动物实验,骨关节炎（OA） z-bo 2020-09-02 425

　　Background: Osteoarthritis (OA) is the most common joint pain and dysfunction syndrome, with varying degrees of functional limitation and poor quality of life. Since most osteoarthritis is irreversible and progressive, the cartilage layer is lost. The ideal treatment for OA is to block cartilage catabolism and promote normal cartilage regeneration. Palliative treatment of osteoarthritis usually focuses on pain and discomfort, improving motor function and preventing further degeneration. The main methods of clinical treatment of OA include the widespread use of non-steroidal anti-inflammatory drugs, analgesics and hyaluronic acid. These symptoms can be relieved in a short period of time, but there is no obvious effect on improving the disease. Therefore, there is an urgent need to develop alternative agents to fundamentally prevent the destruction of cartilage or promote its proper repair. Efforts to find effective cartilage preservation methods using cell sources. The method of expansion and transplantation of isolated chondrocytes is considered the basic solution. The main problem of chondrocyte culture is the loss of hyaline cartilage characteristics. In the dog model, it is reported that the cultured autologous chondrocytes cannot restore normal hyaline cartilage. Platelet-rich plasma (PRP) is defined as plasma with a platelet count greater than 1 x 106 cells/μl, and its platelet content is 4-8 times that of normal plasma. PRP has a variety of growth factors, including platelet-derived growth factor and transforming growth factor-β. PRP has angiogenic, anti-inflammatory and anti-degradation properties. According to reports, PRP transforming growth factor-β and fibroblast growth factor have anabolic effects on cartilage. These factors promote wound healing and extracellular matrix remodeling by stimulating angiogenesis and regulating cell migration and proliferation. Based on previous studies, we speculate that the combination of PRP and adipose mesenchymal stem cells has a synergistic effect on cartilage regeneration. The purpose of this study is to use a canine OA model to study the effects of PRP and adipose-derived mesenchymal stem cells on the morphology and regeneration of articular cartilage during inflammation.

　　Method: PRP preparation: Use the dual rotation method to prepare your own PRP. Collect 50 ml of fresh blood and add 7 ml of citrate dextrose formula A. The blood is then centrifuged at 1200pm for 10 minutes and divided into three layers: plasma, white blood cells and red blood cells. The plasma and buffy coat were separated into a new test tube, and the mixture was centrifuged at 2500 pm for 10 minutes. The supernatant was discarded, leaving only 20% plasma. The collected plasma (PRP) will be tested for a complete blood count and ensure that it is ≥1 x 106 platelets/μL. The prepared PRP was used within 6 hours. Isolation and culture of adipose-derived mesenchymal stem cells: Take out about 15 grams of adipose tissue from the side of the dog’s abdominal wall. Wash with phosphate buffered saline (PBS) several times to remove residual anesthetic and blood. Digest with 0.075% type I collagenase in a 37 degree water bath for 2 hours, and then turn over every 30 minutes. Add an equal volume of DMEM and 10% fetal bovine serum, and then centrifuge at 1200pm for 10 minutes. Remove the supernatant and blood lipids. The cell particles were washed with PBS and filtered through a 100 μm nylon mesh. After centrifugation under the same conditions, the cells were suspended in a 100×20 mm cell culture dish containing a modified low-sugar DMEM medium and 10% fetal bovine serum medium. After 24 hours, non-adherent cells and debris were washed with PBS, and the cell culture medium was changed twice a week. Collect and use fat-derived mesenchymal stem cells between the first and second stages. The established MSC flow cytometry analysis showed that the adipose-derived mesenchymal stem cell differentiation group (CD) 34, CD45 and CD29, CD44 positive.

　　Dog cruciate ligament amputation model: In this experiment, 24 healthy beagle dogs were selected. The dog weighs 7.7 ± 1.1 kg and is 2-3 years old. Under anesthesia, no. 11 Use a scalpel to remove the cruciate ligament on the right hind leg. Use conventional methods to suture connective tissue and skin. Postoperatively, analgesics (tramadol 8 mg/kg, subcutaneous) and antibiotics (enrofloxacin 5 mg/kg, subcutaneous) were given for 3 days. One week after the soft tissues healed, each dog walked for 10 minutes a day for 2 months. After that, treatment was given once a week for one month. Two months later, the dog was killed and a specimen was collected. Application of MSC and PRP: After establishing the canine osteoarthritis model, each group of materials was injected once a week, and the animals were injected intraarticularly for one month. The control group was treated with 1 ml PBS, the PRP group was 1 ml PRP, and the MSC group contained 1x107 in 1 ml PBS. Adipose-derived mesenchymal stem cells, MSC and PRP group (MP) contain 1x107 PRP in 1 ml PRP. The contralateral capsule of the control dog underwent histopathological examination without any treatment material.

