Project is partially funded by Zentrales Innovationsprogramm Mittelstand (ZIM) des Bundesministeriums f&#252;r Wirtschaft und Energie -KF3010902AJ4. The publication fee has been covered by the BG trauma hospital T&#252;bingen, Germany.

NOTE: Immortalized human mesenchymal stromal precursor cells (SCP-1 cells) were used for the experiments. SCP-1 cells were provided by Prof. Matthias Schieker12.
1. Expansion of SCP-1 Cells
<ol>
	<li>Prior to working with the SCP-1 cells, properly clean the working area (designated biosafety cabinet I) with 70% ethanol (v/v) wearing gloves.</li>
	<li>In the cleaned biosafety cabinet prepare an appropriate volume of cell culture medium by mixing the required components as indicated...

<ol>
	<li>Bridwell, K. H., Anderson, P. A., Boden, S. D., Vaccaro, A. R., Wang, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What's+new+in+spine+surgery.">What's new in spine surgery.</a> J Bone Joint Surg Am. 95, 1144-1150 (2013).</li><li>Adams, M. 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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+scientific+considerations+in+total+disc+arthroplasty.">Basic scientific considerations in total disc arthroplasty.</a> Spine J. 4, 219-230 (2004).</li><li>Buck, A. E., Kaps, H. -. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implant+for+surgical+use+in+humans+or+vertebrates.">Implant for surgical use in humans or vertebrates.</a> US8728164 B2. Google Patents. , (2014).</li><li>Kettler, A., Kaps, H. P., Haegele, B., Wilke, H. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomedical+applications+of+titanium+and+its+alloys.">Biomedical applications of titanium and its alloys.</a> JOM. 60, 46-49 (2008).</li><li>Hallab, N., Link, H. D., McAfee, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomaterial+optimization+in+total+disc+arthroplasty.">Biomaterial optimization in total disc arthroplasty.</a> Spine (Phila Pa 1976). 28, 139-152 (2003).</li><li>Gustafsdottir, S. 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P., Leong, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scaffolding+in+tissue+engineering:+general+approaches+and+tissue-specific+considerations.">Scaffolding in tissue engineering: general approaches and tissue-specific considerations.</a> Eur Spine J. 17, 467-479 (2008).</li><li>Navarro, M., Michiardi, A., Castano, O., Planell, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomaterials+in+orthopaedics.">Biomaterials in orthopaedics.</a> J R Soc Interface. 5, 1137-1158 (2008).</li><li>Priyadarshani, P., Li, Y., Yao, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+biological+therapy+for+nucleus+pulposus+regeneration.">Advances in biological therapy for nucleus pulposus regeneration.</a> Osteoarthritis Cartilage. , (2015).</li><li>. Thermofisher Fluorescence Spectraviewer Available from: <a target="_blank" href="https://www.thermofisher.com/de/de/home/life-science/cell-analysis/labeling-chemistry/fluorescence-spectraviewer.html">https://www.thermofisher.com/de/de/home/life-science/cell-analysis/labeling-chemistry/fluorescence-spectraviewer.html</a> (2016)</li></ol>

The scaffold surface plays an important role in its interaction with surrounding tissue in vivo thereby determining implants functional durability. Thus, the bio-compatibility of the scaffold is studied by in vitro assays using cells (SCP1 cell line), when plated on the scaffolds.
Microscopy techniques that function well with thin and optically transparent scaffolds are poorly suited for non-transparent scaffolds to study the biocompatibility. This is mainly because the non-t...

