固定膝关节模型,动物造模 z-bio 2021-11-26 868

　　Abstract: Trauma, ligament reconstruction surgery, and bleeding disorders (such as hemophilia) can cause joint bleeding. The recurrence of bleeding in the joint cavity can cause synovitis and oxidative stress, which can lead to articular cartilage degeneration and arthropathy. Joint fixation is a common method to treat joint fractures with joint bleeding. Although joint bleeding has a negative impact on articular cartilage, there is no consensus on whether reducing joint bleeding can effectively prevent articular cartilage degeneration. The purpose of this study was to investigate the effect of joint bleeding combined with joint fixation on the degeneration of cartilage in the knee joint of rats. Methods: The knee joints of adult male rats were fixed with an internal fixator for 3 days to 8 weeks. The rats were randomly divided into the following groups: fixed blood injection (Im-B) group and fixed saline injection (Im-NS) group. The cartilage is evaluated in two areas: the contact area and the non-contact area. Assess chondrocyte count, modified Mankin score and cartilage thickness. Total RNA was extracted from cartilage in two areas, and the expression of MMP-8, MMP-13, IL-1β and TNF-α was detected by real-time quantitative polymerase chain reaction. Results: Compared with the Im-NS group, the number of chondrocytes in both parts of the Im-B group was significantly reduced. The modified Mankin score of the Im-B group at 4 to 8 weeks was significantly higher than the score of the Im-NS group only in the contact area. Compared with the Im-NS group, the expression of MMP-8 and MMP-13 in the Im-B group increased significantly within 2 to 4 weeks, while the expression of TNF-α increased significantly within 2 to 8 weeks . , IL-1β has no obvious gender difference. The results showed that joint bleeding exacerbated the degradation of articular cartilage caused by fixation. Eliminating joint bleeding or avoiding load may help prevent cartilage degeneration during joint fixation bleeding.

　　Background: Joint bleeding is usually caused by bleeding disorders, such as trauma, large joint surgery, and hemophilia. Repeated joint bleeding has direct or indirect adverse effects on cartilage degeneration. Bleeding causes iron to accumulate in the joint space, forming free radicals near the cartilage, leading to chondrocyte apoptosis and matrix deformation. In addition, iron induces angiogenesis and excessive proliferation of synovial cells, and subsequent inflammation of the synovial membrane causes arthropathy. This destructive event exacerbated by repeated bleeding is caused by a single blood exposure and may lead to the progression of continuous stromal aplasia and joint damage. In hemophilic arthritis, the synovial tissue full of hemosiderin produces pro-inflammatory cytokines, such as interleukin (IL)-1β and tumor necrosis factor (TNF)-α. Matrix metalloproteinases (MMP), osteoarthritis and rheumatoid arthritis. These cytokines also increase the absorption of iron by monocytes and synovial fibroblasts, thereby accelerating the vicious cycle of synovitis. However, methods for inducing joint bleeding, including bone marrow stimulation, have been proven to be an effective method for the treatment of knee cartilage defects through cartilage regeneration. After a fracture or ligament injury, even after proper treatment or surgery, post-traumatic osteoarthritis may occur. According to previous studies, trauma and surgery can lead to articular cartilage degeneration, inflammation, synovial cell hypertrophy, and post-traumatic osteoarthritis. In addition, joint fixation is a common treatment method for wound repair to maintain rest and promote recovery, thereby causing degenerative changes in articular cartilage, leading to the development of osteoarthritis. The blood in the joints will be quickly cleared within 48 hours without fixing the joints. However, one study reported that there was too much blood remaining during joint replacement surgery. Arthrodesis can exacerbate the harmful effects of blood on articular cartilage, which has not been studied. Therefore, this study investigated the effect of joint bleeding combined with joint fixation on the degeneration of articular cartilage in rats.

　　Animals: 12-week-old adult male SD rats were used. The number of rats used in this study was determined based on previous studies. A total of 108 rats were used (histological and immunohistochemical evaluation, n = 72; gene expression analysis, n = 36). Rats were anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg/kg), and a plastic plate and two metal screws were used to fix one knee joint with 150 degrees of flexion at different times. After the operation, the rats were randomly divided into a fixed blood injection group (Im-B) and a fixed saline injection group (Im-NS). Rats in the Im-B group were injected with 50 μL of autologous blood directly from the tail vein to the knee joint. Rats in the Im-NS group were given the same amount of normal saline in the same manner.

