动物造模,大鼠胫骨骨折模型 z-bio 2021-10-11 549

　　Background: Based on the previous model designed by Jackson et al., Bonnarens et al. described a rat fracture model. In this model, an intramedullary nail (IM) is retrogradely implanted through the knee joint before the fracture. Then, a fixed weight fell on the bones on a blunt guillotine, forming a standardized closed mid-femoral fracture. Several studies have reported recurring complications in rat models of femoral fractures using blunt decapitation, including death, misaligned fractures, excessive comminution, and deep infections. In addition, different degrees of fracture healing are particularly difficult in the process of fracture healing. The degree of fracture comminution has an impact on the formation of callus, and the rat fracture model has a certain correlation with the formation of comminuted callus. The fracture was established in a rat tibia fracture model, and the results showed that the comminutedness was small, but the bone mass shifted high, and the needle tip angle was greater than 10°. The use of three-point forceps in the rat tibial fracture model showed minimal edema and death, but there was still a significant shortening rate of tibial callus six months after the fracture, and the needle angle was 80±2°. Due to the limitations of existing fracture models, a reliable fracture model is needed, especially in experimental animals, which is feasible and reproducible.

　　Method: Animals: 10 rats of 30 weeks old and 71 rats of 8 weeks old were used. Three-point bending forceps are used for fractures, as described below. In the in vivo study, a total of twenty-five (n=25), 8-week-old female Sprague-Dawley rats with an average weight of 187.04±10.15 grams were used. In the in vivo study, a total of twenty-five (n=25), 8-week-old female SD rats with an average weight of 187.04±10.15 grams were used. There is a one-week acclimation period before the start of the experiment. After the acclimation period, the rats were fasted for 12 hours before surgery.

　　Surgical operations: All operations are performed under pre-anaesthesia with ketamine and xylazine. After intramuscular injection of ketamine (40 mg/kg) and xylazine (5 mg/kg), rats in each group underwent surgery and radiography. A 4 mm skin incision was used as the surgical incision, and a scalpel blade (#15) was used to perform surgical excision at the knee joint. Expose the tibia with stainless steel, and manually introduce a sharp probe through the attachment of the tibia, the skull to the anterior cruciate ligament and the anterior horn of the meniscus to determine the area of the intramedullary needle entrance. A 23G, 11/2" needle is inserted into the distal tibia, and the penetration depth of the needle is about 22 mm. After the intramedullary nail is completely inserted through the tibia, use a bone cutter to cut off the excess proximal needle. The incision is simply interrupted without absorption Closing with 4/0 nylon sutures. In order to change the fracture of the tibia in rats, by modifying the upper jaw compression jaws of Otto et al., a specially improved three-point bending forceps with a size of 10×0.1 mm was constructed. The three-point bending forceps were changed. The two supporting claws were changed to 7 mm instead of 17 mm. After using the pliers, visually control the position and close the pliers until a crack is heard, and the resistance of the pliers suddenly drops. After that, the pressure is immediately released. Apply the above principles to establish a large Rat tibia or femur fracture model. The left tibia was fractured, and the right tibia on the control side was intact as a control. Each animal was placed in a separate cage to allow free movement after surgery. Tramadol was administered 5 mg/day after surgery. kg was given as analgesia for 3 consecutive days. The animals were euthanized by intraperitoneal injection of 20 mg/kg pentobarbital and radiological observations were performed.

　　General assessment of fracture: Since the fracture is pre-fixed with intramedullary nails in the fractured bone, the fracture is considered stable and does not require additional postoperative external support bandages. The dissected tibia was collected from induced fractures and non-fractured limbs (control). Five rats died in the first, second, third, fourth, and sixth weeks after operation (n=5). The tibia was dissected and the control method was used to determine the scab index and callus area. A 3.1μm-pixel dissecting stereo microscope was used to take anatomical bone images, and the width of the callus (mm) of the tibia fracture healing in rats was measured. With the right tibia of the contralateral rat as a control, the ratio of callus index was measured with IMAGE software. The same software was used to measure the callus area, callus index, pin angle, proximal fracture, full-length fracture, and normal control bone.

　　Radiological evaluation of fractures: Immediately after surgery and once a week after surgery, X-ray machines were used to perform two-angle X-ray photography of the fracture and the intact tibia on the opposite side. The bone healing was objectively evaluated with indexes such as the size of bone joints and callus formation, the degree of bone comminution, needle angle, and X-ray callus index. X-ray callus index is the maximum width of the callus formed in the middle section of the tibia, including the original bone divided by the width of the contralateral control bone. A comminuted fracture is the splitting or breaking of bone into two or more fragments. First, the imaging observation of the two anterior and posterior positions showed no smashing phenomenon. Second, the smaller fragments are crushed, and their size is smaller than the diameter of the nail, that is,<1.1 mm; third, severely crushed (large fragments larger than the diameter of an intramedullary nail).

　　Histological examination of fractures: The animals were sacrificed at 1, 2, 3, 4, and 6 weeks after the operation, and tibia specimens were collected from the dissected limbs every week (n=5). Under the fracture site (the formation of callus is obvious), the bone was cut into 2 mm, fixed in 10% formaldehyde for 24 hours, decalcified in 10% formic acid for 4 days, and then processed and stained with H&E standards.

　　Results: A preliminary study on 30-week-old rats (n=10) showed that tibial fractures were mainly comminuted, oblique, and distal. In 8-week-old rats (n=65), the results showed that the main tibia was non-comminuted, transverse and central axis. Another 8-week-old rat (n=6) was used to compare the types of femur and tibia fractures. The main types of femoral and tibial fractures are transverse and non-comminuted fractures, located in the central axis of the bone. In the in vivo study, all induced fractures were found in the middle third tibia. The average operation time was 3.90±0.55 min. All animals survived the operation without any complications, soft tissue damage at the fracture site or stiffness of the adjacent knee and ankle joints. No signs of necrosis or severe edema were seen at the fracture site. Twenty-four hours after the fracture, the rat showed normal behavior. After 1 to 2 weeks, the rats began to bear weight on the affected limb. After 2 weeks, all rats began to show weight on all four legs. The formation of callus was observed in the first week and gradually decreased in the sixth week. The type of fracture is determined by radiological measurement of the angle between the fracture line and the long axis of the tibia. The main type of fracture is transverse (89.41±2.11). The average length of the proximal fractured bone was 41.02 of that of the intact bone, which was ±3.27%. The average angles of the IM needles in lateral and anteroposterior positions were 167.33±3.67° and 161.47±4.87°, respectively. The formation of callus around the fracture site is clearly visible on the X-ray film. In the third to fourth weeks, the density of the callus increased, and the fracture line was difficult to observe. The fracture line disappeared in the fourth week, and the fracture healed completely. In the first week, the X-ray callus index was almost unmeasurable, and in the second week it was very small and almost invisible. It reached the highest point in the 3rd week, and gradually decreased in the 4th and 6th weeks. Mild comminuted fractures were observed in four animals, and no comminuted fractures were found in the rest. Histological examination after the fracture showed proliferative hematoma and chondrocytes in the first week after the fracture, while the cartilage cell area and cancellous bone increased in the second week. In the third and fourth weeks, cancellous bone increases with the appearance of hypertrophic chondrocytes and bone marrow.

　　Conclusion: The improved three-point bending forceps used in this study are practical to create a reproducible closed fracture with very few postoperative complications in rats.