动物造模 z-bio 2022-08-26 674

　　INTRODUCTION: Surgical treatment of acute Achilles tendon rupture includes early accelerated rehabilitation which shortens the immobilization period, allowing early range of motion and muscle strengthening exercises. Using this method, several studies have demonstrated immediate weight bearing capacity after injury. Experimental models suggest that early rupture and loading of severed Achilles tendon promotes tissue maturation of collagen fibers. Achilles tendon ruptures can be treated not only with mechanical stimulation, such as early weight-bearing and range-of-motion exercises, but also with biological methods such as growth factor therapy. Tendon healing involves interdependent and overlapping inflammatory, proliferative, and remodeling phases. During the initial inflammatory phase, red blood cells and inflammatory cells enter the injury site, and necrotic tissue is phagocytosed by monocytes and macrophages. Growth factors are released, which in turn stimulate angiogenesis and proliferation of fibroblasts, which migrate to the site of injury and trigger type III collagen synthesis. The proliferative phase begins a few days later, when type III collagen synthesis is maximal and remains elevated for approximately 6 weeks. During the subsequent remodeling stage, a higher proportion of type I collagen is synthesized, and tenocytes and collagen fibers are aligned in the direction of stress. Platelet-rich plasma (PRP) includes autologous blood with platelet concentrations above baseline. PRP can promote cell proliferation, chemotaxis, cell differentiation, and angiogenesis by delivering various growth factors contained in alpha granules, thereby promoting the healing process. To our knowledge, the effect of PRP on the three phases of tendon healing (inflammatory, proliferative, and remodeling) has not been investigated. This study aimed to evaluate the effect of PRP on the healing of rabbit Achilles tendon in three stages by histological examination and quantitative assessment. We hypothesized that PRP treatment accelerates the remodeling process by promoting tissue healing by stimulating fibroblast proliferation and collagen production.

　　MATERIALS AND METHODS: Male Japanese white rabbits (n=50) weighed 3 kg, the temperature was 22-24 °C, the light/dark cycle was 12-12 hours, and the water and food were ad libitum.

　　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 described above, 8 models of Achilles tendon rupture were established. The rabbits were anesthetized with sodium pentobarbital (20 mg/kg body weight), and the skin of the right hind limb was shaved. Perform surgery under sterile conditions. A longitudinal skin incision is made at the midline of the Achilles tendon. The collateral ligament is cut in the same direction as the skin incision, and the Achilles and plantar tendons are dissected from the surrounding tissue. The total length of the Achilles tendon is approximately 3 cm. Cut in the middle of the Achilles tendon. The plantaris tendon remains intact as an internal splint for a short distance (0–1 mm). The severed tendon is not sutured. The rabbits were randomly divided into two groups, 25 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. The collateral ligaments and skin are then sutured. After surgery, the ankle joint was immobilized in 60° plantar flexion by percutaneous insertion. Supplementary fixation with plaster. Both were observed using traditional wire mesh cages. Immobilization was maintained postoperatively until the Achilles tendon was harvested.

　　Histological examination: Achilles tendon was taken 1, 2, 3, 4, and 6 weeks after operation. In the control group and the PRP group, the number of samples per sampling was 5. Tissues were fixed in 20% buffered neutral formalin for 3 days, dehydrated, and then embedded in paraffin. Tendon longitudinal sections were stained with hematoxylin and eosin (H&E). The Achilles tendon was sectioned in the longitudinal plane with a thickness of 7 μm by immunohistochemistry. . Sections were incubated with anti-endothelial differentiation CD(31) monoclonal antibody and type I collagen. Immunohistochemical staining was performed using the EnVIEW SUV+ kit. Sections were counterstained with Mayer's hematoxylin. The tendon healing process was compared between the two groups by evaluating the results of H&E staining. Type I collagen immunoreactivity was assessed semi-quantitatively with a scoring system. H&E staining results and CD31 immunoreactivity were quantified by analyzing images of the gap between the Achilles tendon stumps acquired with a BX50 light microscope equipped with a DP72 digital camera. Proliferation was assessed by counting the number of fibroblasts at 400x magnification for 10 fields. To evaluate angiogenesis, we measured the area ratio of CD31-positive cells in 10 areas using LuMaVIEW V.2 image analysis software at 100x original magnification.

　　RESULTS: The average platelet concentration in whole blood was 40.6±9.6×104/μL, and the average platelet concentration in platelets was 105.7±22.8×104/μL, which was about 2.6 times higher than that in whole blood. One week after surgery, the gap between the severed Achilles tendon of the PRP-treated and untreated rabbits was filled with fibroblasts, blood cells and fibrin. However, no increase in collagen fiber density was observed. Collagen fibers were sparse at 2 weeks in the control group; however, in the PRP group, fibers and capillaries were densely present in the interstices. At 3 weeks, collagen fiber density increased in both groups. 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, bundles of collagen fibers aligned in a single direction parallel to the long axis of the tendon. At 6 weeks, collagen fiber bundles were partially oriented along the tendon axis in control rabbits. In PRP-treated rabbits, bundles of collagen fibers aligned parallel to the tendon axis, and they approximated fibers in normal tendons. From week 1, fibroblast migration in both groups gradually increased, but there were more fibroblasts in the PRP group than in the control group at 1 and 2 weeks. At 3 weeks, the number of fibroblasts was high in the control group, but in PRP-treated rabbits, at 3 and 4 weeks, mature cells had more flattened nuclei than fibroblasts with proliferating spindle-shaped nuclei. Indicates a decrease in the number of immature fibroblasts. At 4-6 weeks, fibroblast proliferation 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 was significantly lower at 6 weeks than at 2 weeks. The control group continued to increase at 6 weeks. Type I collagen immunoreactivity was not observed in either group until week 2. At 3 weeks, the signal intensity of the PRP-treated group was higher than that of the control group (score of 3 to 2), indicating that the former tendon was more mature at this time. Therefore, the collagen I immunoreactivity scores at 4 and 6 weeks were 2 and 3 in the control group, respectively, whereas the type I collagen immunoreactivity scores in the PRP-treated rabbits were 3 and 4, respectively. Vascular proliferation was significantly higher in the PRP group than in the control group at 1 and 2 weeks. Vessel diameter increased at 3 and 4 weeks in the control group, as did the number of vessels. At this time point, vessel diameter and vessel number decreased in the PRP group. At 6 weeks, the blood vessels in the control group were smaller in diameter and spread 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 week and 2 weeks. Area ratios were similar between the two groups at 3, 4 and 6 weeks. The PRP group peaked at 2 weeks but remained high at 6 weeks.

　　Conclusion: PRP administration can shorten the inflammatory phase and promote tendon healing in the proliferative phase. Early PRP treatment is initiated in the remodeling phase to promote early healing of the tendon tissue. The findings suggest that PRP combined with early functional rehabilitation therapy promotes healing of the Achilles tendon without tendon lengthening or rerupture, thereby reducing downtime required for work or physical activity.