Experimental study on effect of 3-methyladenine (3-MA) on lymphatic clearance rate and fracture healing

AimTo investigate the effect of 3-methyladenine(3-MA)on lymphatic clearance and fracture healing after lower limb fracture in mice.MethodsC57BL/6 male mice(8-week-old)were randomly divided into: rapamycin (RPM) group, 3-MA group, model group, and sham group, with 6 mice in each group. The RPM group, 3-MA group, and model group established a left tibia intramedullary fixation fracture model and were continuously administered for 14 days, while the sham group only underwent skin incision. The lymphatic clearance rate and tissue swelling rate of the left hind limb were measured on the 1st, 7th, and 14th day respectively. After 14 days, changes in the fracture site were observed through micro-CT scanning, immunohistochemical (IHC) staining of RUNX2, and safranin O-fast green staining.Results① On the 1st, 7th, and 14th day, the lymphatic clearance rate of the 3-MA group was lower than that of the sham group but higher than that of the RPM group and model group. ② On the 1st, 7th, and 14th day, the tissue swelling rate of the 3-MA group was higher than that of the sham group but lower than that of the RPM and model group. ③ Micro-CT scanning showed that in terms of callus volume (VOXBV), bone connection density(Conn-Dens), trabecular number(Tb. N), and trabecular thickness(Tb. Th), the 3-MA group was higher than the model group, while the RPM group was lower than the model group. Additionally, the fracture line in the 3-MA group was not obvious. ④ IHC staining showed that the expression of RUNX2 in the 3-MA group was significantly higher than that in the model and RPM group, while the RPM group was lower than the model group. There were significant statistical differences in the average optical density (AOD) of positive areas among the groups. ⑤ Safranin O-fast green staining showed that the callus in the 3-MA group began to remodel, excess callus tissue was absorbed, chondrocytes decreased, and the number of osteoblasts increased significantly. However, the fracture ends in the RPM group were obviously not healed, and the numbers of cartilage and trabeculae were sparse.Conclusion3-MA can enhance lymphatic clearance after fracture, reduce tissue swelling, and promote fracture healing.