促进软骨再生的逐层Janus碱基纳米基质的制备和表征
Summary
该协议描述了通过依次添加Janus碱基纳米管（JBNts），基质素-3和转化生长因子β-1（TGF-β1）来逐层组装Janus碱基纳米基（JBNm）支架。JBNm 是制造和表征的;此外，它显示出优异的生物活性，促进细胞功能，如粘附、增殖和分化。
Abstract
已经开发了各种生物材料支架来引导细胞粘附和增殖，以期促进 体外 和 体内 使用的特定功能。将生长因子添加到这些生物材料支架中通常是为了提供最佳的细胞培养环境，介导细胞分化及其后续功能。然而，传统生物材料支架中的生长因子通常设计为在植入时释放，这可能导致对周围组织或细胞的意外副作用。在这里，受DNA启发的Janus碱基纳米基质（JBNm）成功地实现了高度本地化的微环境，具有逐层结构，用于自我可持续的软骨组织构建。JBNms由Janus基纳米管（JBNts），基质素-3和通过生物亲和力 转化 生长因子β-1（TGF-β1）自组装而成。JBNm以1：4：10的TGF-β1：matrilin-3：JBNt比例组装，因为这是可以正确组装到逐层结构中的确定比例。首先，将TGF-β1溶液加入到基质素-3溶液中。然后，将该混合物移液数次以确保在加入JBNt溶液之前具有足够的均匀性。这形成了逐层JBNm，在再次移液几次后。通过多种实验表征了JBNm结构、单独JBNts、单独使用基质素-3和单独表征TGF-β1。利用紫外-可见吸收光谱研究了JBNm的形成，并用透射电子显微镜（TEM）观察了JBNm的结构。由于创新的逐层JBNm支架是在分子尺度上形成的，可以观察到荧光染料标记的JBNm。TGF-β1被限制在可注射的JBNm的内层内，可以阻止生长因子向周围区域释放，促进局部软骨形成，促进抗肥大的微环境。
Introduction
组织工程中的支架在为细胞附着和随后的组织发育提供结构支持方面起着至关重要的作用1。通常，没有任何支架的常规组织构建体依赖于细胞培养环境和添加的生长因子来介导细胞分化。此外，这种将生物活性分子添加到支架中通常是指导细胞分化和功能的首选方法2，3。一些支架可以独立地模仿天然组织的生化微环境，而另一些支架可以通过生长因子直接影响细胞功能。然而，研究人员在选择可能对细胞粘附、生长和分化产生积极影响的支架时经常遇到挑战，同时在很长一段时间内提供最佳的结构支持和稳定性4，5.生物活性分子通常松散地结合在支架上，导致这些蛋白质在植入时快速释放，导致它们在不需要的位置释放。这最终导致对非故意靶向的组织或细胞的副作用6，7。
脚手架通常由聚合物材料制成。Janus基础纳米基质（JBNm）是一种仿生支架平台，采用新颖的逐层方法创建，用于自我可持续的软骨组织构建8。这些新型DNA启发的纳米管被命名为Janus碱基纳米管（JBNts），因为它们正确地模仿了细胞外基质（ECM）中胶原蛋白的结构和表面化学。通过添加生物活性分子，如马曲林-3和转化生长因子β-1（TGF-β1），JBNm可以创造一个最佳的微环境，然后可以刺激所需的细胞和组织功能9。
JBNts是源自核碱基腺嘌呤和胸腺嘧啶合成版本的新型纳米管。JBNts通过自组装10形成;六个合成核碱基键合形成一个环，这些环经历π π堆积相互作用，形成长度为200-300μm的纳米管11。这些纳米管在结构上类似于胶原蛋白;通过模拟天然软骨微环境的一个方面，JBNts已被证明为软骨细胞和人间充质干细胞（hMSCs）提供了有利的附着位点11，12，13，14。由于纳米管经过自组装并且不需要任何类型的引发剂（例如紫外线），因此它们作为难以到达的缺陷区域的可注射支架显示出令人兴奋的潜力15。
基质蛋白-3是一种在软骨中发现的结构细胞外基质蛋白。这种蛋白质在软骨生成和适当的软骨功能中起重要作用16，17。最近，它已被纳入生物材料支架，促进软骨形成而不肥大9，18，19。通过将这种蛋白质包含在JBNm中，软骨细胞被吸引到含有与其天然微环境相似成分的支架上。此外，已经表明基曲林-3是软骨细胞20内适当的TGF-β1信号传导所必需的。生长因子作为信号分子发挥作用，导致某种细胞或组织的特定生长。因此，为了实现最佳的软骨再生，基质素-3和TGF-β1是JBNm中必不可少的成分。将TGF-β1添加到逐层支架中可以进一步促进组织结构中的软骨再生。TGF-β1是一种生长因子，用于促进骨软骨缺陷的愈合过程，促进软骨细胞和hMSC增殖和分化21，22。因此，TGF-β1在软骨再生JBNm（J/T/M JBNm）23中起关键作用，促进适当的生长，特别是当它位于JBNm层内时。
如前所述，生长因子通常组装在支架的外部，没有特定的掺入方法。在这里，凭借精确设计的生物材料的纳米结构，JBNm被开发用于特定靶向目标细胞和组织。JBNm由粘附在内层JBNt表面上的TGF-β1和粘附在外层24，25的JBNt表面上的基质素-3组成。TGF-β1 掺入逐层结构的内层允许沿 JBNm 纤维的高度局部微环境，从而产生稳态组织结构，蛋白质12 的释放速度要慢得多。JBNm的可注射性使其成为未来各种生物材料应用的理想软骨组织结构26。
Protocol
1. JBNts的合成 利用先前发表的方法制备JBNt单体，涉及多种化合物的合成12。 使用反相柱用高效液相色谱（HPLC）合成粗JBNt单体后纯化粗单体。使用溶剂A：100%水，溶剂B：100%乙腈和溶剂C：HCl水溶液，pH = 1。使用3毫升/分钟的流速。在7.2分钟收集HPLC中获得的最大峰。 2. JBNt/matn1/TGF-β1的制造（视频1） <p class="jove_conte…
Representative Results
按照实验方案，成功合成了JBNts，并用紫外-可见吸收和透射电镜进行了表征。JBNm 是一种可注射的固体支架，经过快速仿生过程。在生理环境中将JBNts加入TGF-β1/matrilin-3溶液的混合物中后，形成固体白色网状支架，表明JBNm组装成功，如图 1所示。这在表征方法中得到了证明。 在生理条件下，基曲林-3由于其等电点27 而带负电（<strong class=…
Discussion
