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.

<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. A., Dolan, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intervertebral+disc+degeneration:+evidence+for+two+distinct+phenotypes.">Intervertebral disc degeneration: evidence for two distinct phenotypes.</a> J Anat. 221, 497-506 (2012).</li><li>Schizas, C., Kulik, G., Kosmopoulos, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disc+degeneration:+current+surgical+options.">Disc degeneration: current surgical options.</a> Eur Cell Mater. 20, 306-315 (2010).</li><li>Lewis, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleus+pulposus+replacement+and+regeneration/repair+technologies:+present+status+and+future+prospects.">Nucleus pulposus replacement and regeneration/repair technologies: present status and future prospects.</a> J Biomed Mater Res B Appl Biomater. 100, 1702-1720 (2012).</li><li>Cunningham, B. 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. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomechanical+behavior+of+a+new+nucleus+prosthesis+made+of+knitted+titanium+filaments.">Biomechanical behavior of a new nucleus prosthesis made of knitted titanium filaments.</a> SAS J. 1, 125-130 (2007).</li><li>Nerurkar, N. L., Elliott, D. M., Mauck, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+design+criteria+for+intervertebral+disc+tissue+engineering.">Mechanical design criteria for intervertebral disc tissue engineering.</a> J Biomech. 43, 1017-1030 (2010).</li><li>Elias, C. N., Lima, J. H. C., Valiev, R., Meyers, M. 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. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+cytological+profiling+assay+to+measure+diverse+cellular+states.">Multiplex cytological profiling assay to measure diverse cellular states.</a> PLoS One. 8, e80999 (2013).</li><li>Bocker, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introducing+a+single-cell-derived+human+mesenchymal+stem+cell+line+expressing+hTERT+after+lentiviral+gene+transfer.">Introducing a single-cell-derived human mesenchymal stem cell line expressing hTERT after lentiviral gene transfer.</a> J Cell Mol Med. 12, 1347-1359 (2008).</li><li>Ehnert, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transforming+growth+factor+beta1+inhibits+bone+morphogenic+protein+(BMP)-2+and+BMP-7+signaling+via+upregulation+of+Ski-related+novel+protein+N+(SnoN):+possible+mechanism+for+the+failure+of+BMP+therapy.">Transforming growth factor beta1 inhibits bone morphogenic protein (BMP)-2 and BMP-7 signaling via upregulation of Ski-related novel protein N (SnoN): possible mechanism for the failure of BMP therapy.</a> BMC Med. 10, 101 (2012).</li><li>Morgan, S. P., Rose, F. R., Matcher, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Optical Techniques in Regenerative Medicine. , (2013).</li><li>Vielreicher, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Taking+a+deep+look:+modern+microscopy+technologies+to+optimize+the+design+and+functionality+of+biocompatible+scaffolds+for+tissue+engineering+in+regenerative+medicine.">Taking a deep look: modern microscopy technologies to optimize the design and functionality of biocompatible scaffolds for tissue engineering in regenerative medicine.</a> J R Soc Interface. 10, 20130263 (2013).</li><li>Curtis, A., Wilkinson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Topographical+control+of+cells.">Topographical control of cells.</a> Biomaterials. 18, 1573-1583 (1997).</li><li>Niu, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+imaging+of+endothelial+cells+in+bioengineered+blood+vessels:+the+impact+of+crosslinking+of+the+scaffold.">Fluorescent imaging of endothelial cells in bioengineered blood vessels: the impact of crosslinking of the scaffold.</a> J Tissue Eng Regen Med. , (2014).</li><li>Chan, B. 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 authors declare that they have no competing interests. No portion of the work has been or is currently under consideration for publication or has been published elsewhere.

