Objectives: To evaluate the immunogenicity and osteogenic ability of animal-derived bone graft material decellularized with supercritical carbon dioxide.
Methods: Porcine femurs were randomly divided into two groups after preliminary treatment, and decellularized with conventional method (conventional control group) or supercritical carbon dioxide (experimental group). Clearance rate of galactose-α-1, 3-galactose (α-Gal) and residual DNA of the two groups were analyzed to assess the immunogenicity of the xenogenic materials. Clearance rate of α-Gal was determined by enzyme-linked immunosorbent assay and residual DNA was detected by fluorescence method. Nine SPF-grade male athymic nude mice of 6 weeks old were randomly divided into experimental group, conventional control group and positive control group. Samples were implanted over biceps femoris muscle of athymic nude mice, the explants were collected 4 weeks post implantation, hematoxylin and eosin (HE) staining and immunohistochemistry were applied to determine the osteogenic ability and bone tissue-associated protein expressions of the implants.
Results: The clearance rates of α-Gal antigen in the experimental group and the conventional control group were (99.09±0.26)% and (30.18±2.02)%, respectively (t=58.67, P<0.01). The residual DNA of the experimental group, the conventional control group and the positive control group were (13.49±0.07) ng/mg, (15.20±0.21) ng/mg and (14.70±0.17) ng/mg, the residual DNA in the experimental group was significantly lower than that in the conventional control group (t=－13.41, P<0.01) and the positive control group (t=－11.30, P<0.01). HE staining showed that multiple bone formation centers with active osteogenesis and rich bone marrow were observed in experimental group 4 weeks after implantation, only a small number of bone formation centers were observed in the conventional control group and the positive control group, with no obvious osteoblasts present. Immunohistochemistry results indicated that the expressions of alkaline phosphatase, Runt-related transcription factor 2, typeⅠcollagen and osteocalcin in the experimental group showed an increasing trend compared with those in the conventional control group and the positive control group.
Conclusions: Compared with clinically used demineralized bone matrix and bone graft material decellularized with conventional method, bone graft material decellularized with supercritical carbon dioxide exhibits lower immunogenicity and better osteogenic ability.
目的: 评价采用超临界二氧化碳脱细胞后动物源性骨修复材料的免疫原性和成骨能力。方法: 将猪股骨前处理后随机分成两组，分别使用常规方法（对照组）和超临界二氧化碳（实验组）进行脱细胞处理。通过酶联免疫吸附试验和荧光染色法检测常规对照组和实验组半乳糖-α-1，3-半乳糖（α-Gal）抗原清除率和DNA残留量。9只6周龄SPF级雄性裸鼠随机分成实验组、常规对照组和阳性对照组，在裸鼠股二头肌肌间隙中植入不同骨组织，术后4周取材，分别用苏木精-伊红（HE）染色和免疫组织化学检测植入部位诱导成骨能力及成骨相关蛋白表达。结果: 实验组和常规对照组α-Gal抗原清除率分别为（99.09±0.26）%和（30.18±2.02）%，差异有统计学意义（t=58.67，P<0.01）。实验组、常规对照组和阳性对照组的DNA残留量分别为（13.49±0.07）、（15.20±0.21）和（14.70±0.17）ng/mg，其中实验组DNA残留量显著低于常规对照组（t=－13.41，P<0.01）和阳性对照组（t=－11.30，P<0.01）。HE染色结果显示，实验组术后4周观察到植骨部位有多个骨生发中心伴成骨活跃、骨髓丰富；常规对照组和阳性对照组仅观察到少量骨生发中心，未见明显的成骨细胞。免疫组织化学结果提示，实验组成骨相关蛋白碱性磷酸酶、Runt相关转录因子2、Ⅰ型胶原蛋白和骨钙蛋白的表达量均有较常规对照组和阳性对照组增加的趋势。结论: 经超临界二氧化碳脱细胞处理的骨组织比临床上常用的同种脱钙骨基质和常规方法处理的骨修复材料具有更低的免疫原性和更好的诱导成骨能力。.
Keywords: Bone repair; Decellularized matrix scaffold; Supercritical carbon dioxide; Xenotransplantation.