Xenogeneic bone matrix immune risk assessment using GGTA1 knockout mice
Xenogeneic bone matrix immune risk assessment using GGTA1 knockout mice
复制标题
使用 GGTA1 敲除小鼠进行异种骨基质免疫风险评估
DOI：
10.1080/21691401.2018.1493489
复制
发表时间：
2018-01-01
影响因子：
5.8
通讯作者：
Wang, Chengbin
中科院分区：
文献类型：
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作者：
Shao, Anliang;Ling, You;Wang, Chengbin
同种异体骨移植用于替代疗法已经在临床数据中得到了可靠的证明。然而，适用于同种异体移植的人类供体骨供应短缺，这有利于寻找合适的替代品，如异种移植。α - gal抗原相关的异种移植物免疫风险直接影响到生物材料的安全性和有效性，限制了其在临床中的应用。免疫风险可以通过在移植前消耗或破坏抗gal抗体来预防。因此，如何评估骨替代物的免疫风险，选择可靠的动物实验模型就显得尤为重要。在本研究中，我们制备了冻干骨替代物（T1）和广豪生物技术骨替代物（T2，动物源性α - gal抗原降低的生物材料），旨在评估异种骨替代物对GGTA1基因敲除小鼠的免疫风险。首次采用ELISA法在体外检测T1和T2的α - gal抗原含量。将骨替代物分别皮下植入GGTA1基因敲除小鼠2周、4周和12周。检测小鼠血清总抗体水平、抗α - gal抗体水平、炎性细胞因子和脾淋巴细胞表面分子水平，并对皮肤和胸腺进行组织学分析，系统评价T1和T2骨代用品引起的小鼠免疫应答。体外实验结果显示，T1骨替代物中α - gal抗原表位的数量明显高于T2骨替代物，T2骨替代物中α - gal抗原的清除率达到55.6%左右。体内抗体水平结果显示，T1骨代用品组总IgG、IgM、IgA和抗α - gal IgG水平显著高于T2组和对照组，而T2组与对照组相比，这些指标均无显著变化。炎症因子方面，T1骨代用品IL-4、IL-12P70、IL-10水平明显高于T2组和对照组，T2组与对照组相当。三组（T1、T2和对照组）脾淋巴细胞表面分子水平在实验期间均未见变化。病理结果显示，T2组炎症反应较T1组轻，这与炎症细胞因子水平一致。上述结果表明抗原去除过程有效降低了T2骨替代物中α - gal抗原的含量，在体内引起的免疫反应较小，可作为骨愈合材料。本研究还表明，GGTA1敲除小鼠可作为评估动物源性生物材料免疫风险的常规工具。
Homeotransplantation of bones for replacement therapy have been demonstrated reliably in clinical data. However, human donor bones applicable for homeotransplantation are in short supply, which facilitates the search for suitable alternatives, such as xenografts grafts. The alpha-Gal antigen-related immune risk of xenografts directly affects the safety and effectiveness of the biomaterials and limits their applications in the clinic. The immune risk can be prevented by depletion or breaking anti-Gal antibody prior to transplant. Therefore, how to assess the immune risk of the bone substitutes and select the reliable animal research model become extremely important. In this study, we prepared lyophilized bone substitutes (T1) and Guanghao Biotech bone substitutes (T2, animal-derived biomaterials with alpha-Gal antigen decreased), aimed to assess the immune risk of xenografts bone substitutes on GGTA1 knockout mice. The alpha-Gal antigen contents of T1 and T2 were firstly detected by ELISA method in vitro. The bone substitutes were then implanted subcutaneously into GGTA1 knockout mice for 2, 4 and 12weeks, respectively. The total serum antibody levels, anti-alpha-Gal antibody levels, inflammatory cytokine and splenic lymphocyte surface molecules were detected and histology analysis of skin and thymus were performed to systematically evaluate the immune response caused by the T1 and T2 bone substitutes in mice. In vitro results showed that the amount of alpha-Gal epitopes in T1 bone substitutes was significantly higher than T2 bone substitutes, and the clearance rate of alpha-Gal antigen in T2 bone substitutes achieved about 55.6%. Results of antibody level in vivo showed that the T1 bone substitutes group possessed significantly higher total IgG, IgM, IgA and anti-alpha-Gal IgG levels than T2 and control group, while T2 group showed no significant changes of these indexes compared with control. In terms of inflammatory cytokines, T1 bone substitutes showed evidently higher levels of IL-4, IL-12P70 and IL-10 than T2 and control, while T2 group was comparable to control. No changes in the levels of splenic lymphocyte surface molecules were found in the three groups (T1, T2 and control group) during the experimental periods. The pathological results demonstrated that the inflammatory response in T2 group was lighter than the T1 group, which was in accordance with the inflammatory cytokines levels. The above results indicated that the process of antigen removal effectively reduced the alpha-Gal antigens content in T2 bone substitutes, which caused little immune response in vivo and could be used as bone healing materials. This study also demonstrated that GGTA1 knockout mice can be used as a routine tool to assess the immune risk of animal-derived biomaterials.