Our study demonstrated that joint hemorrhage exacerbated articular cartilage degeneration in a rat immobilized knee model. Joint hemorrhage progression decreased the number of chondrocytes, independent of the cartilage area. Iron deposition in and inflammation of the synovium persisted until 8 weeks after blood injection, and joint hemorrhage increased the expression of MMP-8 and MMP-13 throughout the cartilage. Furthermore, joint hemorrhage aggravated cartilage degeneration induced by joint immobilization in the contact area. The differences in the contact and non-contact areas of the immobilized knee model can be attributed to the degree of mechanical stress or residual mobility [15, 27, 28]. Patellar mobility was not restricted, and it could move slightly in the medial or lateral direction. In the femoral non-contact area adjoined to the patella, immobility rigidity was less than that in the contact area.
Mechanical stress on cartilage determines the delicate balance between cartilage growth and breakdown . Some studies have reported that mechanical stress induces cartilage degeneration, whereas others have shown that mild and moderate loading can stimulate cartilage matrix synthesis [29-31]. Joint hemorrhage, when combined with mechanical loading, exerts harmful effects on cartilage matrix turnover and integrity . In our immobilized knee model, significant decreases in the number of chondrocytes were observed in both contact and non-contact areas of the Im-B group. However, cartilage degeneration was severe only in the contact area of the Im-B group. A previous study showed that iron-induced synovitis and hydroxy radicals cause chondrocyte apoptosis , but we did not assess it. The results of this study suggested that chondrocyte death by apoptosis or necrosis was stimulated by hemorrhage, regardless of mechanical stress. Thus, avoidance of mechanical stress may prevent the progression of cartilage degeneration. In clinical practice of hemophilic arthropathy treatment, articular cartilage is damaged symmetrically and broadly . However, there have been no studies to evaluate the changes in cartilage thickness due to joint hemorrhage. A previous study assessed cartilage thickness using a rat immobilized knee model  and reported that joint immobilization induced cartilage thickening due to cartilage regeneration induced by compressive and shear force at the joint. In this study, cartilage thickness was not altered by joint hemorrhage and immobilization. The difference might be due to the dual effects of cartilage regeneration and degeneration caused by joint immobilization and hemorrhage, immobilization periods, or portion of cartilage assessed in the study.
Iron deposition was observed for up to 8 weeks in the Im-B group. Jansen et al. described opsonization of injected red blood cells into the joint cavity and recognition as foreign by macrophages and synoviocytes . Additionally, the concentration of red blood cells in the joint cavity decreased to less than 5% within 48 h; however, this time course was sufficient to adversely affect the cartilage and synovial tissues . Furthermore, Onoda et al. reported iron deposition for 8 weeks in the synovial membrane and capsule, employing the same model used in this study . The results were consistent with our findings, indicating that immobilization could inhibit hemosiderin absorption and prolong hemosideric inflammation. The synovitis score was slightly but not significantly higher in the Im-B group than in the Im-NS group, and CD68-positive cells were primarily observed in the synovial membrane, indicating that iron deposition potentially induces synovial inflammation. Furthermore, CD68-positive cells tended to be more numerous in the Im-B group than in the Im-NS group at 4 weeks, and this number was similar in both groups at 8 weeks. The effect of joint hemorrhage on inflammation reduced after 4 weeks, after which immobilization was considered as the main cause of inflammation [25, 33].
MMP-8 and MMP-13 act as collagenases to cleave type Ⅱ collagen, which is the basis of articular cartilage . MMP-8 is expressed in neutrophils, osteoarthritic chondrocytes, articular chondrocytes, and synovial fibroblasts . It interacts with inflammatory cytokines and contributes to chronic inflammatory diseases. In contrast, MMP-13 is the only collagenase implicated in the degradation of collagenous matrices, and has a higher enzyme activity than other MMPs in osteoarthritis . MMP-13 is known to be involved in hemophilic arthropathy and rheumatoid arthritis . Our study revealed that joint hemorrhage significantly increased the expression of MMP-8 and MMP-13 compared with that in the control group, regardless of mechanical stress. The findings indicated that even a single joint hemorrhage could lead to a higher expression of MMP-8 and MMP-13, which can cause cartilage breakdown. MMP-13 expression increased at 2 weeks in the contact area and at 4 weeks in the non-contact area of the Im-B group compared with that in the Im-NS group. Mechanical stress and strain have been associated with MMP-13 activation and synthesis [37, 38], and the difference in the time of expression in our study could be due to the influence of mechanical stress.
There are numerous studies on inflammatory cytokines such as IL-1α, IL-1β, and TNF-α in joint disorders, including osteoarthritis , rheumatoid arthritis, posttraumatic osteoarthritis , and hemophilic arthropathy [2, 6]. These cytokines are detected in the synovial fluid, synovial membrane, and cartilage , affecting chondrocytes and resulting in tissue destruction . It is considered that TNF-α promotes acute inflammation, whereas IL-1 plays a pivotal role in sustaining inflammation and cartilage destruction . IL-1β and TNF-α regulate MMP activation , and the activation of IL-1β and TNF-α originally precedes that of MMPs . In our study, TNF-α expression in the contact area was higher in the Im-B group than the Im-NS group at 2 weeks, which could lead to the subsequent MMP expression.
Mechanical stress activates TNF-α expression . Joint hemorrhage also increases TNF-α expression [6, 41], which might strengthen the effect of mechanical stress . Conversely, TNF-α expressions in the non-contact area was higher in the Im-B group than in the Im-NS group at 4 or 8 weeks. It might be due to chronic inflammation induced by prolonged deposition of hemosiderin without mechanical stress . Additionally, there was no significant difference in IL-1β expression between the hemorrhage and control groups. We speculate that the effect of immobilization on IL-1β gene expression might exceed that of hemorrhage.
The results of this study may have clinical implications. Joint hemorrhage exacerbated cartilage degeneration induced by joint immobilization. Drainage of a joint hemorrhage or avoidance of loading may help prevent cartilage degeneration during joint immobilization with a hemorrhage, and this should be assessed in future studies.
This study had some limitations. First, the actual amount of blood administered into the joint was not assessed. Second, we did not quantify protein expression related to the pathogenesis. Third, gene expression was not evaluated within 2 weeks. Fourth, the synovium was not assessed by PCR, and therefore, we could not show the active synovitis and its influence on cartilage. Fifth, the number of rats was not determined on the basis of the statistical power. Finally, we did not include a normally loaded control with blood injection, which made the difference between the effects of hemorrhage with or without loading unclear; this necessitates further studies.