Cobalt chloride-induced hypoxic environment accelerates knee cartilage degeneration in New Zealand rabbits

Abstract

BACKGROUND: Cobalt chloride solution is commonly used to induce osteoarthritis cell models in vitro. However, its ability to construct animal models of osteoarthritis by intra-articular injection remains unknown. OBJECTIVE: To investigate the effect of intra-articular injection of different concentrations of cobalt chloride solution on cartilage degeneration in the knee joint. METHODS: Thirty-six healthy adult male New Zealand rabbits were randomly divided into four groups: low, medium and high dose cobalt chloride groups and control group. The right hind knee was intra-articularly injected with 100, 200, and 300 μmol/(L·kg) of cobalt chloride, while the left hind knee served as the control knee and was injected with an equal amount of normal saline. At 4, 8 and 12 weeks after operation, four rabbits were killed respectively. The cartilage on the surface of the femur was exposed for gross morphological observation, and then the cartilage tissues were taken for hematoxylin-eosin staining, safranine O-fast green staining, the Osteoarthritis Research Society International scoring, and immunohistochemical staining of interleukin 1 and tumor necrosis factor α, to determine cartilage degeneration in various aspects. RESULTS AND CONCLUSION: (1) Gross observation: At the same postoperative time point, with the increase of cobalt chloride concentration, cartilage degeneration showed a progressive aggravation trend, and the high-dose cobalt chloride group even involved the deep layer of cartilage and subchondral bone; under the same concentration of cobalt chloride, with the prolongation of modeling time, cartilage degeneration progressed progressively. (2) Hematoxylin-eosin staining, safranine O-fast green staining, and Osteoarthritis Research Society International scoring showed that at the same postoperative time point, with the increase of cobalt chloride concentration, the cartilage surface gradually became rough, the superficial layer became thinner, and the destruction aggravated, and the Osteoarthritis Research Society International score gradually increased (P < 0.05); under the same concentration of cobalt chloride, with the prolongation of modeling time, the arrangement of chondrocytes tended to be disordered, polarity was lost, and the destruction of superficial cartilage and subchondral bone progressively aggravated, and the Osteoarthritis Research Society International score gradually increased (P < 0.05). (3) Immunohistochemistry showed that at the same postoperative time point, with the increase of cobalt chloride concentration, cartilage degeneration aggravated, intracellular brown particles increased, and the positive expression of interleukin 1 and tumor necrosis factor α increased (P < 0.01); under the same concentration of cobalt chloride, with the prolongation of modeling time, cartilage destruction and fissures aggravated, and the positive expression of interleukin 1 and tumor necrosis factor α increased (P < 0.01). This experiment successfully established an osteoarthritis model of New Zealand rabbits induced by intra-articular injection of cobalt chloride solution, preliminarily verified the stability and reliability of the animal model, and also proved that with the increase of modeling concentration and the prolongation of modeling time, cartilage degeneration progressed progressively.

1. Introduction

Knee osteoarthritis is a chronic, degenerative joint disease characterized by knee pain, stiffness, and limited function, and in severe cases, joint deformity and muscle atrophy, severely affecting patients' quality of life and mobility [1-4]. Animal models are an effective means to study the occurrence, development, diagnosis, and treatment of chronic diseases in modern medicine. Establishing effective and reliable animal models is an important method to explore the specific pathogenesis of osteoarthritis [5]. Numerous studies have shown that cobalt chloride is the most commonly used chemical substance for inducing cellular hypoxia and hypoxic models in vitro [6-9].

For instance, Li Xinyi et al. [10] successfully established an in vitro hypoxia model of primary nucleus pulposus cells using cobalt chloride solution and studied the effects of different concentrations of cobalt chloride on cell proliferation, apoptosis, and expression of hypoxia-related genes and proteins. Li Xiaojuan et al. [11] treated chondrocytes from the spinal growth plate with cobalt chloride to establish an in vitro hypoxia model and further explored the role of the HIF-1α/YAP signaling pathway in maintaining the chondrocyte phenotype. Li Xiaofeng et al. [12] used different concentration gradients of cobalt chloride solution to treat rat bone marrow mesenchymal stem cells to create a hypoxia model, and then studied the mechanism of mangiferin on apoptosis and hypoxia protection.

However, through the above studies, it is found that cobalt chloride, as a reliable and efficient chemical inducer, is often used to establish in vitro cell hypoxia models and has achieved satisfactory results. But whether it can be used as an injectable preparation injected into the knee joint cavity to induce a hypoxic environment in chondrocytes to accelerate cartilage degeneration and the occurrence of osteoarthritis has not been studied. This experiment adopts the method of intra-articular injection of different concentrations of cobalt chloride solution into the knee joints of New Zealand rabbits to observe the effects on knee cartilage degeneration, aiming to establish a reliable chemical-induced osteoarthritis animal model and provide a new approach for the study of osteoarthritis pathogenesis and drug treatment.