基于状的不均匀 Nanopatterns 对局部控制表面粘附的研究--一种直接软骨分化的方法
1Institute for Bioengineering of Catalonia (IBEC), The Barcelona Institute of Science and Technology (BIST), 2Department of Engineering Electronics,University of Barcelona (UB), 3Networking Biomedical Research Center (CIBER), 4Instituto de Investigacin Biomédica de Málaga (IBIMA), Department of Organic Chemistry,Universidad de Málaga (UMA), 5Andalusian Centre for Nanomedicine and Biotechnology-BIONAND, 6Unidad de Bioingeniería Tisular y Terapia Celular (GBTTC-CHUAC), Grupo de Reumatolog ía, Instituto de Investigación Biomèdica de A Coruña (INIBIC), Complexo Hospitalario Universitario de A Coruña (CHUAC), Sergas,Universidade da Coruña (UDC), 7Institució Catalana de Recerca i Estudis Avançats (ICREA), 8Instituto de Investigación Biomédica de Málaga (IBIMA), Department of Cell Biology, Genetics and Physiology,Universidad de Málaga (UMA)
Summary
本文介绍了一种获得基于状的不均匀 nanopatterns 的方法, 该法允许对局部精氨酸-甘氨酸 (RGD) 的表面密度进行纳米控制, 并将其用于细胞黏附和软骨分化的研究。
Abstract
细胞黏附和分化的条件是由纳米尺度配置的细胞外基质 (ECM) 成分, 当地浓度有一个主要的影响。在这里, 我们提出了一种方法, 以获得大规模不均匀 nanopatterns 的精氨酸-甘氨酸-天冬酸 (RGD) 官能化状, 允许纳米控制的局部 RGD 表面密度。Nanopatterns 是由不同初始浓度的溶液中的状表面吸附形成的, 其特征是水接触角 (CA)、X 射线光电子能谱 (XPS) 和扫描探针显微镜技术, 如扫描隧道显微镜 (STM) 和原子力显微镜 (AFM)。用 AFM 图像, 利用最小间距离的概率等高线图测量了 RGD 的局部表面密度, 并与细胞黏附反应和分化相关。这里介绍的 nanopatterning 方法是一个简单的过程, 可以以直接的方式扩展到大面积的表面积。因此, 它完全符合细胞培养协议, 并可适用于其他配体, 发挥浓度依赖性的影响细胞。
Introduction
在这里, 我们描述一个简单和通用的状的 nanopatterning 程序, 以获得细胞培养表面, 允许在纳米尺度的局部粘附控制。已报告了 ECM 组织的纳米尺度细节,1,2,3和细胞黏附表面的 nanopatterning 提供了深刻的洞察力的细胞要求与黏附力4, 5。实验使用胶束光刻基 nanopatterns 揭示了阈值约 70 nm 的 RGD 肽 nanospacing, 细胞粘附明显延迟超过此值6,7,8 ,9。这些研究还突出了局部配体密度对细胞黏附力的影响大于9,10,11。
在形态发生过程中, 细胞与周围环境的相互作用触发了第一次分化事件, 直到最终形成复杂的组织结构。在这个框架内, nanopatterned 表面被用来解决最初的细胞表面相互作用对形态发生的影响。在β型 Ti-40Nb 合金中横向间距为 68 nm 的光刻基 RGD nanopatterns 有助于维持未分化的干细胞的表型12, 而 RGD 95 和150纳米之间的 nanospacings 则增强了间充质干细胞 (MSCs) 向脂/成骨13,14,15和软骨命运16。此外, 通过向信号元件修饰的自组装大分子, 可以通过提供信号提示的纳米结构调节来指示细胞的黏附力和分化程度17。在这方面, 状的沉积与细胞相互作用的基在他们的外球面18,19,20到表面已经被用来研究细胞黏附力21,22,形态学23,24和迁移事件25,26。然而, 在这些研究中缺乏表面表征, 使得很难在状表面构型和细胞反应之间建立任何相关性。
在低离子强度的溶液中, 状吸附到低电荷表面时, 可以得到状 nanopatterns 的液样顺序和定距。27在此属性的基础上, 本文给出了一种在低带电表面上获得大尺度不均匀 nanopatterns 的方法, 它允许对局部 RGD 表面密度进行纳米尺度的 RGD 控制。水接触角 (CA), x 射线光电子能谱 (XPS) 和扫描探针显微镜技术 (STM 和 AFM nanopatterns) 表明, 局部配体密度可以调整修改初始状浓度的解决方案。利用最小间距离的概率等高线图, 用 AFM 图像对局部 RGD 表面密度进行量化, 然后与细胞实验相关联。与其他 nanopatterning 技术4相比, 基于状的 nanopatterning 非常简单, 可以很容易地扩展到较大的表面区域, 从而与单元格区域性应用程序完全兼容。Nanopatterns 被用作生物活性基质, 以评估局部 RGD 表面密度对细胞黏附力的影响28和对成人骨髓间充质干细胞的软骨诱导,29。我们的结果表明, RGD 状基 nanopatterns 维持细胞生长, 细胞粘附增强了高局部 RGD 表面密度。在分化实验中, 细胞对基质的中间粘附倾向于 MSC 缩合和早期软骨分化。由于容易与状外周组可以修改, 这里所描述的方法可以进一步扩展到其他 ECM 配体, 发挥浓度依赖性的影响细胞。
