动态力学加载下MC3T3-E1细胞在3D打印三维仿生复合支架上的成骨效应研究  

Osteogenesis effect of dynamic mechanical loading on MC3T3-E1 cells in threedimensional printing biomimetic composite scaffolds

作　　者：宋秀钢 李晖[1] 李瑞欣 袁清献 刘迎节[1,2,3] 程威 张西正 SONG Xiugang1,2,3, LI Hui1, LI Ruixin3, YUAN Qingxian3,4, LIU Yingjie1,2,3, CHENG Wei1,2,3, ZHANG Xizheng3(1. Department of Orthopedics, Tianjin Medical University General Hospital, Tianjin, 300052, P.R.China 2. Department of Clinical Medicine of Graduate School, Tianjin Medical University, Tianjin, 300070, P,R.China 3. Institute of Medical Equipment, Academy of Military Medical Science, Tianjin, 30016I, P.R.China 4. Department of Biomechanics of the Institute of Mechanics, Tianjin University of Technology, Tianjin, 300384, P.R.Chin)

机构地区：[1]天津医科大学总医院骨科,天津300052 [2]天津医科大学研究生院临床医学系,天津300070 [3]中国人民解放军军事医学科学院卫生装备研究所,天津300161 [4]天津市口腔医院中心实验室 [5]天津理工大学机械学院生物力学系,天津300384

出　　处：《中国修复重建外科杂志》2018年第4期448-456,共9页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：国家自然科学基金资助项目(31370942、31470935、11432016)

摘　　要：目的观察动态力学加载对低温3D打印联合冷冻干燥法制备的三维仿生复合支架材料内MC3T3-E1细胞增殖、分化、成骨特异性基因表达的影响。方法将丝素蛋白、Ⅰ型胶原、羟基磷灰石按质量比3∶9∶2混合后,采用低温3D打印联合冷冻干燥技术制备三维仿生复合支架;大体观察支架外形,Micro-CT观察支架孔径及孔隙率,测定吸水膨胀率以及应力、应变、弹性模量。取MC3T3-E1细胞接种至三维仿生复合支架,随机分成两组:实验组培养期间行动态力学加载,每天1次、每次15 min、频率1 Hz、应变3 500με;对照组不作力学加载。培养7、14 d,分别对细胞-支架复合物行HE染色及扫描电镜观察细胞在支架上生长情况;Western blot以及实时荧光定量PCR检测Ⅰ型胶原、BMP-2、骨钙素(osteocalcin,OCN)蛋白及m RNA表达。结果制备的三维仿生复合支架为白色立方体网格,Micro-CT检测见支架材料内部呈孔隙网状结构,孔隙连通性良好;大孔径直径为(506.37±18.63)μm,微孔径直径为(62.14±17.35)μm,孔隙率为97.70%±1.37%,吸水膨胀率为1 341.97%±64.41%。力学测试示,支架压缩至10%时,支架压缩位移为(0.376±0.004)mm、压缩应力为(0.016±0.002)MPa、弹性模量为(162.418±18.754)k Pa。复合培养7、14 d,两组HE染色及扫描电镜均见细胞在支架内部生长,主要分布在支架孔壁周围,其中实验组细胞较对照组增多,且细胞由梭形变为扁平状。除200倍镜下14 d时两组细胞计数差异无统计学意义(t=–2.024,P=0.080)外,其余不同放大倍数(40、100、400倍)下各时间点实验组细胞数均显著高于对照组(P<0.05)。实时荧光定量PCR检测示:实验组培养7、14 d时Ⅰ型胶原、OCN m RNA相对表达量显著高于对照组(P<0.05),但BMP-2 m RNA相对表达量与对照组比较差异无统计学意义(P>0.05)。Western blot检测示,实验组培养7、14 d时Ⅰ型胶原、BMP-2、OCN蛋白相对表达量均显著高于对照组(P<0.ObjectiveTo observe the effect of dynamic mechanical loading on the proliferation, differentiation, and specific gene expression of MC3T3-E1 cells that on three-dimensional （3D） biomimetic composite scaffolds prepared by low temperature 3D printing technology combined with freeze-drying.MethodsThe silk fibroin, collagen type Ⅰ, and nano-hydroxyapatite （HA） were mixed at a mass ratio of 3∶9∶2 and were used to prepare the 3D biomimetic composite scaffolds via low temperature 3D printing technology combined with freeze-drying. General morphology of 3D biomimetic composite scaffold was observed. Micro-CT was used to observe the pore size and porosity of the scaffolds, and the water swelling rate, stress, strain, and elastic modulus were measured. Then, the MC3T3-E1 cells were seeded on the 3D biomimetic composite scaffolds and the cell-scaffold composites were randomly divided into 2 groups. The experimental group was subjected to dynamic mechanical loading （3 500 με, 1 Hz, 15 minutes per day）; the control group was not subjected to loading treatment. After 7 days and 14 days, the cell-scaffold composites of 2 groups were harvested to observe the growth of cells on the scaffolds by HE staining and scanning electron microscope. And the gene and protein expressions of collagen type Ⅰ, BMP-2, and osteocalcin （OCN） were measured by real-time fluorescent quantitative PCR and Western blot.ResultsThe 3D biomimetic composite scaffold was a white cubic grid. Micro-CT detection showed the pore network structure in the scaffold material with good pore connectivity. The diameters of large pore and micro-aperture were （506.37±18.63） μm and （62.14±17.35） μm, respectively. The porosity was 97.70%±1.37%, and the water absorption swelling rate was 1 341.97%±64.41%. Mechanical tests showed that the compression displacement of the scaffold was （0.376±0.004） mm, the compressive stress was （0.016±0.002） MPa, and the elastic modulus was （162.418±18.754） kPa when the scaffold was compr

关 键 词：丝素蛋白 胶原 羟基磷灰石 MC3T3-E1细胞 力学加载 

分 类 号：R318.08[医药卫生—生物医学工程] R68[医药卫生—基础医学]