本研究的目标是开发一种仿生支架平台JBNm，以克服依赖细胞培养环境介导细胞分化的传统组织结构的局限性。JBNm 是一种逐层结构支架，用于自我可持续的软骨组织结构。创新设计基于新型DNA启发的纳米材料JBNts。JBNm由JBNts30，TGF-β1和matrilin-3组成，通过一种新的逐层技术组装，其中支架的自组装在分子水平上得到控制。通过zeta电位测量、紫外-可见吸收、TEM和荧光光谱分析对JBNm?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项工作得到了NIH资助7R01AR072027和7R03AR069383，NSF职业奖1905785，NSF 2025362和康涅狄格大学的支持。这项工作也得到了NIH拨款S10OD016435的部分支持。
Materials
| 10 % Normal Goat Serum | Thermo Fisher | 50062Z | Agent used to block nonspecific antibody binding actions during staining. |
| 24-well plate | Corning | 07-200-740 | 24-well plate used for comparative cell culture. |
| 384-Well Black Untreated Plate | Thermo Fisher | 262260 | 384-well plate used for absorption measurements. |
| 8-well chambered coverglass | Thermo Fisher | 155409PK | 8-well coverglass used for comparative cell culture. |
| 96-well flat bottom | Corning | 07-200-91 | 96-well plate used for comparative cell culture. |
| 96-Well Plate non- treated | Thermo Fisher | 260895 | 96-well plate used for comparative cell culture and analysis. |
| Agarose Gel | Sigma-Aldrich | A9539 | Hydrogel used for cell culture. |
| Agarose Gel | Sigma Aldrich | A9539 | Hydrogel used as an environment for cell culture. |
| Alexa Fluor Microscale Protein Labeling Kit | Thermo Fisher | A30006 (488) and A30007 (555) | Fluorescent dye used to label proteins. |
| Anti-Collagen X Antibody | Thermo Fisher | 41-9771-82 | Antibody used to stain collagen-X. |
| Bio-Rad PCR Machine | Bio-Rad | Equipment used to perform PCR on samples. | |
| C28/I2 Chondrocyte Cell Line | Cells used to analyze proliferative abilities of various samples. | ||
| Cell Counting Kit 8 | Milipore Sigma | 96992 | Cell proliferation assay. |
| Cell Profiler | Broad Institute | Software used to analyze cell images. | |
| Cryostat Microtome | Equipment used to produce thin segments of samples for use in staining and microscopy. | ||
| DAPI | Invitrogen | D1306 | Blue fluorescent stain that binds to adenine-thymine DNA regions. |
| Disposable cuvettes | FISHER Scientific | 14-955-128 | Container used for spectrophotometry. |
| DMEM Cell Culture Medium | Thermo Fisher | 10566032 | Media used to support cellular growth. |
| Fetal Bovine Serum | GIBCO | A4766801 | Serum used in cell culture medium to support cell growth. |
| Fluoromount-G Mounting Medium | Thermo Fisher | 00-4958-02 | Solution used to mount slides for immunostaining. |
| Formaldehyde | Compound used to fix samples prior to microtoming. | ||
| Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody | Thermo Fisher | A16110 | Antibody used for protein staining. |
| Human Mesenchymal Stem Cells | LONZA | PT-2501 | Cells used to analyze differentiative abilities of various samples. |
