School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031, China.
Institute for Medical Device Control, National Institutes for Food and Drug Control, Beijing 102629, China.
Biomed Res Int. 2022 Jan 17;2022:7950834. doi: 10.1155/2022/7950834. eCollection 2022.
In this study, -Gal epitope-deficient (GGTA1 knockout (GTKO)) mice were used to assess the immunological risks of xenogeneic dural patch by comparing with raw material.
The xenogeneic dural patch (T2) was prepared from bovine pericardium (T1, raw material) through decellularization and carboxymethyl chitosan (CMCS) coating. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) were used to characterize the collagen fibers and surface microstructural changes in the T1 and T2 samples. The remnant -Gal epitopes and DNA of implants were detected by standardized method. T1 and T2 were implanted subcutaneously into GTKO mice for 4 and 12 weeks, respectively, and the negative control group (Con) was only performed sham operation. The total serum antibody, anti-Gal antibody, and splenic lymphocyte subtypes were analyzed by ELISA or flow cytometry, and histological analysis of implant-tissue was performed by H&E and Masson stain.
TEM and Sirius red staining showed that the collagen fibers in the dural patch were closely arranged, and SEM showed that a loose three-dimensional structure was successfully constructed on the surface of the dural patch after CMCS coating. The remnant DNA in T2 was 24.64 ± 8.73 ng/mg (dry weight), and clearance of -Gal epitope was up to 99.83% compared to T1. The significant increases in serum total IgM, anti-Gal IgG, and anti-Gal IgM at 4 weeks and the significant changes in anti-Gal IgG and spleen lymphocyte at 12 weeks were observed in the T1 group, but no significant change was observed in the T2 group, compared to the control group. Histological semiquantitative analysis showed severe cell and tissue responses at 4 weeks and a moderate response at 12 weeks in the T1 group, while a moderate response at 4 weeks and a slight response at 12 weeks in the T2 group.
The results demonstrated that the xenogeneic dural patch has a lower and acceptable immunological risk compared to the raw material and control, respectively. On the other hand, it was suggested that GTKO mice are useful experimental model for immunological risk assessment of animal tissue-derived biomaterials.
本研究通过与原材料比较，利用缺乏β-半乳糖苷酶表位的（GGTA1 敲除（GTKO））小鼠评估异种硬膜补片的免疫风险。
通过脱细胞化和羧甲基壳聚糖（CMCS）涂层，从牛心包（T1，原材料）制备异种硬膜补片（T2）。透射电子显微镜（TEM）和扫描电子显微镜（SEM）用于表征 T1 和 T2 样品中的胶原纤维和表面微观结构变化。采用标准化方法检测植入物中残留的β-半乳糖苷酶表位和 DNA。将 T1 和 T2 分别皮下植入 GTKO 小鼠 4 周和 12 周，阴性对照组（Con）仅进行假手术。通过 ELISA 或流式细胞术分析总血清抗体、抗 Gal 抗体和脾淋巴细胞亚型，并通过 H&E 和 Masson 染色对植入物组织进行组织学分析。
TEM 和 Sirius red 染色显示，硬膜补片中的胶原纤维排列紧密，SEM 显示 CMCS 涂层后，硬膜补片表面成功构建了疏松的三维结构。T2 中的残留 DNA 为 24.64±8.73ng/mg（干重），与 T1 相比，β-半乳糖苷酶表位清除率高达 99.83%。与对照组相比，T1 组在 4 周时血清总 IgM、抗 Gal IgG 和抗 Gal IgM 显著增加，12 周时抗 Gal IgG 和脾淋巴细胞明显变化，但 T2 组无明显变化。组织学半定量分析显示，T1 组在 4 周时细胞和组织反应严重，12 周时反应中度，T2 组在 4 周时反应中度，12 周时反应轻度。
结果表明，与原材料和对照组相比，异种硬膜补片的免疫风险较低且可接受。另一方面，建议 GTKO 小鼠是评估动物组织衍生生物材料免疫风险的有用实验模型。
