恒河猴体内构建组织工程骨的生物学安全性评价

Biological safety of tissue-engineered bone constructed in rhesus

目的:对恒河猴体内构建的组织工程骨进行生物学安全性评价。方法:实验于2003-05/2004-12在解放军第一军医大学完成。雄性恒河猴6只,体质量4～6kg,分别抽取实验动物的骨髓,体外培养骨髓基质干细胞并向成骨细胞诱导,将诱导后的骨髓基质干细胞以5×109L-1的浓度与可吸收羟基磷灰石复合,体外培养3d后回植入恒河猴背阔肌异位构建组织工程骨,分别于术前、术后2,4,12周4个时间点进行长期毒性试验(观察植入后动物的生理状况及血生化指标在术后各时间点与术前的变化)、肌内植入试验(观察炎性浸润、纤维包囊形成及有无异形细胞)、遗传毒性试验(观察嗜多染红细胞,记录微核数)等检测。结果:纳入恒河猴6只,均进入结果分析。①体内构建组织工程骨后,动物精神、行为、活动、摄食及粪便无异常,无血尿,体质量逐渐增加。术后2,4,12周和术前相比,外周血象、血生化指标差异均无显著性意义(P>0.05),②各时间点骨髓相均增生活跃、形态正常;植入试验2周周围肌肉可见少量炎性细胞浸润,4,12周未见明显炎性细胞和异型细胞。③各组骨髓涂片在镜下难以见到嗜多染红细胞,均未发现有微核形成。结论:恒河猴体内构建组织工程骨具有良好的生物学安全性。

AIM： To evaluate the biological safety of tissue-engineered bone constructed in rhesus. METHODS： The experiment was conducted in the First Military Medical University of Chinese PLA between May 2003 and December 2004. Bone marrow stromal cells of six male rhesuses of 4-6 kg were cultured and induced into osteoblast in vitro, then the cell suspension was mixed with absorbable hydroxyapatite at concentration of 5×10^9 L^-1. Three days later, the biomaterials mixed with cells were implanted into the latissimus dorsi muscle of rhesuses for the heterotopic construction of tissue-engineered bone. Long-term toxicity （changes of physiological condition and blood biochemical indexes before and each time point after the implantation）, muscle implanting test （inflammatory infiltration, fibrous encapsulation formation and allotype cells）, and genotoxicity （polychromatic erythrocytes, and number of micronuclei） were performed before and 2, 4, and 12 weeks after implantation. RESULTS： Six rhesuses were involved in the result analysis. （1）After the tissue-engineered bone was constructed in vivo, the physiological status including mental, behavior, activity, ingestion and dejecture of rhesus kept well, and the body mass was increased gradually; additionally, no blood urine was found. Compared with those before implantation, the peripheral blood picture, blood biochemical indexes had no significant difference at 2, 4 and 12 weeks after the operation （P 〉 0.05）. （2）The myelogram at each time point showed active proliferation and normal appearance, and a small quantity of inflammatory cells were observed in muscle around 2 weeks after implantation, but no inflammatory cells or allotype cells were found at 4, and 12 weeks. （3）Polychrematic erythrecyte was hardly observed in marrow smear, neither the micrenuclei in each group. CONCLUSION： Tissue-engineered bone constructed in rhesus has good biological safety.