[1]孙静,卫丹,朱禹达,等.海藻酸钠水凝胶中粘附位点及空间构建促进MG63细胞铺展及成骨分化[J].中国材料进展,2013,(10):623-630.[doi:10.7502/j.issn.1674-3962.2013.10.07]
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海藻酸钠水凝胶中粘附位点及空间构建促进MG63细胞铺展及成骨分化()
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中国材料进展[ISSN:1674-3962/CN:61-1473/TG]

卷:
期数:
2013年第10期
页码:
623-630
栏目:
特约研究论文
出版日期:
2013-10-31

文章信息/Info

作者:
孙静卫丹朱禹达钟美玲左一聪范红松*
四川大学国家生物医学材料工程技术研究中心,成都610064
Author(s):
The construct of cell adhesion sites and degradation space in alginate hydrogels to enhance spreading and osteogenic differentiation of MG-63 cells
关键词:
水凝胶明胶微球RGD细胞铺展成骨分化
DOI:
10.7502/j.issn.1674-3962.2013.10.07
摘要:
细胞粘附与铺展是三维水凝胶基质中贴壁依赖型细胞存活所必须的两个条件,将细胞粘附位点的引入和凝胶中细胞铺展空间的构建相结合,本文提出了同时含有RGD多肽和明胶微球的粘附型大孔水凝胶模型,以促进细胞在其中的铺展与分化。该模型采用光交联海藻酸钠水凝胶为基础,同时引入RGD多肽和明胶微球,通过RGD多肽的共价接枝为细胞粘附提供前提,利用明胶微球在37℃下的快速降解性,为细胞的进一步增殖和铺展以及分化提供所需空间。结果显示,明胶微球的加入提高了凝胶的力学性能,同时降低了凝胶的溶胀率。RGD和明胶微球的引入能够很好地支持MG-63细胞在其中的增殖、粘附与铺展,并显著提高其碱性磷酸酶活性,上调成骨相关基因(BMP-2,COL-I和OCN)的表达。而在不含微球的RGD-ALG和ALG凝胶中,细胞铺展及成骨分化均受到很大抑制。
Abstract:
Adhesion and spreading of anchorage-dependent cells are two essential factors for their subsequent settlement and commitment in 3D hydrogel substrate. An adhesive macroporous alginate hydrogel system was suggested here to overcome the low cell affinity and high cell constraint of hydrogel substrates for cells encapsulated in them. RGD peptide was grafted onto the alginate molecule to support cell adhesion and gelatin microspheres were introduced into photocrosslinked alginate hydrogels to create macropores by their degradation at 37℃ for cell spreading and facilitate cell ingrowth as well as prolonged cell survival within hydrogels. With the incorporation of gelatin microspheres, the hybrid alginate hydrogels exhibited significantly enhanced mechanical properties and decreased swelling ratios. MG-63 cells in alginate hydrogel cooperated with RGD and gelatin microspheres exhibited a rapid proliferation and spread gradually with prolonged culture time, and their osteogenic differentiation was greatly facilitated, such as the high expression of BMP-2, COL-I and OCN genes. While in the pure alginate hydrogel (ALG) and hydrogels only capable of cell affinity (RGD-ALG), cells could just exhibit either a rounded shape or a spreading morphology with a very limited degree, and the osteogenic differentiation was also inhibited

参考文献/References:

[1].方法16。4℃,pH8条件下,海藻酸钠上的羟基和甲基丙烯酸酐发生酯化反应,透析后冷冻干燥得到甲基丙烯酸接枝率为50%的甲基丙烯酸化海藻酸钠(MAA)。配制3 wt%的MAA/TEOA(含0.5% I2959)溶液,按质量比为RGD:MAA=0.385:1加入RGD多肽(GCGYGRGDSPG),室温下反应1h后,加入与MAA等体积的明胶微球,混合均匀后加入到模具中,7.9 mW/cm2紫外光照下交联得到粘附型的大孔海藻酸钠水凝胶。
[2].1.4 凝胶性能测试
[3].采用电子万能材料试验机(Shenzhen SANS Testing Machine)测试了凝胶的压缩强度和压缩模量,传感器为200N,试样为8mm×2mm 的圆柱形,加载速度为0.3 mm/min,每组试样平行测五次。将凝胶化 24h 的试样按 0.1g/mL 加入PBS中浸泡,通过测定其质量随时间变化规律考察各组凝胶在PBS中的溶胀率。溶胀率计算公式如下: S=(w-w0)/w0×100%,其中 S为溶胀率, w为材料吸水后的质量, w0 为凝胶的初始质量.
[4].1.5 MG-63细胞—海藻酸钠凝胶体外培养
[5].将MG-63 细胞悬液按1×107 cells/mL密度分别接种于凝胶前溶液中,溶液固化成凝胶后加入DMEM培养基,置37℃, 5% CO2 培养箱内培养。
[6].体外培养1,3,5天后,加入0.5mg/ml MTT, 37℃孵育4 h后加入DMSO振荡, 酶联免疫检测仪在490 nm波长测定吸光度(optical density, OD)。
[7].通过FDA/PI染色,激光共聚焦显微镜观察MG-63细胞在凝胶内的形态和分布。
[8].采用ALP试剂盒(Sensolyte pNPP Alkaline Phosphatase Assay Kit, AnaSpec)检测细胞内碱性磷酸酶(ALP)的活性。
[9].荧光定量PCR检测成骨相关基因的表达:将载有细胞的凝胶经液氮速冻研磨后,用TRIZOL常规提取总RNA,经逆转录试剂盒(Bio-Rad, USA)反转录成cDNA。RT-qPCR在六色梯度实时定量PCR仪(Bio-Rad, CFX960, USA)上进行,试剂盒为SsoFastTM EvaGreen Supermix (Bio-Rad, USA)。实验选择骨形态发生蛋白-2(BMP-2),I型胶原(COL-I),骨钙素(OCN)和GAPDH基因,其引物序列见表1。BMP-2,COL-I,OCN和GAPDH mRNA的表达水平表示为阈值(CT),用2△△Ct值法来计算相对表达量,看家基因GAPDH的表达作为内参来归一化结果,所有样品分析三次。

更新日期/Last Update: 2013-10-11