Chronic back pain is a multifactorial disease. The interest in a minimally invasive treatment option for the degenerative disc disease has grown since the 1950s. Until today, multi-segmental fusion of the spinal column is the most widely used treatment. Since, this method often leads to limitations in the mobility of the affected segment1,2, exploration of the arthroplasty era became a wide interest. Significant advancements in total disc replacement and nucleus replacement has become a good alternative to treat chronic back pain1. Despite the huge progress, none of the methods has been clinically evaluated. The less rigid nucleus implants represent a promising alternative to total disc replacement, provided that the annulus fibrosus is intact3,4. However, the currently present nucleus implants on the market are often associated with complications like changes in vertebral body, dislocation, vertical height loss of the disc and the lack of necessary associated mechanical rigidity5. In order to overcome the current drawbacks, a novel nucleus implant made of knitted titanium wires has been successfully developed6. Due to the unique knitted structure, this newly developed scaffold has shown distinguished biomechanical characteristics, e.g., damping feature, pore size, loading capacity and reliability7. Aiming to test the biocompatibility of this novel nucleus implant, depicted severe limitations in the (optical) analysis techniques attributed to the non-transparent nature of the implant.
In order to test the biocompatibility, cell-metal interaction plays a prominent role8-10. An interaction between the cells and the scaffold is necessary for the stabilization and hence for the better implant integration within the host system. However, an increasing ingrowth depth might alter the mechanical properties of the scaffold. Aiming to investigate whether the scaffold surface provides a base for cell attachment, proliferation and differentiation or whether the metal affects cell viability, it is important to troubleshoot the common well-known problem of imaging cells on/in non-transparent and opaque scaffolds. In order to overcome this limitation several fluorescent based techniques were explored. Companies provide a large range of fluorophores to visualize living cells, cellular compartments, or even specific cellular states11. Fluorophores for this experiment were chosen with the help of the online tool spectral viewer in order to best fit our fluorescent microscope.
The developed strategy for the analysis of the adherent cells behavior on/in the non-transparent knitted titanium scaffold involves the following: 1) fluorescent (green fluorescent protein/GFP) labeling of the osteochondro-progenitor cells to allow tracking of the cells on the scaffold, 2) measuring the viability (mitochondrial activity) of the cells, and 3) visualizing cell-cell and cell-material interactions within the scaffold. The procedure has the advantage that it can be easily transferred to other adherent cells and other non-transparent or opaque scaffold. Furthermore, viability and ingrowth pattern can be monitored over several days, thus it can be used with limited amounts of scaffold material or cells.
The present study demonstrates the successful use of our current protocol to measure the cell viability and visualize in-growth pattern of osteochondro-progenitor cells on/in the non-transparent knitted titanium scaffold. Furthermore, the developed protocols might be used in order to determine the scaffold impurities and to check cleaning protocols.

