新型生物活性接骨螺钉材料对成骨细胞生物学行为影响的体外研究  

In vitro study on new bioactive bone screws affecting biological behavior of osteoblasts

作　　者：苏保[1] 蒋电明[1] 李吉东[2] 安洪[1] 邬均[1] 祁小桐[1] 

机构地区：[1]重庆医科大学附属第一医院骨科,400016 [2]四川大学分析测试中心

出　　处：《中华创伤杂志》2014年第10期1055-1060,共6页Chinese Journal of Trauma

基　　金：重庆市科委科技攻关资助项目（CSTC,2009AB5080）

摘　　要：目的,研究用于制备新型生物活性接骨螺钉的纳米羟基磷灰石／聚酰胺66／玻璃纤维（nano—hydroxyapatite／polyamide66／glassfiber，n-HA／PA66／GF）对成骨细胞生物学行为的影响，为生物螺钉后期临床应用提供实验依据。方法,制备圆片状n-HA／PA66／GF及其浸提液；取小鼠成骨细胞与n-HA／PA66／GF浸提液共培养，采用直接接触实验观察细胞生长变化情况；MTT实验检测细胞增殖情况；流式细胞仪检测细胞凋亡率；ELISA法测定细胞骨钙素含量；Tran-swell小室实验观察细胞迁移能力；扫描电镜观察细胞黏附和生长情况；细胞免疫荧光检查细胞骨架及肌动蛋白纤维表达。结果,直接接触实验表明，n-HA／PA66／GF对成骨细胞无明显细胞毒性。MTT检测n-HA／PA66／GF浸提液与成骨细胞共培养2，4，6d后，吸光度（A）值分别为0.96±0.14，1.54±0.15，2.39±0.27。结果显示，成骨细胞数量随着培养时间延长而增多（P〈0.05）。流式细胞检测结果证实，n-HA／PA66／GF能使更多成骨细胞进入S期而对细胞凋亡率无明显影响，成骨细胞骨钙素分泌随着共培养时间延长而增多。Transwell实验显示，含浸提液和FBS的α-MEM培养基小室穿过半透膜的细胞数分别为（8.73±3.26）个／视野、（9.47±3.29）个／视野，差异无统计学意义（P〉0.05）。扫描电镜观察n-HA／PA66／GF表面的成骨细胞形态规整，紧紧黏附在材料表面，逐渐呈现复层生长。免疫荧光结果表明，材料浸提液对细胞骨架及肌动蛋白纤维的分布无明显影响。结论,新型生物螺钉n-HA／PA66／GF具有良好的成骨细胞相容性，对细胞生长、增殖、分泌、黏附、周期和骨钙素分泌等均有一定调节作用。Objective, To detect the effect of a new bioactive bone screws composed of nanohydroxyapatite/polyamid 66/glass fiber （n-HA/PA66/GF） biomaterial on biological behavior of osteoblasts with a view to a later clinical application of the screws. Methods, Mice osteoblasts were co-cul- tured with n-HA/PA66/GF biomaterial or its extract. Cell growth was detected under a microscope, cell proliferation using MTY assay, apoptosis by flow cytometry, osteocalcin by ELISA method, cell migration using Transwell invasion assay, cell adhesion and growth by scanning electron microscope, and cytoskeleton and actin filament distribution using immunofluorescene. Results, Direct contact test demonstrated n-HA/PA66/GF material had no obvious cytotoxicity to osteoblasts. Extract of n-HA/PA66/GF material stimulated osteoblast proliferative activity over time with absorbance value of 0.96±0.14, 1.54±0.15, and 2.39±0.27 respectively after 2, 4, and 6 hours of coculture （P 〈 0.05）. The n-HA/PA66/GF material induced more osteoblasts to entering S period without obvious influence on apoptosis rate and promoted osteocalcin secretion. Migrated cells in medium supplied with n-HA/PA66/GF material or FBS was 8.73±3.26 and 9.47±3.29 in each visual field, but there was no significant difference （ P 〉 0.05 ）. Osteoblasts had a regular form on the surface of n-HA/PA66/GF material, closely adhered to the surface, and reproduced and aggregated with each other to form stratified cell layers. However, n- HA/PA66/GF material exhibited no obvious influence on distribution of cytoskeleton and actin filament. Conclusion, The new n-HA/PA66/GF screws has excellent cytoeompatibility with positive regulatory effect on cell growth, proliferation, secretion, adhesion, cycle, and osteocalcin secretion.

关 键 词：细胞生物学 成骨细胞 纳米羟基磷灰石/聚酰胺66/玻璃纤维 

分 类 号：R318.08[医药卫生—生物医学工程]