摘要
[目的]探讨低氧培养条件对多孔钽-成骨细胞形态学、增殖能力以及COL-Ⅰ、OC蛋白表达的影响。[方法]选用MG63成骨细胞原代培养及传代。将第3代细胞以1×10~6/L^(-1)细胞浓度接种于多孔钽材料进行复合培养。采用三气培养箱,以20%氧体积分数作为常氧环境,氧体积分数2%作为缺氧环境,同时设1%及3%氧体积分数作为缺氧反应对比条件。实验分组:将多孔钽-成骨细胞复合物分为4组:常氧钽组(对照组),3%缺氧钽组、2%缺氧钽组及1%缺氧钽组(实验组)。CCK-8法检测多孔钽-成骨细胞复合培养第1、3、5、7、9、11 d各组成骨细胞生长和增殖状态;扫描电镜观察缺氧对多孔钽-成骨细胞复合物形态特征影响;透射电镜观察缺氧条件下成骨细胞超微结构改变;免疫细胞化学法检测缺氧各组成骨细胞COL-Ⅰ、OC蛋白的表达。[结果]CCK-8法检测显示缺氧条件下各实验组与对照组比较,细胞随培养时间的延长,成骨细胞生长及增殖速度明显放慢,除第1 d外,其余各时间点OD值差异均有统计学意义(P<0.05);各实验组组间比较除第3 d外,其余时间点各组间比较差异均存在统计学意义(P<0.05),扫描电镜显示在缺氧条件下多孔钽-成骨细胞对缺氧反应表现为体积变小,伪足少而细,只分泌极少量的细胞外基质覆盖于多孔钽表面。透射电镜显示:在缺氧条件下钽-成骨细胞复合培养与常氧培养的成骨细胞相比,线粒体、内质网扩张,空泡样变性,线粒体嵴断裂或消失。免疫细胞化学检测结果得出:缺氧条件下各实验组成骨细胞COL-Ⅰ、OC蛋白表达较对照组显著降低(P<0.05)。[结论]多孔钽-成骨细胞复合物在缺氧条件下成骨细胞增殖能力降低,细胞形态及超微结构改变,功能降低以及成骨蛋白COL-Ⅰ、OC表达降低。提示:监测骨缺损局部氧分压将有助于三维结构骨移植材料内部成骨细胞生长及骨形成量。
[Objective] To investigate the effect of hypoxia on the morphology, proliferation, and COL-Ⅰ and OC pro- tein expression of osteoblasts growing in porous tantalum scaffolds in vitro. [Methods] MG63 primary osteoblasts were cul- tured and passaged. The 3rd generation of cells were seeded into porous tantalum scaffolds at 1 × 10^6 cells/L and cultured in Tri- Gas incubators. The ceils were divided into a normal control group cultured with 20% oxygen volume fraction and three treat- ment groups cultured with 3%, 2%, or 1% oxygen volume fraction. The growth and proliferation of osteoblasts in porous tanta- lum scaffolds at 1^st, 3^rd, 5^th, 7^th, and 11^th days were tested using CCK-8. The morphological changes of osteoblasts upon hy- poxia were examined by scanning electron microscope （SEM） . Transmission electron microscope （TEM） was used to examine the ultrastructure of the osteoblasts. The protein ex- pression of COL-Ⅰ and OC in the osteoblasts was examined by immunocytochemistry. [Results] Under hypoxia, compared with the normal control group, the treatment groups had a significantly lower OD value at the above time points except the Ft day （P 〈0.05） . This indicated the growth and proliferating rates decreased under hypoxia over the time of incubation. There were also significant differences between treatment groups at all the time points exc, ept the 3^rd day （P 〈0.05） . The SEM results showed that the hypoxia treatment groups had smaller cell volumes, fewer and thinner pseudopods, and an extremely small amount of extraeellular matrix. The TEM examination showed that compared with the normal control group the treatment groups had mitochondria and endoplasmic reticulum expansion, vacuolar degeneration, and breakage or disappearance of the mitochondria cristae. The re- suits of immunocytochemistry showed that under low-oxygen conditions, the osteoblasts had significantly lower protein expres- sion of COL-Ⅰ and OC （P 〈0.05） . [Conclusions] The osteoblasts growing in porous tantalum scaffolds under low-oxygen conditions have reduced proliferating ability, abnormal cell morphology, uhrastructure, and function, and reduced protein expression of COL-Ⅰ and OC. This indicates that monitoring local osteoblast oxygen tension is beneficial for improving osteo- hlast growth and bone formation in three-dimensional bone substitute materials.
作者
史伟
甘洪全
王茜
任原
张辉
王辉
李琪佳
王志强
Sill Wei GAN Hong-quan WANG Qian et al(Department of Orthopaedics, Affiliated Hospital of North China University of Science and Technology, Tangshan Hebei, 063000, Chin)
出处
《中国矫形外科杂志》
CAS
CSCD
北大核心
2017年第8期729-736,共8页
Orthopedic Journal of China
基金
国家科技部科技支撑课题资助项目(编号:2012BAE06B03)
河北省科技支撑资助项目(编号:16277776D)
河北省医学科学研究重点课题计划(编号:20160225)
华北理工大学博士科研启动基金资助项目
