动物造模,急性跟腱断裂 z-bo 2022-02-19 424

　　Introduction: Surgical treatment of acute Achilles tendon rupture includes early accelerated rehabilitation, which shortens the fixation period and allows early range exercise and muscle strengthening exercises. Using this method, multiple studies have shown the ability to carry weight immediately after injury. The experimental model showed that early fracture and loading of the severed Achilles tendon promoted the tissue maturation of collagen fibers. Achilles tendon rupture can be treated not only by mechanical stimulation, such as early weight-bearing and range of motion exercises, but also by biological methods such as growth factor therapy. Tendon healing includes interdependent and overlapping periods of inflammation, proliferation, and remodeling. In the initial inflammation phase, red blood cells and inflammatory cells enter the injury site, and the necrotic tissue is swallowed by monocytes and macrophages. Growth factors are released, which in turn stimulates angiogenesis and proliferation of fibroblasts, migrates to the injury site and triggers the synthesis of type III collagen. The proliferative phase begins a few days later, at this time, the synthesis of type III collagen is at its maximum and remains elevated for approximately 6 weeks. In the subsequent remodeling stage, the proportion of type I collagen synthesized is higher, and tendon cells and collagen fibers are aligned in the direction of stress. Platelet-rich plasma (PRP) includes autologous blood, and the platelet concentration is higher than the baseline value. PRP can promote cell proliferation, chemotaxis, cell differentiation and angiogenesis by delivering a variety of growth factors contained in alpha particles, thereby promoting the healing process. As far as we know, the effect of PRP on the three stages of tendon healing (inflammatory, proliferative, and remodeling) has not been studied. This study aims to evaluate the effect of PRP on the healing of rabbit Achilles tendon in three stages through histological examination and quantitative evaluation. We hypothesize that PRP treatment promotes tissue healing by stimulating fibroblast proliferation and collagen production, thereby accelerating the remodeling process.

　　Materials and methods: Male Japanese white rabbits (n=50) weigh 3 kg, temperature 22-24℃, light/dark cycle for 12-12 hours, free drinking and eating.

　　Preparation of PRP: Four male Japanese white rabbits were used for PRP production. PRP was prepared by the autologous platelet system according to the manufacturer's instructions.

　　Achilles tendon rupture model: As mentioned above, 8 Achilles tendon rupture models were established. The rabbits were anesthetized with sodium pentobarbital (20 mg/kg body weight), and the skin of the right hind limb was shaved. The operation is performed under aseptic conditions. Make a longitudinal skin incision at the midline of the Achilles tendon. Cut the collateral ligament in the same direction as the skin incision, and dissect the Achilles and plantar tendons from the surrounding tissues. The total length of the Achilles tendon is about 3 cm. Cut in the middle of the Achilles tendon. The plantaris tendon is kept intact as an internal splint, keeping a short distance (0 to 1 mm). Cut the tendon without suture. The rabbits were randomly divided into two groups, 25 rabbits in each group. In the control group, only Achilles tendon rupture was performed; in the PRP group, 0.3 g of PrP was placed in front of the tendon stump immediately after the tendon was cut. Then suture the collateral ligaments and skin. After the operation, the ankle joint is fixed at 60° plantar flexion through percutaneous insertion. Use plaster to supplement the fixation. All were observed with a traditional steel mesh cage. Keep it fixed after the operation until the Achilles tendon is obtained.

