动物造模,膝关节多发性缺损,软骨细胞移植 z-bo 2022-10-24 424

　　Background: Since articular cartilage cannot heal naturally, there is no cure for methods that can regenerate hyaline cartilage in a consistent and predictable manner. Some recognized cartilage repair methods include minimally invasive (MFX) and matrix-induced autologous chondrocyte transplantation (MACI). MFX can provide short-term clinical improvement, but due to the nature of fibrocartilage and fibrous repair tissue, this improvement tends to worsen after 2-5 years. MACI has similar clinical improvements but is more durable than MFx. However, the limitations of MACI treatment are the need for two separate surgical procedures and the high cost of cell culture. Recently, the use of cartilage chips has become a potential treatment method. Published research focuses on juvenile and autologous cartilage chips combined with scaffolding or embedded in fibrin glue, but long-term results are yet to be studied. Developing new surgical therapies and testing existing therapies requires large animal models that must be cost-effective, reliable, and predictable. Miniature pigs have previously been used in cartilage repair research. The bone reaches maturity at 18 months and is characterized by the closure of the distal femoral growth plate and the maturation of cartilage. A defect with a diameter of 5 mm has proven to be a critical dimension. Bone maturation is important to prevent the overestimation of repair response due to internal healing. The size of the animal and the size of the joints make minipigs an attractive animal model for cartilage research. For histological evaluation and reliable translation of the healing response in animal models, the imaging must be comparable to humans. In addition, the use of animals with multiple cartilage defects is economically and ethically important, because using multiple defects can reduce the number of animals used by researchers. The repair effect of multiple joint injuries has not been studied. The purpose of this study is to verify the following: 1) To verify whether the existing clinical repair technology is used to use miniature pigs as a preclinical model for cartilage and osteochondral repair; 2) 1 We study two knee defects (double defect knees) Compared with the repair effect of knee defects, whether it will affect the repair effect.

　　Method: The experimental design is shown in the figure. Two independent research hypotheses were tested. All animals underwent bilateral knee surgery. The first study showed that every knee has a defect. Use 10 miniature pigs (5 males and 5 females). Five different treatment methods and two different blank controls were used to test the applicability and repair response of each knee joint defect. In the second study, each knee had 2 defects, while 6 male minipigs had 2 defects in each knee. The treatment effects of adjacent defect, single knee defect and double knee defect were compared. The average age of miniature pigs is 21 months (18.5-23.7 months). The average weight is 41.9 kg (32.4-47 kg). Anesthesia: Azaperone (0.1ml/Kg) and hematoxylin (0.1ml/Kg) were administered subcutaneously to induce anesthesia and sedation. Etomidate (0.25 ml/Kg) was given through the ear vein for general anesthesia, and a tracheal tube (specification 6.5) was inserted. To maintain general anesthesia, use sevoflurane (3%) and fentanyl (0.175ml/kg/h). Before surgery, the animals were treated with prophylactic antibiotics (penicillin 0.03 ml/kg).

