非透明的支架成像细胞活性 - 使用新型针织钛种植体为例
Summary
这里，我们提出一个基于荧光的成像技术来检测细胞生存力上的非透明钛脚手架以及检测的脚手架杂质影子。这个协议进行故障排除在非透明支架成像细胞 – 细胞或细胞 – 金属相互作用的缺点。
Abstract
Intervertebral disc degeneration and disc herniation is one of the major causes of lower back pain. Depletion of extracellular matrix, culminating in nucleus pulposus (NP) extrusion leads to intervertebral disc destruction. Currently available surgical treatments reduce the pain but do not restore the mechanical functionality of the spine. In order to preserve mechanical features of the spine, total disc or nucleus replacement thus became a wide interest. However, this arthroplasty era is still in an immature state, since none of the existing products have been clinically evaluated.
This study intends to test the biocompatibility of a novel nucleus implant made of knitted titanium wires. Despite all mechanical advantages, the material has its limits for conventional optical analysis as the resulting implant is non-transparent. Here we present a strategy that describes in vitro visualization, tracking and viability testing of osteochondro-progenitor cells on the scaffold. This protocol can be used to visualize the efficiency of the cleaning protocol as well as to investigate the biocompatibility of these and other non-transparent scaffolds. Furthermore, this protocol can be used to show adherence pattern of cells as well as cell viability and proliferation rates on/in the scaffold. This in vitro biocompatibility testing assay provides a propitious tool to analyze cell-material interaction in non-transparent and opaque scaffolds.
Introduction
慢性背部疼痛是一种多因素疾病。自20世纪50年代的椎间盘退行性疾病的微创治疗选择的兴趣增加。直到今天，在脊柱的多节段的融合是最广泛使用的治疗方法。因为，这种方法常常导致在受影响的段1,2的移动性限制，关节造形术时代的勘探成为广泛的兴趣。总椎间盘置换髓核置换显著进步已成为治疗慢性背痛1一个很好的选择。尽管巨大的进步，没有一种方法已被临床评估。少刚性核植入物代表一个有前途的替代全椎间盘置换，只要该纤维环是完好3,4。然而，市场上的当前存在的核植入物通常伴有并发症像在椎体，错位，盘和叔垂直高度损耗变化他缺乏必要的相关机械刚性5。为了克服目前的缺点，提出针织钛线的新型核植入物已研制成功6。由于独特的编织结构，这种新开发的脚手架已经显示出杰出的生物力学性能， 如阻尼特性，孔径，承载能力和可靠性7。在归因于植入物的非透明性的（光学）分析技术旨在测试此新颖核植入物的生物相容性，描绘严重限制。
为了测试生物相容性，细胞-金属相互作用中起着突出的作用8-10。细胞和支架之间的相互作用是必要的稳定，从而为在主机系统内的更好的植入物的整合。然而，越来越长入深度可能改变支架的机械性能。旨在不变拟tigate所述支架表面是否提供了用于细胞附着，增殖和分化，或碱金属是否影响细胞生存力，它解决在非透明和不透明支架成像上/细胞的常见公知的问题是重要的。为了克服此限制几个荧光灯为基础的技术进行了探讨。公司提供大范围的荧光的可视化活细胞，细胞器，甚至是特定的细胞状态11。此实验的荧光团进行，以最好地适合我们的荧光显微镜与在线工具光谱观看者的帮助下选择。
上/在非透明针织钛支架贴壁细胞行为的分析开发策略涉及以下内容：osteochondro祖细胞的1）的荧光（绿色荧光蛋白/ GFP）标记，以允许在细胞的跟踪脚手架，2）测定存活率（线粒体chondrial活性）的细胞，以及3）可视化的细胞 – 细胞和所述支架内的细胞物质的相互作用。该过程具有的优点在于它可以很容易地转移到其它贴壁细胞和其他非透明或不透明的支架。此外，生存力和向内生长模式可以在几天进行监控，因此，它可以用有限数量的支架材料或细胞的使用。
本研究证明的成功使用我们的当前协议来测量细胞活力和可视化在生长上/ osteochondro祖细胞的图案在非透明针织钛支架。此外，开发的协议可能，以便确定所述支架的杂质，并检查清洗协议一起使用。
Protocol
Representative Results
Discussion
在支架表面起着其相互作用与体内周围组织，从而确定植入官能耐久性重要的作用。因此，支架的生物相容性，通过使用细胞（SCP1细胞系） 的体外测定，对支架镀时的研究。
与薄和光学透明支架功能良好的显微技术适合较差的非透明支架研究生物相容性。这主要是由于非透明支架防止未经显著失真15穿透光。为了部分解决这些问题，我们在此建立在/在?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
项目受到Zentrales Innovationsprogramm的Mittelstand（ZIM）DES Bundesministeriums献给Wirtschaft UND科特布斯-KF3010902AJ4资助。出版费用已涵盖的BG创伤医院德国蒂宾根。
Materials
| 6/24/48 well plates, T25/ T75 culture flask | Greiner Bio-One GmbH | * |
| * 24 well plates | Greiner Bio-One GmbH | CELLSTAR 662 160 |
| * 48 well plates | Corning Incorporated USA | 3548 |
| * 6 well plates | Falcon | 353046 |
| * T25 | Greiner Bio-One GmbH | 690 175 |
| * T75 | Greiner Bio-One GmbH | 658 175 |
| Acetic acid, purum ≥ 99,0 % | Carl Roth | 3738.4 |
| Acetone | Carl Roth | 5025.1 |
| Axioplan-2 | Carl Zeiss, Germany | |
| Biological safety cabinets | Thermo Scientific | safe 2020 |
| Calcein acetoxymethyl ester (calcein AM) | Sigma | 17783 |
| Cell Culture Incubtator | Binder, Tuttlingen, Germany | 9040-0078 |
| Filter unit (0.22µm) | Millipore, IRL | SLGP033RS |
| Centrifuges 5810 R And 5417 R | Thermo Fisher Scientific, NY | Megafuge 40R |
| Dimethylsulfoxid (DMSO) | Carl Roth | 4720.2 |
| Dulbecco’s PBS without Ca & Mg | Sigma | H15-002 |
| Ethanol 99 % | SAV liquid prod. GmBH | 475956 |
| Ethidium homodimer | Sigma | 46043 |
| EVOS Fluorescence imaging system | Life technologies | AMF4300 |
| Fetal Bovine Serum (FCS) | Gibco | 10270-106 |
| Hemocytometer | Hausser Scientific, PA, USA | |
| Hoechst 33342 | Sigma | 14533-100MG |
| Knitted titanium nucleus implant | Buck co & KG,Germany | |
| MEM Alpha Modification with Glutamine w/o nucleoside | Sigma | E15-832 |
| Omega microplate Reader | BMG Labtech,Germany | FLUOstar Omega |
| Penicillin/Streptomycin | Sigma | P11-010 |
| Resazurin sodium salt | Sigma | 199303-1G |
| Sulforhodamine B sodium salt | Sigma | S1402-1G |
| Test tube rotator | Labinco B.V.,The Netherlands | Model LD-76 |
| TRIS (hydroxymethyl) aminomethan | Carl Roth | AE15.1 |
| Triton | Carl Roth | 3051.2 |
| Trypan Blue 0.5 % | Carl Roth | CN76.1 |
| Trypsin/EDTA | Sigma | L11-004 |
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