Protocol
1. 承印物的制备 在云母基底上退火 1.4 x 1.1 cm Au (111)。 将 Au (111) 衬底放在玻璃陶瓷滚刀上, 用丁烷火焰将其退火3分钟, 使基板在氩气气氛下冷却。对每个 Au (111) 衬底重复此步骤。注: 退火后应立即使用 Au (111) 衬底。 制备聚 (l-乳酸) (PLLA) 涂层玻璃基板。 切下并冲洗玻璃片。 切割显微镜幻灯片18幻灯片 1.25 cm x 1.25 cm 与钻石…
Representative Results
我们提出了一种 nanopatterning 的方法, 允许在纳米尺度上处理表面附着 (图 1)。RGD-Cys-D1 的化学结构如图 1A所示。状在导电金 (111) 表面进行了图案, 用于高分辨率的 STM 特征。解决方案中的低状浓度 (最高为 10-5% w/w) 呈现的隔离状直径为 4-5 nm (图 1B), 而高度填充的状聚合体在较高浓度 (<strong class…
Discussion
在所述协议的开发过程中, 应考虑一些关键步骤。第一个是指用扫描探针显微镜技术 nanopattern 表征。为了可视化 nanopatterns, 图案产生的表面必须有一个粗糙值低于状的平均直径, 这是在 4-5 nm 的测量, 由 STM (图 1B)。此外, 应该考虑到高分辨率 STM 成像限制在导电基板, 在这种情况下 Au (111)。在自旋涂层后, 从滑轨的拐角处提升聚合物, 可以使用生物相容胶进行校正。
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Disclosures
The authors have nothing to disclose.
Acknowledgements
作者承认 Oriol 字体巴赫和阿尔伯特 G. 卡洛斯·卡斯塔诺在dmin量化中的帮助。他们也承认先进的数字显微镜单位在生物医学研究所 (IRB 巴塞罗那), 让作者在他们的处所录制的视频。这项工作得到了西班牙网络生物医学研究中心 (CIBER) 的支持。CIBER 是由《国家研究 & 发展 & 计划2008-2011、倡议赫尼奥2010、Consolider 方案、CIBER 行动和干杯第三研究所资助的一项倡议, 并得到欧洲区域开发基金的支持。这项工作得到了大学和创新、大学和加泰罗尼亚自治区加泰罗尼亚的企业的研究委员会 (2014 SGR 1442) 的支持。它还由 OLIGOCODES 项目资助 (No。MAT2012-38573-C02) 和 CTQ2013-41339-P, 由西班牙经济和竞争力部授予, 除了区域 V-西班牙-葡萄牙 2014-2020 POCTEP (0245_IBEROS_1_E)。C.R.P. 承认西班牙经济和竞争力部给予的财政支持 (No。IFI15/00151)。
Materials
| Gold (111) on mica. 1.4×1.1 cm | Spi Supplies | 466PS-AB | |
| Glass micro slides, plain | Corning | 2947-75×25 | |
| Deionized water | Millipore | 18MΩ cm | |
| Ethanol 96% | PanReac | 131085.1212 | |
| L-Lactide/DL-Lactide copolymer | Corbion | 95/05 molar ratio | |
| 1,4 – dioxane | Sigma-Aldrich | 296309-1L | |
| Silicone oil, high temperature | Acros Organics | 174665000 | |
| Spinner | Laurell | WS-650MZ-23NPP/Lite | |
| Tissue culture laminar flow hood | Telstar | Bio II Advance | Class II biological safety cabinet |
| Filter unit | Millex-GP | SLGP033RB | 0.22 µm |
| Syringe 10 mL | Discardit | 309110 | |
| Atomic Force microscope | Veeco Instruments | Dimension 3000 AFM instrument | |
| Silicon AFM probes | Budget Sensors | Tap300AI-G | Resonant Freq. 300 kHz, k = 40 N/m |
| Scanning tunneling microscope | Molecular Imaging | PicoSPM microscope | |
| Pt0.8:Ir0.2 wire | Advent | PT671012 | Diameter 0.25 mm |
| WSxM 4.0 software | Nanotec electronica | ||
| Optical contact angle (CA) system | Dataphysics | ||
| SCA20 software | Dataphysics | ||
| X-ray photoelectron spectrometer | Physical Electronics | Perkin-Elmer PHI 5500 Multitechnique System | |
| Fibronectin from bovine plasma | Sigma-Aldrich | F1141-1MG | 1.0 mL solution |
| Dulbecco's Phosphate Buffer Saline (DPBS) | Gibco | 21600-10 | Powder |
