Abstract: BACKGROUND: Natural biomaterials such as allogeneic materials are highly susceptible to bacterial contamination during the production process. Among them, bacterial endotoxin released by Gram-negative bacteria can cause human fever, coagulation, shock, and even death. There is no relevant research on the method of endotoxin removed from allogeneic materials while maintaining their natural structure and biological activity. 
OBJECTIVE: To explore the method and effect of endotoxin removed from allogeneic materials and other natural biological materials and to control the residual H2O2.
METHODS: The endotoxin was removed from allogeneic bone materials by H2O2/ultrasound. The influence of ultrasound and the concentration of H2O2 on the endotoxin removal process was explored. The concentration of H2O2 was set as 1%, 2%, 3%, 4%, and 5% by mass. The power of ultrasound was set as 80, 120, 140, 160, and 200 W. The duration of ultrasound was 20, 30, 40, 50, and 60 minutes. The temperature was 30, 35, 40, 45, and 50 ℃. The optimal combination of conditions for treating allogeneic bone materials was selected by endotoxin content detection for subsequent experiments. The microstructure, H2O2 residue, and cytotoxicity of allogeneic bone materials after endotoxin removal were detected, and the effects of different concentrations of H2O2 residue on the survival rate of L929 mouse fibroblasts were explored. 
RESULTS AND CONCLUSION: (1) According to the results of the endotoxin removal process, when the concentration of H2O2 was 4%, the power of ultrasound was 200 W, with the duration of ultrasound of 50 minutes, and the temperature 50 ℃, it would effectively remove endotoxin from allogeneic bone materials. The optimal combination was selected to remove endotoxins from allogeneic bone materials. (2) After endotoxin removal, the residual H2O2 content of allogeneic bone materials was less than 47.551 mg/kg after being placed in a 60 ℃ water bath and changing the solution 6 times, which had no effect on the viability of L929 mouse fibroblasts. (3) Optical microscopy and scanning electron microscopy observations exhibited that the use of H2O2 solution in conjunction with ultrasonic treatment had no effect on the structure of allogeneic bone materials. (4) The results indicate that the H2O2 solution in conjunction with ultrasonic treatment has the characteristics of efficient and rapid removal of endotoxins, which can ensure the safety of allogeneic bone materials.

Key words: allogeneic material, endotoxin removal, ultrasound, hydrogen peroxide, combined treatment, engineered bone material