中国组织工程研究 ›› 2025, Vol. 29 ›› Issue (28): 5985-5993.doi: 10.12307/2025.471

• 组织工程骨材料 tissue-engineered bone • 上一篇    下一篇

去除同种异体骨中内毒素的方法及效果

侯立存,胡  凯,邵怡然   

  1. 上海亘从生物医用材料研究中心,上海市   201201
  • 收稿日期:2024-06-05 接受日期:2024-07-27 出版日期:2025-10-08 发布日期:2024-12-07
  • 作者简介:侯立存,女,1986年生,河北省邢台市人,汉族,工程师,主要从事生物医用材料的研究与转化方面的研究。

Methods and effects on endotoxin removal in allogeneic bone

Hou Licun, Hu Kai, Shao Yiran   

  1. Shanghai Gencong Biomedical Materials Research Center, Shanghai 201201, China
  • Received:2024-06-05 Accepted:2024-07-27 Online:2025-10-08 Published:2024-12-07
  • About author:Hou Licun, Engineer, Shanghai Gencong Biomedical Materials Research Center, Shanghai 201201, China

摘要:


文题释义:

内毒素:是革兰阴性菌细胞壁的重要组成部分,化学本质是脂多糖,主要由多糖O-特异性抗原、核心多糖、类脂A 3个部分组成,约占细菌细胞壁的3/4,当细菌细胞死亡、裂解或人工破坏菌体后才会释放到环境中,又称之为“热原”。内毒素能够诱导动物体内单核巨噬细胞合成并释放多种炎症递质,参与机体的免疫调节反应,严重时可导致脓毒血症或休克。
同种异体材料:作为植入性医疗器械产品,在生产过程中对内毒素进行控制至关重要,进而保证产品的安全性。


背景:天然生物材料(如同种异体材料)生产过程中极易受到细菌污染,其中革兰阴性菌释放出的细菌内毒素可以引起人体发热、凝血、休克,严重的会导致死亡。如何去除同种异体材料中的内毒素,同时又能保证材料的天然结构和生物活性,目前尚未有相关报道。

目的:探究去除同种异体材料等天然生物材料中内毒素的方法及效果,并对H2O2残留进行控制。
方法:采用H2O2溶液协同超声处理方法去除同种异体骨材料的内毒素,研究超声条件及H2O2浓度对内毒素去除效果的影响程度,其中H2O2浓度分别设定为1%,2%,3%,4%,5%,超声功率分别设定为80,120,140,160,200 W,超声时间分别设定为20,30,40,50,60 min,超声水浴温度分别设定为30,35,40,45,50 ℃,通过内毒素含量检测选择最优的组合条件处理同种异体骨材料,进行后续实验。对去除内毒素后同种异体骨材料的微观结构、H2O2残留及细胞毒性进行检测,同时探讨不同浓度H2O2残留对L929小鼠成纤维细胞存活率的影响。

结果与结论:①从内毒素去除效果来看,实验条件为H2O2浓度4%、超声功率200 W、超声时间50 min、超声水浴温度50 ℃时,为去除同种异体骨材料中内毒素的最佳条件,选择该最优组合去除同种异体骨材料的内毒素;②去除内毒素处理后同种异体骨材料在60 ℃水浴、换6次液后H2O2残留量小于47.551 mg/kg,对L929小鼠成纤维细胞活性无影响;③光学显微镜及扫描电镜观察显示,采用H2O2溶液协同超声处理对同种异体骨材料的结构无影响;④结果表明,H2O2溶液协同超声处理方式具有高效快速去除内毒素的特点,可保证同种异体骨材料的安全性。

https://orcid.org/0009-0000-2601-4556 (侯立存) 

中国组织工程研究杂志出版内容重点:生物材料;骨生物材料;口腔生物材料;纳米材料;缓释材料;材料相容性;组织工程

关键词: 同种异体材料, 内毒素去除, 超声, 过氧化氢, 联合处理, 工程化骨材料

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

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