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		<tr height="21">
			<td height="21" style="height:21px;width:275px;">6/24/48 well plates, T25/ T75 culture flask</td>
			<td style="width:205px;">Greiner Bio-One GmbH</td>
			<td style="width:144px;">*</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:275px;">* 24 well plates</td>
			<td style="width:205px;">Greiner Bio-One GmbH</td>
			<td style="width:144px;">CELLSTAR 662 160</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:275px;">* 48 well plates</td>
			<td style="width:205px;">Corning Incorporated USA</td>
			<td style="width:144px;">3548</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:275px;">* 6 well plates</td>
			<td style="width:205px;">Falcon</td>
			<td style="width:144px;">353046</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:275px;">* T25</td>
			<td style="width:205px;">Greiner Bio-One GmbH</td>
			<td style="width:144px;">690 175</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:275px;">* T75</td>
			<td style="width:205px;">Greiner Bio-One GmbH</td>
			<td style="width:144px;">658 175</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:275px;">Acetic acid, purum &#8805; 99,0 %</td>
			<td style="width:205px;">Carl Roth</td>
			<td style="width:144px;">3738.4</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:275px;">Acetone</td>
			<td style="width:205px;">Carl Roth</td>
			<td style="width:144px;">5025.1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Axioplan-2&#160;</td>
			<td style="width:205px;">Carl Zeiss, Germany</td>
			<td style="width:144px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Biological safety cabinets</td>
			<td style="width:205px;">Thermo Scientific</td>
			<td style="width:144px;">safe 2020</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:275px;">Calcein acetoxymethyl ester (calcein AM)</td>
			<td style="width:205px;">Sigma</td>
			<td style="width:144px;">17783</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:275px;">Cell Culture Incubtator</td>
			<td style="width:205px;">Binder, Tuttlingen, Germany</td>
			<td style="width:144px;">9040-0078</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:275px;">Filter unit (0.22&#181;m)</td>
			<td style="width:205px;">Millipore, IRL</td>
			<td style="width:144px;">SLGP033RS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Centrifuges 5810 R And 5417 R</td>
			<td style="width:205px;">Thermo Fisher Scientific, NY</td>
			<td style="width:144px;">Megafuge 40R</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:275px;">Dimethylsulfoxid (DMSO)</td>
			<td style="width:205px;">Carl Roth</td>
			<td style="width:144px;">4720.2</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:275px;">Dulbecco&#8217;s PBS without Ca &#38; Mg</td>
			<td style="width:205px;">Sigma</td>
			<td style="width:144px;">H15-002</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:275px;">Ethanol 99 %</td>
			<td style="width:205px;">&#160;SAV liquid prod. GmBH</td>
			<td style="width:144px;">475956</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:275px;">Ethidium homodimer</td>
			<td style="width:205px;">Sigma</td>
			<td style="width:144px;">46043</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">EVOS Fluorescence imaging system</td>
			<td style="width:205px;">Life technologies</td>
			<td style="width:144px;">AMF4300</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:275px;">Fetal Bovine Serum (FCS)</td>
			<td style="width:205px;">Gibco</td>
			<td style="width:144px;">10270-106</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Hemocytometer</td>
			<td style="width:205px;">Hausser Scientific, PA, USA</td>
			<td style="width:144px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:275px;">Hoechst 33342</td>
			<td style="width:205px;">Sigma</td>
			<td style="width:144px;">14533-100MG</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Knitted titanium nucleus implant</td>
			<td style="width:205px;">Buck co &#38; KG,Germany</td>
			<td style="width:144px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:275px;">MEM Alpha Modification with Glutamine w/o nucleoside</td>
			<td style="width:205px;">Sigma</td>
			<td style="width:144px;">E15-832</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Omega microplate Reader</td>
			<td style="width:205px;">BMG Labtech,Germany</td>
			<td style="width:144px;">FLUOstar Omega</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:275px;">Penicillin/Streptomycin</td>
			<td style="width:205px;">Sigma</td>
			<td style="width:144px;">P11-010</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:275px;">Resazurin sodium salt</td>
			<td style="width:205px;">Sigma</td>
			<td style="width:144px;">199303-1G</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:275px;">Sulforhodamine B sodium salt</td>
			<td style="width:205px;">Sigma</td>
			<td style="width:144px;">S1402-1G</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Test tube rotator</td>
			<td style="width:205px;">Labinco B.V.,The Netherlands</td>
			<td style="width:144px;">Model LD-76</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:275px;">TRIS (hydroxymethyl) aminomethan</td>
			<td style="width:205px;">Carl Roth</td>
			<td style="width:144px;">AE15.1</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:275px;">Triton</td>
			<td style="width:205px;">Carl Roth</td>
			<td style="width:144px;">3051.2</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:275px;">Trypan Blue 0.5 %</td>
			<td style="width:205px;">Carl Roth</td>
			<td style="width:144px;">CN76.1</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:275px;">Trypsin/EDTA</td>
			<td style="width:205px;">Sigma</td>
			<td style="width:144px;">L11-004</td>
		</tr>
	</tbody>
</table>

imaging cell viability on non-transparent scaffolds &#8212; using the example of a novel knitted titanium implant

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.

Preliminary results showed that the described novel nucleus implant not only has good damping features but also is biocompatible with SCP-1 cells. During the production process of the implant, it comes in contact with strong corrosive and toxic substances (lubricant, mordant, electro-polishing solution). With the help of indirect fluorescent staining techniques we were able to visualize remaining impurities and consequently optimize a cleaning protocol showing significant reduction in sub...

Watch this Scientific Journal Video about Imaging Cell Viability on Non-transparent Scaffolds Ã¢â‚¬â€ Using the Example of a Novel Knitted Titanium Implant at JoVE.com

Imaging Cell Viability on Non-transparent Scaffolds ÃƒÂ¢Ã¢â€šÂ¬Ã¢â‚¬Â Using the Example of a Novel Knitted Titanium Implant

Here we present a fluorophore based imaging technique to detect cell viability on a non-transparent titanium scaffold as well as to detect glimpses of the scaffold impurities. This protocol troubleshoots the drawback of imaging cell-cell or cell-metal interactions on non-transparent scaffolds.

Imaging Cell Viability on Non-transparent Scaffolds &#8212; Using the Example of a Novel Knitted Titanium Implant

Intervertebral disc degeneration and disc herniation is one of the major causes of lower back pain. Depletion of extracellular matrix, culminating in ...

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