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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	<tbody>
		<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>
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		<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>
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			<td height="20" style="height:20px;width:275px;">* 6 well plates</td>
			<td style="width:205px;">Falcon</td>
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		<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>
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			<td height="20" style="height:20px;width:275px;">Acetic acid, purum &#8805; 99,0 %</td>
			<td style="width:205px;">Carl Roth</td>
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			<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>
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			<td height="21" style="height:21px;width:275px;">Biological safety cabinets</td>
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			<td style="width:144px;">safe 2020</td>
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			<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>
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			<td height="20" style="height:20px;width:275px;">Ethanol 99 %</td>
			<td style="width:205px;">&#160;SAV liquid prod. GmBH</td>
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			<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 â€” Using the Example of a Novel Knitted Titanium Implant at JoVE.com

Imaging Cell Viability on Non-transparent Scaffolds &#8212; 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.

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

imaging-cell-viability-on-non-transparent-scaffolds-using-example

Research

JoVE Journal

Bioengineering

11.3K Views.  Eberhard Karls Universität Tübingen. 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.

Video: Imaging Cell Viability on Non-transparent Scaffolds — Using the Example of a Novel Knitted Titanium Implant

Density Gradient Multilayered Polymerization (DGMP): A Novel Technique for Creating Multi-compartment, Customizable Scaffolds for Tissue Engineering

Complex tissue culture matrices, in which types and concentrations of biological stimuli (e.g. growth factors, inhibitors, or small molecules) or matrix structure (e.g. composition, concentration, or stiffness of the matrix) vary over space, would enable a wide range of investigations concerning how these variables affect cell differentiation, migration, and other phenomena. The major challenge in creating layered matrices is maintaining the structural integrity of layer interfaces without diffusion of individual components from each layer1. Current methodologies to achieve this include photopatterning2-3, lithography4, sequential functionalization5, freeze drying6, microfluidics7, or centrifugation8, many of which require sophisticated instrumentation and technical skills. Others rely on sequential attachment of individual layers, which may lead to delamination of layers9. 
DGMP overcomes these issues by using an inert density modifier such as iodixanol to create layers of varying densities10. Since the density modifier can be mixed with any prepolymer or bioactive molecule, DGMP allows each scaffold layer to be customized. Simply varying the concentration of the density modifier prevents mixing of adjacent layers while they remain aqueous. Subsequent single step polymerization gives rise to a structurally continuous multilayered scaffold, in which each layer has distinct chemical and mechanical properties. The density modifier can be easily removed with sufficient rinsing without perturbation of the individual layers or their components. This technique is therefore well suited for creating hydrogels of various sizes, shapes, and materials.
A protocol for fabricating a 2D-polyethylene glycol (PEG) gel, in which alternating layers incorporate RGDS-350, is outlined below. We use PEG because it is biocompatible and inert. RGDS, a cell adhesion peptide11, is used to demonstrate spatial restriction of a biological cue, and the conjugation of a fluorophore (Alexa Fluor 350) enables us to visually distinguish various layers. This procedure can be adapted for other materials (e.g. collagen, hyaluronan, etc.) and can be extended to fabricate 3D gels with some modifications10.

Density Gradient Multilayered Polymerization DGMP: A Novel Technique for Creating Multi-compartment, Customizable Scaffolds for Tissue Engineering

Here we describe a unique strategy for creating biocompatible, layered matrices with continuous interfaces between distinct layers for tissue engineering. Such a scaffold could provide an ideal customizable environment to modulate cell behavior by various biological, chemical or mechanical cues

Complex tissue culture matrices, in which types and concentrations of biological stimuli (e.g. growth factors, inhibitors, or small molecules) or matrix ...

