Bioactive Nanofibers Instruct Cells to Proliferate and Differentiate During Enamel Regeneration
Bioactive Nanofibers Instruct Cells to Proliferate and Differentiate During Enamel Regeneration
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DOI：
10.1359/jbmr.080705
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发表时间：
2008-12-01
影响因子：
6.2
通讯作者：
Snead, Malcolm L.
中科院分区：
文献类型：
--
作者：
Huang, Zhan;Sargeant, Timothy D.;Snead, Malcolm L.
在牙齿发育过程中，外胚层衍生的成纤维细胞通过合成用于控制细胞的复杂蛋白混合物来与基质相互作用和羟基磷灰石结晶岩的习惯，从而产生牙釉质。使用体外细胞和器官培养系统，我们研究了人工生物活性纳米结构对成成木细胞的影响，其长期目标是开发基于细胞的牙齿再生策略。我们使用了含有肽基序Arg-Gly-Asp或“ RGD”（缩写的BRGD-PA）的分支肽两亲分子，已知在生理环境中自我组装成纳米纤维中，这些纳米纤维在其表面上显示出这种生物学信号的高密度。在PA水凝胶中培养了成纤维细胞样细胞（LS8线）和原代搪瓷器官上皮（EOE）细胞，并将PA注入小鼠胚胎牙齿的搪瓷器官上皮。通过定量的实时PCR和免疫检测技术检测到氨基蛋白蛋白，蛋白质细胞蛋白，整联蛋白α5和整联蛋白α6的表达。我们使用BRDU标记和使用αAlizarin红色S染色的生物矿化测定法进行了细胞增殖测定法，并使用定量Ca2+测量结果进行了生物矿化测定。在细胞培养模型中，成纤维细胞样细胞（LS8）和原代EOE细胞对BRGD-PA纳米结构做出反应，并具有增强和更大的amelegenin，amelogoentin，ameloblastin和整合素表达水平。在器官培养模型中BRGD-PA注射的部位，我们观察到EOE细胞的增殖，分化为成成木细胞，这是通过其表达搪瓷特异性蛋白的表达来证明的。超微结构分析表明，在形成细胞外基质中的纳米纤维与参与牙釉质形成和再生的EOE细胞接触。这项研究表明，使用牙釉质蛋白的BRGD-PA纳米纤维参与整联蛋白介导的细胞结合与基质的结合，并通过传递指导性信号以形成搪瓷。
During tooth development, ectoderm-derived ameloblast cells create enamel by synthesizing a complex protein mixture serving to control cell to matrix interactions and the habit of hydroxyapatite crystallites. Using an in vitro cell and organ culture system, we studied the effect of artificial bioactive nanostructures on ameloblasts with the long-term goal of developing cell-based strategies for tooth regeneration. We used branched peptide amphiphile molecules containing the peptide motif Arg-Gly-Asp, or "RGD" (abbreviated BRGD-PA), known to self-assemble in physiologic environments into nanofibers that display on their surfaces high densities of this biological signal. Ameloblast-like cells (line LS8) and primary enamel organ epithelial (EOE) cells were cultured within PA hydrogels, and the PA was injected into the enamel organ epithelia of mouse embryonic incisors. The expression of amelogenin, ameloblastin, integrin alpha 5, and integrin alpha 6 was detected by quantitative real-time PCR and immunodetection techniques. We performed cell proliferation assay using BrdU labeling and a biomineralization assay using Alizarin red S staining with quantitative Ca2+ measurements. In the cell culture model, ameloblast-like cells (LS8) and primary EOE cells responded to the BRGD-PA nanostructures with enhanced proliferation and greater amelogenin, ameloblastin, and integrin expression levels. At the site of injection of the BRGD-PA in the organ culture model, we observed EOE cell proliferation with differentiation into ameloblasts as evidenced by their expression of enamel specific proteins. Ultrastructural analysis showed the nanofibers within the forming extracellular matrix, in contact with the EOE cells engaged in enamel formation and regeneration. This study shows that BRGD-PA nanofibers present with enamel proteins participate in integrin-mediated cell binding to the matrix with delivery of instructive signals for enamel formation.