| Human Mesenchymal Stem Chondrogenic Medium | LONZA | PT-3003 | Cell medium used to promote chondrogenic differentiation. |
| ImageJ | National Institutes of Health | Image analysis software used in conjunction with microscopy. | |
| itaq Universal SYBR Green One-Step Kit | BioRad | 1725150 | Kit used for PCR. |
| Janus-base nanotubes (JBNts) | Nanotube made from synthetic nucleobases to act as cell scaffolding tool. | ||
| LaB6 20-120 kV Transmission Electronic Microscope | Tecnai | Equipment used to perform transmission electron microscopy on a sample. | |
| MATLAB | MathWorks | Statistical software used for modeling and data analysis. | |
| Matrilin-3 | Fisher Scientific | 3017MN050 | Structural protein used as adhesion sites for chondrocytes. |
| NanoDrop Spectrophotometer | Thermo Fisher | Equipment used to measure absorption values of a sample. | |
| Nikon A1R Spectral Confocal Microscope | Nikon | A1R HD25 | Confocal microscope used to analyze samples. |
| Number 1.5 Chamber Coverglass | Thermo Fisher | 152250 | Environment for sterile cell culture and imaging. |
| Optimal Cutting Temperature Compound Reagent | Compound used to embed cells prior to microtoming. | ||
| Paraformaldehyde | Thermo Scientific | AAJ19943K2 | Compound used to fix cells. |
| PDC-32G Plasma Cleaner | Harrick Plasma | Cleaner used to prepare grids prior to transmission electron microscopy. | |
| penicillin-streptomycin | GIBCO | 15-140-148 | Antibiotic agent used to discourage bacterial growth during cell culture. |
| Phosphate Buffered Saline | Thermo Fisher | 10010023 | Solution used to wash cell medium and act as a buffer during experimentation. |
| Rhodamine-phalloidin | Invitrogen | R415 | F-Actin red fluorescent dye. |
| Rneasy Plant Mini Kit | QIAGEN | 74904 | Kit used to filter and homogenize samples during RNA extraction. |
| Sucrose Solution | Solution used to process samples prior to microtoming. | ||
| TGF beta-1 Human ELISA Kit | Invitrogen | BMS249-4 | Assay kit used to determine the presence of TGF-β1 in a sample. |
| TGF-β1 | PEPROTECH | 100-21C | Growth factor used for the stimulation of chondrogenic differentiation and proliferation. |
| Triton-X | Invitrogen | HFH10 | Compound used to lyse cells not fixed during staining process. |
| TRIzol Reagent | Thermo Fisher | 15596026 | Reagent used to isolate RNA. |
| Zetasizer Nano ZS | Malvern Panalytical | Equipment used to measure zeta-potential values of a sample. |
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Citer Cet Article
Landolina, M., Yau, A., Chen, Y. Fabrication and Characterization of Layer-By-Layer Janus Base Nano-Matrix to Promote Cartilage Regeneration. J. Vis. Exp. (185), e63984, doi:10.3791/63984 (2022).