the cartilage and subchondral bone of the knee joint, and tartrate-resistant acid phosphatase staining was used to observe the activity and distribution of osteoclasts in the subchondral bone of the knee joint.
RESULTS AND CONCLUSION: (1) Compared with the sham-operated groups, the bone mineral density of the distal femur in the model group decreased significantly at 4 weeks after operation (P < 0.000 1), and remained stable at 8 weeks after operation. Compared with the sham-operated group, the bone mineral density of the subchondral bone in the two model groups decreased significantly at 4 weeks after operation (P < 0.000 1), and showed an increasing trend at 8 weeks after operation. Compared with the sham-operated groups, the bone volume fraction and the number of bone trabeculae in the two model groups were significantly decreased (P < 0.01), and the separation of bone trabeculae was significantly increased (P < 0.01). Compared with the osteoarthritis-osteoporosis group, the osteoporosis-osteoarthritis group had a significantly lower bone volume fraction (P < 0.000 1) and a significantly higher trabecular separation (P < 0.001) at 4 weeks after operation, but there was no significant difference between the two model groups at 8 and 12 weeks after operation. (2) Compared with the sham-operated groups, the serum levels of inflammatory factors tumor necrosis factor-α and interleukin-1β in the two model groups were significantly increased (P < 0.01), and there were sighificant differences between the two model groups. The osteoprotegerin/receptor activator of nuclear factor kappa B ligand ratio was significantly down-regulated (P < 0.01), and there was no significant difference between the two model groups. (3) The structure of bone trabeculae was complete and continuous, the cartilage surface was smooth and the tide line was clear in the sham-operated groups, while the number of bone trabeculae was reduced, the cartilage surface was uneven, and the chondrocytes were unevenly distributed in the model groups at 4 weeks after operation. At 8 weeks after operation, the trabecular structure was broken, the joint space was reduced, and the tide line was interrupted. At 12 weeks after operation, proteoglycan was severely lost, and the red signal was weakened or completely faded. (4) The results of tartrate-resistant acid phosphatase staining showed that the positive rate in the sham-operated groups was evenly distributed, mainly distributed on the surface of bone trabeculae, without abnormal aggregation. The positive rate in the subchondral bone of the model groups increased, and abnormal aggregation of osteoclasts or invasion of cartilage calcification layer could be seen in some areas. To conclude, bilateral ovariectomy followed by modified Hulth method for 4 weeks can stably establish the animal model of osteoporosis and osteoarthritis comorbidities, with the early stage of comorbidities at 4 weeks postoperatively, and the middle to late stage of comorbidities at 8-12 weeks postoperatively.

Key words: osteoporosis, osteoarthritis,  animal model, rats, comorbidity, castration, Hulth method