Hao Feng, Pan Kaifeng, Huang Liuyun, Chen Xuhong, Wei Haikun, Chen Xianhua, Zhang Jianfeng
Zhejiang Decellmatrix Biotech Co., Ltd., Hangzhou 310018, China.
Department of Orthopedic Surgery, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou 310016, China.
Zhejiang Da Xue Xue Bao Yi Xue Ban. 2024 Dec 25;53(6):772-778. doi: 10.3724/zdxbyxb-2024-0108.
To evaluate the immunogenicity and osteogenic ability of animal-derived bone graft material decellularized with supercritical carbon dioxide.
Porcine femurs were randomly divided into two groups after preliminary treatment, and decellularized with conventional method (control group) or supercritical carbon dioxide (experimental group). Allogenic demineralized bone matrix was used as positive control. Clearance rate of galactose-α-1, 3-galactose (α-Gal) antigen was determined by enzyme-linked immunosorbent assay and residual DNA was detected by a fluorescence method. Nine SPF-grade male athymic nude mice of 6 weeks old were randomly divided into experimental, control and positive control groups. 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.
The clearance rates of α-Gal antigen in the experimental group and the control group were (99.09±0.26)% and (30.18±2.02)%, respectively (=58.67, 0.01). The residual DNA of the experimental, control and positive control groups were (13.49±0.07), (15.20±0.21) and (14.70±0.17) ng/mg. The residual DNA in the experimental group was significantly lower than that in the control group (=－13.41, <0.01) and positive control group (=－11.30, <0.01). HE staining results showed that multiple bone formation centers with active osteogenesis and rich bone marrow were observed in experimental group 4 weeks after implantation, but only a small number of bone formation centers were observed in the control and positive control groups, with no obvious osteoblasts present. Immunohistochemistry results indicated that the expressions of alkaline phosphatase, Runt-related transcription factor 2, collagen typeⅠand osteocalcin in the experimental group showed an increasing trend compared with those in the control and positive control groups.
Compared with clinically used allogenic 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.
评估经超临界二氧化碳脱细胞处理的动物源性骨移植材料的免疫原性和成骨能力。
猪股骨经初步处理后随机分为两组，分别采用常规方法脱细胞（对照组）或超临界二氧化碳脱细胞（实验组）。同种异体脱矿骨基质用作阳性对照。采用酶联免疫吸附测定法测定半乳糖-α-1,3-半乳糖（α-Gal）抗原清除率，采用荧光法检测残留DNA。将9只6周龄的SPF级雄性无胸腺裸鼠随机分为实验组、对照组和阳性对照组。将样本植入无胸腺裸鼠的股二头肌上方。植入后4周收集植入物。应用苏木精-伊红（HE）染色和免疫组织化学方法测定植入物的成骨能力和骨组织相关蛋白表达。
实验组和对照组的α-Gal抗原清除率分别为（99.09±0.26）%和（30.18±2.02）%（=58.67，<0.01）。实验组、对照组和阳性对照组的残留DNA分别为（13.49±0.07）、（15.20±0.21）和（14.70±0.17）ng/mg。实验组的残留DNA显著低于对照组（=-13.41，<0.01）和阳性对照组（=-11.30，<0.01）。HE染色结果显示，实验组植入后4周可见多个成骨活跃且骨髓丰富的骨形成中心，而对照组和阳性对照组仅见少量骨形成中心，未见明显成骨细胞。免疫组织化学结果表明，与对照组和阳性对照组相比，实验组碱性磷酸酶、 runt相关转录因子2、Ⅰ型胶原和骨钙素的表达呈上升趋势。
与临床使用的同种异体脱矿骨基质及常规方法脱细胞的骨移植材料相比，经超临界二氧化碳脱细胞的骨移植材料免疫原性更低，成骨能力更强。