Wide-field Fluorescent Microscopy and Fluorescent Imaging Flow Cytometry on a Cell-phone

Fluorescent microscopy and flow cytometry are widely used tools in biomedical research and clinical diagnosis. However these devices are in general relatively bulky and costly, making them less effective in the resource limited settings. To potentially address these limitations, we have recently demonstrated the integration of wide-field fluorescent microscopy and imaging flow cytometry tools on cell-phones using compact, light-weight, and cost-effective opto-fluidic attachments. In our flow cytometry design, fluorescently labeled cells are flushed through a microfluidic channel that is positioned above the existing cell-phone camera unit. Battery powered light-emitting diodes (LEDs) are butt-coupled to the side of this microfluidic chip, which effectively acts as a multi-mode slab waveguide, where the excitation light is guided to uniformly excite the fluorescent targets. The cell-phone camera records a time lapse movie of the fluorescent cells flowing through the microfluidic channel, where the digital frames of this movie are processed to count the number of the labeled cells within the target solution of interest. Using a similar opto-fluidic design, we can also image these fluorescently labeled cells in static mode by e.g. sandwiching the fluorescent particles between two glass slides and capturing their fluorescent images using the cell-phone camera, which can achieve a spatial resolution of e.g. ~ 10 &mu;m over a very large field-of-view of ~ 81 mm2. This cell-phone based fluorescent imaging flow cytometry and microscopy platform might be useful especially in resource limited settings, for e.g. counting of CD4+ T cells toward monitoring of HIV+ patients or for detection of water-borne parasites in drinking water.

We review our recent results on the integration of fluorescent microscopy and imaging flow cytometry tools on a cell-phone using compact and cost-effective opto-fluidic attachments. These cell-phone based micro-analysis devices might be useful for cytometric analysis, such as performing various cell counting tasks as well as for high-throughput screening of e.g., water samples in resource limited settings.

Fluorescent microscopy and flow cytometry are widely used tools in biomedical research and clinical diagnosis. However these devices are in general ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain central aspects of cellular physiology. To easily combine cell patterning with Multi-Electrode Arrays (MEAs) and silicon-based &#8216;lab on a chip&#8217; technologies, a microfabrication-compatible protocol is required. We describe a method that utilizes deposition of the polymer parylene-C on SiO2&#160;wafers. Photolithography enables accurate and reliable patterning of parylene-C at micron-level resolution. Subsequent activation by immersion in fetal bovine serum (or another specific activation solution) results in a substrate in which cultured cells adhere to, or are repulsed by, parylene or SiO2 regions respectively. This technique has allowed patterning of a broad range of cell types (including primary murine hippocampal cells, HEK 293 cell line, human neuron-like teratocarcinoma cell line, primary murine cerebellar granule cells, and primary human glioma-derived stem-like cells). Interestingly, however, the platform is not universal; reflecting the importance of cell-specific adhesion molecules. This cell patterning process is cost effective, reliable, and importantly can be incorporated into standard microfabrication (chip manufacturing) protocols, paving the way for integration of microelectronic technology.

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

This protocol describes a microfabrication-compatible method for cell patterning on SiO2. A predefined parylene-C design is photolithographically printed on SiO2 wafers. Following incubation with serum (or other activation solution) cells adhere specifically to (and grow according to the conformity of) underlying parylene-C, whilst being repulsed by SiO2 regions.

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain ...

A Novel Method for Localizing Reporter Fluorescent Beads Near the Cell Culture Surface for Traction Force Microscopy

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When the substrate is coated with an extracellular matrix protein, cells adhere to the gel and apply forces, causing the gel to deform. The deformation depends on the cell traction and the elastic properties of the gel. If the deformation field of the surface is known, surface traction can be calculated using elasticity theory. Gel deformation is commonly measured by embedding fluorescent marker beads uniformly into the gel. The probes displace as the gel deforms. The probes near the surface of the gel are tracked. The displacements reported by these probes are considered as surface displacements. Their depths from the surface are ignored. This assumption introduces error in traction force evaluations. For precise measurement of cell forces, it is critical for the location of the beads to be known. We have developed a technique that utilizes simple chemistry to confine fluorescent marker beads, 0.1 and 1 &#181;m in diameter, in PA gels, within 1.6 &#956;m of the surface. We coat a coverslip with poly-D-lysine (PDL) and fluorescent beads. PA gel solution is then sandwiched between the coverslip and an adherent surface. The fluorescent beads transfer to the gel solution during curing. After polymerization, the PA gel contains fluorescent beads on a plane close to the gel surface.