　　Histological examination: Achilles tendons were taken 1, 2, 3, 4, and 6 weeks after surgery. In the control group and the PRP group, the number of samples taken per sample was 5. The tissue was fixed in 20% buffered neutral formalin for 3 days, dehydrated, and then embedded in paraffin. The longitudinal section of the tendon was stained with hematoxylin and eosin (H&E). The Achilles tendon was sliced in a longitudinal plane by immunohistochemistry, with a thickness of 7 μm. . The sections were incubated with CD (31) monoclonal antibody against endothelial cell differentiation and type I collagen. EnVIEW SUV+ kit was used for immunohistochemical staining. The sections were counterstained with Mayer's hematoxylin. By evaluating the results of H&E staining, the tendon healing process of the two groups was compared. The type I collagen immune response was evaluated semi-quantitatively with a scoring system. The H&E staining results and CD31 immunoreactivity were quantified by analyzing the images of the gap between the Achilles tendon stumps acquired by a BX50 optical microscope equipped with a DP72 digital camera. The proliferation was evaluated by counting the number of fibroblasts for 10 fields at 400 times magnification. In order to evaluate angiogenesis, we used LuMaVIEW V.2 image analysis software to measure the area ratio of CD31-positive cells in 10 areas at 100 times the original magnification.

　　Result: The average platelet concentration in whole blood is 40.6±9.6×104/μL, and the average platelet concentration of platelets is 105.7±22.8×104/μl, which is about 2.6 times that of whole blood. One week after the operation, the gap between the severed Achilles tendon of PRP-treated and untreated rabbits was filled with fibroblasts, blood cells and fibrin. However, no increase in collagen fiber density was observed. In the control group, collagen fibers were sparse at 2 weeks; however, in the PRP group, fibers and capillaries were densely present in the gaps. At 3 weeks, the density of collagen fibers in both groups increased. At 4 weeks, the fiber bundles were not oriented along the tendon axis, and fibroblasts continued to proliferate in the control group. In contrast, in PRP-treated rabbits, the collagen fiber bundles are aligned in a single direction parallel to the long axis of the tendon. At 6 weeks, the collagen fiber bundles were partially oriented along the tendon axis to the control rabbits. In PRP-treated rabbits, collagen fiber bundles are arranged parallel to the tendon axis, and they are close to the fibers in normal tendons. From the first week, the migration of fibroblasts in the two groups gradually increased, but at 1 and 2 weeks, there were more fibroblasts in the PRP group than in the control group. At 3 weeks, the number of fibroblasts in the control group was high, but in PRP-treated rabbits, mature cells had more flattened nuclei than spindle-shaped nuclei proliferating fibroblasts at 3 and 4 weeks. Shows that the number of immature fibroblasts is reduced. At 4-6 weeks, the proliferation of fibroblasts continued in the control group, but during this period, the number of fibroblasts was significantly reduced, and more fibroblasts with flat nuclei were observed in the PRP group. In PRP-treated rabbits, the number of fibroblasts at 6 weeks was significantly lower than at 2 weeks. The control group continued to increase at 6 weeks. Until the second week, no type I collagen immunoreactivity was observed in both groups. At 3 weeks, the signal intensity of the PRP treatment group was higher than that of the control group (a score of 3 to 2), indicating that the former tendon was more mature at this time. Therefore, the type I collagen immune response scores at 4 and 6 weeks were 2 and 3 of the control group, while the type I collagen immune response scores of the PRP-treated rabbits were 3 and 4, respectively. At 1 and 2 weeks, the vascular proliferation of the PRP group was significantly higher than that of the control group. The blood vessel diameter of the control group increased at 3 and 4 weeks, and the number of blood vessels also increased. At this point in time, the diameter and number of blood vessels in the PRP group decreased. At 6 weeks, the blood vessels of the control group were smaller in diameter and scattered throughout the healing tendon. However, in the PRP group, blood vessels were only observed between collagen fiber bundles. The area of CD31 positive cells in the PRP group was significantly higher than that in the control group at 1 and 2 weeks. At 3, 4 and 6 weeks, the area ratios between the two groups were similar. The PRP group reached a peak at 2 weeks, but still maintained a high level at 6 weeks.

　　Conclusion: PRP administration can shorten the inflammation period and promote the healing of tendons in the proliferative period. Early PRP treatment was started in the remodeling stage to promote early healing of tendon tissue. The results of the study show that PRP combined with early functional rehabilitation can promote the healing of the Achilles tendon without the need for tendon lengthening or re-rupture, thereby reducing the downtime required for work or sports activities.