　　Organization preparation: Based on previous research, prepare the sample to be evaluated. The rats were euthanized by intraperitoneal injection of excessive sodium pentobarbital, then perfused and fixed with 4% paraformaldehyde (PFA) 0.1 μM phosphate buffered saline (PBS, pH 7.4) through the aorta. Subsequently, the tissue around the knee joint was excised and kept in the same fixative at 4°C for 24 hours. The fixed samples were decalcified with 10% ethylenediaminetetraacetic acid (EDTA) at 4°C for 2 months. After dehydration with ethanol and xylene solution, the sample was embedded in paraffin. For evaluation, a 5μm sagittal section was cut in the medial con area of the knee joint. Histological evaluation: hematoxylin-eosin staining and sarcosin O staining are used to evaluate cartilage degeneration, chondrocyte count and cartilage thickness in the contact and non-contact areas of the femur and tibia (postoperatively), 1 day and 3 days, respectively , And 1 week, 2 weeks, 4 weeks, 8 weeks after surgery, n =? 6 / per group). A modified Mankin histological grading scheme was used to assess cartilage degeneration. When calculating cartilage cells in a given field of view, cartilage thickness refers to the distance between the cartilage surface and the osteochondral joint. Use the synovial score system to assess synovitis (including vascular nu, synovial hyperplasia, and subsynovial inflammation). In addition, Perls Prussian blue staining (2, 4, and 8 weeks after surgery) assessed the deposition of hemosiderin.

　　Immunohistochemistry: The paraffin sections of each period were degreased and soaked in 0.3% hydrogen peroxide. Cluster of differentiation 68 (CD68) is used as a marker for type A synovial cells. At room temperature, 3% H2O2 dissolved in PBS inactivated endogenous peroxidase for 20 minutes. The slides were incubated with mouse anti-mouse CD68 antibody at 4°C for 24 hours. Use goat anti-mouse CD68 immunoglobulin (IgG) as the secondary antibody and incubate at room temperature for 30 minutes. The final detection step is to use DAB containing 0.1μm imidazole and 0.03% H2O2 as a coupler. Use Karachi hematoxylin for counterstaining. The negative control uses normal mouse IgG as the primary antibody. CD68 positive cells were counted in the joint space and posterior synovial capsule.

　　Gene expression analysis: After euthanasia, use a scalpel and bone clamp to obtain cartilage in the contact and non-contact areas of the femur and tibia. Immediately immerse the collected cartilage sample in 1 ml QIAzol. The sample was ground and homogenized with Polytron. Extract total RNA and synthesize complementary DNA (cDNA). Results: Histological evaluation: Figure 1 shows the histological characteristics of femoral cartilage stained with hematoxylin and eosin. Articular cartilage degeneration occurred in both Im-B and Im-NS groups. After fixation, degenerative changes gradually appeared, showing degenerative changes in the contact area (Figure 1a-h). After three days of fixation and intra-articular administration, chondrocyte swelling and loss of tangential cells were observed, especially in the Im-B group (Figure 1a and B). The cell layer was irregular at 1 week (Figure 1c and d). The staining intensity of cells in the Im-B group was more severe and decreased within 4 weeks (Figure 1e and f). In the Im-B group, irregular cartilage surface was observed in the second week. Cell cloning and cell loss occurred in the 8th week (Figure 1g and h). Figure 1 (i-p) shows the deterioration of the non-contact area. Similar cell structures were observed in the contact area and the non-contact area, but the cartilage degeneration in the non-contact area was less than that in the contact area. In both groups, the staining intensity of crocin O was observed to decrease at 8 weeks. The Im-B group is more serious than the Im-NS group. The Mankin score of the Im-B group was significantly higher than that of the Im-NS group at 4 weeks, while the tibia was significantly higher than that of the Im-NS group at 4 and 8 weeks. There was no significant change in the non-contact area, nor was there a significant difference between the Im-B group and the Im-NS group. Compared with the Im-NS group, the number of chondrocytes in the femoral contact area of the Im-B group was significantly reduced at the 3rd and 8th weeks, while the number of chondrocytes in the tibial contact area were at the 3rd, 1st and 8th weeks, respectively. In the non-contact area of the Im-B group, the number of femoral and tibial chondrocytes decreased significantly on the 3rd day and the 1st week. There is no change in cartilage thickness. Fixation of contact and non-contact areas and intra-articular administration do not affect cartilage thickness. At all stages, the score of synovitis in the Im-B group was slightly higher than that in the Im-NS group, but there was no significant difference.

　　Pearl Prussian blue staining evaluates iron deposits. Blood injection can cause joint bleeding, but there is no iron ore deposit in the Im-NS group. The presence of iron ore on the synovial and meniscus surface of the Im-B group continued until 8 weeks after the administration. CD68-positive cells were found in both the Im-B and Im-NS groups, and they were mainly distributed in the synovium. The number of CD68-positive cells in the Im-B and Im-NS groups increased slightly between the 4th week and the 8th week, but there was no significant difference. Compared with the Im-NS group, the expression of MMP-8 and MMP-13 in the femur of the Im-B group increased significantly at the second week, and the expression of MMP-8 in the tibia at the second week. Compared with the Im-NS group, the expression of MMP-8 and MMP-13 in the femur and tibia of the Im-B group increased significantly within 2 weeks. There was no significant difference in IL-1β expression. After 2 weeks, compared with the Im-NS group, the expression of TNF-α in the femoral contact area of the Im-B group increased significantly. Compared with the Im-NS group, the expression of the Im-B group increased significantly in the non-contact area of the femur at the 8th week and in the non-contact area of the tibia at the 4th and 8th weeks.

　　Conclusion: Joint bleeding exacerbates cartilage degeneration caused by joint fixation. Eliminating joint bleeding or avoiding load may help prevent cartilage degeneration during joint fixation bleeding.