　　Surgery: Local analgesics act on the skin and tissues around the joints (Zerokine, 10ml, 20mg/ml). Mark the distal rod of the bone and the tibial tubercle, and make a 5 cm midline skin incision directly on the into bone ligament. Expose the bone ligament and obtain an incision of the ella bone ligament from the distal end of the bone to the tibial tubercle. The incision exposes the con interstitial cavity and the pulley. In minipigs with one knee joint injury, cartilage or osteochondral defects were found about 2 cm in the trochlear groove near the femur between were. There are double defects of cartilage in the knee joint, one is located in the trochlear groove, about 2 cm in the con fossa, and the other osteochondral defect is located outside the trochlear groove, about 1 cm in the medial bone groove. Overview of Cartilage Defects Using a 6mm skin biopsy punch and curette, carefully peel off the cartilage until all cartilage, including the calcified cartilage layer, is removed, and no subchondral bleeding is seen. Use a hollow 6 mm drill bit to create an osteochondral defect with a diameter of 6 mm and a depth of 8 mm. Perform slow manual drilling to avoid thermal damage to the tissue. After the treatments shown in the table, suture the pat bone ligament, subcutaneous tissue and skin. After the operation, the animals were given flunixin meglumine 1.1 mg/kg orally for 5 consecutive days with analgesic and anti-inflammatory effects. As shown in Table 1, a single knee joint defect was randomly divided into 7 groups. Two knee defects were randomly divided into autologous bone graft (ABG) or autologous double tissue graft (ADTT). Magnetic Resonance Imaging (MRI): To evaluate the signal strength of the repaired tissue surface, subchondral bone tissue and repaired tissue, scan each miniature pig with MRI and use a 3T whole-body MRI scanner before surgery. Use 3 to 6 months after surgery. .. Use the following sequence: double echo steady state (DESS, 3D imaging field of view (FOV)) 151 x 180 x 90 mm; acquisition matrix 216 x 256 x TR128; TR13.5; TE5.0; flip angle 28°; acquisition time 5 minutes 25 seconds), space (3D imaging field of view 98 x 180 x 78 mm, acquisition matrix 140 x 256 x TR112, TR1000, TE40.0, ETL52, acquisition time 3 minutes and 57 seconds), MPRAGE (3 size imaging field of view It is 120×183×99.8 mm, the acquisition matrix is 168×256×TR128, TR2200, TE2.65TI900, and the acquisition time is 3 minutes 49 seconds). All 3D imaging sequences use the 0.7x0.7x0.7 mm isotropic voxel method. Computed tomography (CT): CT scans were performed on each animal at 3 and 6 months after surgery to assess the cartilage and cartilage damage of bone repair, histological repair and the presence of subchondral bone cysts. I chose Micro CT because of its clinical importance. In order to achieve the highest isotropic resolution of 0.6 x 0.6 x 0.6 mm, the following scanning parameters are technically applied: 120kV, 200mAs, layer thickness 0.6mm, pitch coefficient 0.5mm and 307mm field of view, 0.3mm filter or b40f or b80f increment. To determine the number of bone defects, draw the region of interest (ROI) corresponding to the bone defect on a 2D slide, and then use the OsiriXROI volume tool to calculate the volume of the bone defect. By comparing the remaining defect volume with the original perforation defect, the bone defect repair rate was calculated. Histology: Six months after the operation, the animals were sacrificed and a complete picture of the defect was taken. The sample is dehydrated with increasing ethanol concentration (70-96%) at 4°C. Before removing 2-isopropanol and xylene, impregnate methyl methacrylate (MMA) at 4°C. At -20°C, MMA will accelerate the polymerization of N'N-dimethyl-p-toluidine. Use a microtome to cut the defective sample into 10 μm continuous slices. The samples were stained with hematoxylin and eosin (HE), toluidine blue, and saffron. The first study evaluated the sample size of each treatment group based on cell morphology, glycosaminoglycan (GAG) staining and tissue surface repair. In the second study, two experienced employees used the ICRSII scoring system to blindly evaluate the repair response.

　　Animal welfare: The trained zoo keepers will carefully observe each animal 3 times a day under the supervision of the veterinarian. When the animal breeder thinks that the miniature pig is uncomfortable or uncomfortable, whether it is raised alone or when interacting with the animal breeder, its overall appearance, appetite, clinical symptoms (body temperature, feces, body temperature, feces, etc.) are based on the animal's class and behavior For further evaluation, each category scored 1-4. Mild lines, loss of appetite and decreased mobility were observed on the 4th day after surgery. Results: In the first 5 days of the analgesic treatment, the animals did not swell, their appetite decreased, and their activity levels did not decrease. The surgical method of pat bone ligament provides the opportunity to make full contact with the inner and outer pulley surfaces. The inner and outer surfaces of the pulley are very suitable for 6 mm defects.

　　First study: No complete regeneration of hyaline cartilage was found in any treatment group. The cartilage treatment group was effective, but the surface of the repaired tissue was submerged and GAG positive staining was rare. The worst result was the discovery of a void, mainly fibrous tissue (Figure 3a), and the best result was the combination of vitreous tissue and fibrocartilage in the MACI group. Between 3 months and 6 months after surgery, there were significant differences in the defects. The opaque tissue in the MACI group was smooth and slightly submerged. The worst result of the osteochondral treatment group also found a defect in which the main fibrous tissue was empty. The best effect of osteochondral repair was in the ADTT group, and it was mainly found that vitreous tissue and fibrocartilage were dominant. MRI showed that the surface of the hollow defect was obviously sunken, and the defect tissue in the ADTT group was filled with healthy cartilage. CT imaging showed a significant increase in bone mass from 3 months to 6 months, indicating continuous regeneration of subchondral bone. The amount of subchondral bone increased from 3 months to 6 months, an average of 36%. Compared with the level of natural subchondral bone, the average bone loss after six months was only 0.06 cm (SD±0.04).

　　Second study: There was no significant difference in knee joints between ADTT and ABG treatment groups. There was no significant difference in ICRSII scores between adjacent defects.

　　Conclusion: Based on the results of this study, the author proposed miniature pigs as a suitable animal model for cartilage repair. After 6 months of clinically relevant treatment, the histology is similar to the findings of humans. CT imaging is essential to confirm the monitoring of subchondral bone repair, and it is recommended that all osteochondral treatments be evaluated. Facts have proved that male miniature pigs with double knee defects are the most economical method.