Scoring: me line scoring: Surgery line scoring is measured once a month before and after surgery. All dogs walked normally before the operation and could not be petted. Using the previous scoring system, 0, no obvious softness, 1, slight lateral weight transfer, but soft when walking and walking. 4. Unable to bear weight when walking; 5. Unable to bear weight when walking and standing. Three veterinarians assessed the degree of the line. Determination of local compressive strength: After taking the animal samples, the in vitro compressive strength (0.2 mm) of the joint surface of the femur and tibia was tested by computer. The measurement points are the medial thigh and the central area of the tibia con. Histology: The articular cartilage of the lateral con of the femur and the central area of the tibia taken was taken from the knee joints of each group. Fix them with 10% neutral buffered formalin (NBF), and decalcify with decalcification solution (24.4% formic acid and 0.5N sodium hydroxide) for 14 days (mixed decalcification solution for 14 consecutive days, 1 day a day) . Has been swapped twice). Trim the articular cartilage of each femur and tibia longitudinally. Embed paraffin sections (3-4μm), and stain the cartilage tissue with Sirius Scarlet dye. ECM composition analysis: freeze-dry the femoral and tibial articular cartilage parts of each knee joint to obtain dry weight. The fragments are then digested and used for glycosaminoglycan and collagen analysis. The methyl methylene blue sulfated GAG was measured with an ultraviolet-visible spectrophotometer to determine the GAG content. Erlich's hydroxyproline content can measure collagen content. Use the following formula (μg hydroxyproline x dilution factor)/0.13 =μg collagen to convert the hydroxyproline content to the collagen content. Human meniscus hydroxyproline accounts for about 13% of the amino acid content of collagen. ECM-related chondrocyte gene mRNA expression: real-time fluorescent quantitative PCR technology to detect femoral and tibial articular cartilage j to monitor the expression of SOX9 gene and proteoglycan messenger RNA. BrdU uptake measurement: Intraperitoneal injection of 5-brodeoxyuridine-labeled proliferating cells to evaluate the effects of MSC and PRP or their combination (MP) on the proliferation of dog knee joint cells.. Inject BrdU 50 mg/kg into the dog's intraperitoneal cavity, It was dissolved in saline to 1 ml/kg, and the animals were sacrificed 72 hours later. BrdU incorporation was detected by immunohistochemical staining with anti-BrdU antibody on the section. Immunohistochemistry: Purify the primary antibody with avidin-biotin-peroxidase complex, and observe the immunoreactivity of BrdU as a cell proliferation marker. Using mesenchymal stem cells, PRP or conjugate, caspase-3, truncated poly(ADP) ribose polymerase (PARP) and tumor necrosis factor (TNF) treated with femoral and tibial surface cartilage tissue)-I observed The impact on α. Cyclooxygenase (COX) 2, interleukin (IL)-1β interferon (IFN)-γ, inducible nitric oxide synthase (iNOS) immune response.

　　 Bone Histomorphometry: In order to observe more detailed histopathological changes, Safranin O stained the articular cartilage damage and evaluated it using the Mankin scoring system. The higher the score, the more severe the OA. The computer-based automatic image analyzer can analyze the thickness of the femoral and tibial articular cartilage on the prepared longitudinally cut samples. The histological area of each group of six thighs and tibial joint areas was further analyzed. More than 20% of immune responses are considered positive.

　　Result: The me row score of the MP group was lower than that of the other groups, and there was no significant difference between the two groups. At 2 and 3 months after treatment, the me line scores of the PRP and MP groups were significantly lower than before treatment. The local compressive strength of the articular cartilage of the femur and tibia in the control group was significantly lower than that of the sham operation group. However, the treatment of the three materials significantly improved the local compressive strength. The local compressive strength of MP group was higher than other treatment groups. Compared with the sham operation group, the Mankin score of the control group was significantly improved. It has been found that compared with the control group, the Mankin scores of all three test materials in the articular cartilage of the femur and tibia are significantly reduced. In particular, the dog in the MP treatment group had the lowest Mankin score. The thickness of articular cartilage of MSC and PRP was higher than that of the control group. Compared with the PRP group, the MP group had a more obvious effect on the joint surface. Compared with the sham operation group, the cytokine content of the control group was significantly reduced. However, compared with the control group, each treatment group increased significantly. Real-time fluorescent quantitative PCR detection of ECM related genes, aggrecan, Sox9 gene expression is lower than the sham operation group. However, these three test materials significantly improved the expression of these down-regulated genes, and the MP group showed the strongest gene expression. Compared with the sham operation group, the BrdU-positive cells in the femoral and tibial articular cartilage of the control group were significantly reduced. There are significant differences between each treatment group and the control group. The proliferation of chondrocytes in the MP group was significantly higher than that in the MSC or PRP group. Compared with the sham operation group, the Caspase-3 and PARP immunopositive cells in the control group increased significantly. However, the treatment of these three test materials resulted in a significant reduction in the number of these cells, with the highest percentage of cells in the MP group. The cells stained with tumor necrosis factor α, COX-2, IL-1β, iNOS and IFN-γ in the femoral cartilage and tibial cartilage of the control group were significantly increased, which was significantly different from the sham operation group. These pro-inflammatory cytokines had less cartilage increase in the treatment group than in the control group.

　　 Conclusion: This study shows that the combination of MSC and PRP has a beneficial synergistic effect on OA through ECM synthesis, chondrocyte proliferation and anti-inflammatory response. Therefore, the combination therapy of MSC and PRP may be very useful as an inflammation modulator for the treatment of OA irreversible joint degeneration.