Traditional techniques for fabricating polyacrylamide (PA) gels containing fluorescent probes involve sandwiching a gel between an adherent surface and a glass slide. Here, we show that coating this slide with poly-D-lysine (PDL) and fluorescent probes localizes the probes to within 1.6 &#181;m from the gel surface.

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When ...

Viability of Bioprinted Cellular Constructs Using a Three Dispenser Cartesian Printer

Tissue engineering has centralized its focus on the construction of replacements for non-functional or damaged tissue. The utilization of three-dimensional bioprinting in tissue engineering has generated new methods for the printing of cells and matrix to fabricate biomimetic tissue constructs. The solid freeform fabrication (SFF) method developed for three-dimensional bioprinting uses an additive manufacturing approach by depositing droplets of cells and hydrogels in a layer-by-layer fashion. Bioprinting fabrication is dependent on the specific placement of biological materials into three-dimensional architectures, and the printed constructs should closely mimic the complex organization of cells and extracellular matrices in native tissue. This paper highlights the use of the Palmetto Printer, a Cartesian bioprinter, as well as the process of producing spatially organized, viable constructs while simultaneously allowing control of environmental factors. This methodology utilizes computer-aided design and computer-aided manufacturing to produce these specific and complex geometries. Finally, this approach allows for the reproducible production of fabricated constructs optimized by controllable printing parameters.

A Cartesian bioprinter was designed and fabricated to allow multi-material deposition in precise, reproducible geometries, while also allowing control of environmental factors. Utilizing the three-dimensional bioprinter, complex and viable constructs may be printed and easily reproduced.

Tissue engineering has centralized its focus on the construction of replacements for non-functional or damaged tissue. The utilization of ...

Selective Cell Elimination from Mixed 3D Culture Using a Near Infrared Photoimmunotherapy Technique

Recent developments in tissue engineering offer innovative solutions for many diseases. For example, tissue engineering using induced pluripotent stem cell (iPS) emerged as a new method in regenerative medicine. Although this tissue regeneration is promising, contamination with unwanted cells during tissue cultures is a major concern. Moreover, there is a safety concern regarding tumorigenicity after transplantation. Therefore, there is an urgent need for eliminating specific cells without damaging other cells that need to be protected, especially in established tissue. Here, we present a method for specific cell elimination from a mixed 3D cell culture in vitro with near infrared photoimmunotherapy (NIR-PIT) without damaging non-targeted cells. This technique enables the elimination of specific cells from mixed cell cultures or tissues.

Eliminating specific cells without damaging other cells is extremely difficult, especially in established tissue, yet there is an urgent need for a cell elimination method in the tissue engineering field. Here, we present a method for specific cell elimination from a mixed 3D cell culture using near infrared photoimmunotherapy (NIR-PIT).

Recent developments in tissue engineering offer innovative solutions for many diseases. For example, tissue engineering using induced pluripotent stem ...