| Mouse embryo fibroblasts | ATCC | ATCC CRL-1658 | NIH/3T3 |
| Dulbecco's modified eagle medium (DMEM) liquid high glucose | Gibco | 11960044 | liquid high glucose, no glutamine, 500 mL |
| Fetal Bovine Serum (FBS) | Gibco | 16000044 | 500 mL |
| L-Glutamine | Invitrogen | 25030 | 200 mM (100X) |
| Penicillin-streptomycin | Invitrogen | 15140 | |
| Sodium pyruvate | Invitrogen | 11360039 | 100 mL |
| T75 culture flasks | Nunclon | 156499 | |
| Trypsin | Life Technologies | 25200072 | 0,25% EDTA |
| Centrifuge | Hermle Labortechnik | Z 206 A | |
| Non-tissue culture treated plate, 12 well | Falcon | 351143 | Non-adherent |
| Adipose-derived hMSCs | ATCC | ATCC PCS-500-011 | Cell vial 1 mL |
| MSC basal medium | ATCC | ATCC PCS-500-030 | |
| MSC growth kit | ATCC | ATCC PCS-500-040 | Low serum |
| Chondrocyte differentiation tool | ATCC | ATCC PCS-500-051 | |
| Formalin solution | Sigma-Aldrich | HT5011-15ML | neutral buffered, 10% |
| Ammonium chloride | Sigma-Aldrich | A9434-500G | for molecular biology, suitable for cell culture, ≥99.5% |
| Saponin | Sigma-Aldrich | 47036-50G-F | for molecular biology, used as non-ionic surfactant, adjuvant |
| Bovine Serum Albumin (BSA) | Sigma-Aldrich | A3059-50G | |
| Rabbit monoclonal [Y113] anti-paxillin antibody | Abcam | ab32084 | Diluted 1:200 |
| Mouse monoclonal [1F5] anti-collagen alpha-1 XX chain | Acris Antibodies | AM00212PU-N | Diluted: 1:400 |
| Alexa Fluor 488-conjugated goat anti-mouse IgG (H+L) secondary antibody | Invitrogen | A10667 | 2 mg/mL. Diluted 1:1000 |
| Alexa Fluor 568-conjugated goat anti-rabbit IgG (H+L) secondary antibody | Invitrogen | A11036 | 2 mg/mL. Diluted 1:1000 |
| Hoechst 33342 | Thermo Fisher | H3570 10ML | 10 mg/mL. Diluted 1:1000 |
| Cover glass 24×24 mm | Deltalab | D102424 | |
| Fluoromount | Sigma-Aldrich | F4680-25ML | |
| Epifluorescence Microscope | Nikon | Eclipse E1000 upright microscope | with a CCD camera |
| Confocal Microscope | Leica Microsystems | Leica SPE Upright Confocal Microscope | |
| ImageJ 1.50g freeware | http://imgej.nih.gov/ij | ||
| MATLAB software | The MATHWORKS, Inc. | ||
| OriginPro 8.5 software | OriginLab Coorporation |
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Cite This Article
Casanellas, I., Lagunas, A., Tsintzou, I., Vida, Y., Collado, D., Pérez-Inestrosa, E., Rodríguez-Pereira, C., Magalhaes, J., Gorostiza, P., Andrades, J. A., Becerra, J., Samitier, J. Dendrimer-based Uneven Nanopatterns to Locally Control Surface Adhesiveness: A Method to Direct Chondrogenic Differentiation. J. Vis. Exp. (131), e56347, doi:10.3791/56347 (2018).