Neonatal Cardiac Scaffolds: Novel Matrices for Regenerative Studies

The only definitive therapy for end stage heart failure is orthotopic heart transplantation. Each year, it is estimated that more than 100,000 donor hearts are needed for cardiac transplantation procedures in the United States1-2. Due to the limited numbers of donors, only approximately 2,400 transplants are performed each year in the U.S.2. Numerous approaches, from cell therapy studies to implantation of mechanical assist devices, have been undertaken, either alone or in combination, in an attempt to coax the heart to repair itself or to rest the failing heart3. In spite of these efforts, ventricular assist devices are still largely used for the purpose of bridging to transplantation and the utility of cell therapies, while they hold some curative promise, is still limited to clinical trials. Additionally, direct xenotransplantation has been attempted but success has been limited due to immune rejection. Clearly, another strategy is required to produce additional organs for transplantation and, ideally, these organs would be autologous so as to avoid the complications associated with rejection and lifetime immunosuppression. Decellularization is a process of removing resident cells from tissues to expose the native extracellular matrix (ECM) or scaffold. Perfusion decellularization offers complete preservation of the three dimensional structure of the tissue, while leaving the bulk of the mechanical properties of the tissue intact4. These scaffolds can be utilized for repopulation with healthy cells to generate research models and, possibly, much needed organs for transplantation. We have exposed the scaffolds from neonatal mice (P3), known to retain remarkable cardiac regenerative capabilities,5-8 to detergent mediated decellularization and we repopulated these scaffolds with murine cardiac cells. These studies support the feasibility of engineering a neonatal heart construct. They further allow for the investigation as to whether the ECM of early postnatal hearts may harbor cues that will result in improved recellularization strategies.

In these studies, we provide methodology for novel, neonatal, murine cardiac scaffolds for use in regenerative studies.

The only definitive therapy for end stage heart failure is orthotopic heart transplantation.  Each year, it is estimated that more than 100,000 donor ...

Determination of Zeta Potential via Nanoparticle Translocation Velocities through a Tunable Nanopore: Using DNA-modified Particles as an Example

Nanopore technologies, known collectively as Resistive Pulse Sensors (RPS), are being used to detect, quantify and characterize proteins, molecules and nanoparticles. Tunable resistive pulse sensing (TRPS) is a relatively recent adaptation to RPS that incorporates a tunable pore that can be altered in real time. Here, we use TRPS to monitor the translocation times of DNA-modified nanoparticles as they traverse the tunable pore membrane as a function of DNA concentration and structure (i.e., single-stranded to double-stranded DNA).
TRPS is based on two Ag/AgCl electrodes, separated by an elastomeric pore membrane that establishes a stable ionic current upon an applied electric field. Unlike various optical-based particle characterization technologies, TRPS can characterize individual particles amongst a sample population, allowing for multimodal samples to be analyzed with ease. Here, we demonstrate zeta potential measurements via particle translocation velocities of known standards and apply these to sample analyte translocation times, thus resulting in measuring the zeta potential of those analytes.
As well as acquiring mean zeta potential values, the samples are all measured using a particle-by-particle perspective exhibiting more information on a given sample through sample population distributions, for example. Of such, this method demonstrates potential within sensing applications for both medical and environmental fields.

Here we use a polyurethane tunable nanopore integrated into a resistive pulse sensing technique to characterize nanoparticles surface chemistry via the measurement of particle translocation velocities, which can be used to determine the zeta potential of individual nanoparticles.

Nanopore technologies, known collectively as Resistive Pulse Sensors (RPS), are being used to detect, quantify and characterize proteins, molecules and ...

Fabrication of Decellularized Cartilage-derived Matrix Scaffolds

Osteochondral defects lack sufficient intrinsic repair capacity to regenerate functionally sound bone and cartilage tissue. To this extent, cartilage research has focused on the development of regenerative scaffolds. This article describes the development of scaffolds that are completely derived from natural cartilage extracellular matrix, coming from an equine donor. Potential applications of the scaffolds include producing allografts for cartilage repair, serving as a scaffold for osteochondral tissue engineering, and providing in vitro models to study tissue formation. By decellularizing the tissue, the donor cells are removed, but many of the natural bioactive cues are thought to be retained. The main advantage of using such a natural scaffold in comparison to a synthetically produced scaffold is that no further functionalization of polymers is required to drive osteochondral tissue regeneration. The cartilage-derived matrix scaffolds can be used for bone and cartilage tissue regeneration in both in vivo and in vitro settings.

Decellularized cartilage-derived scaffolds can be used as a scaffold to guide cartilage repair and as a means to regenerate osteochondral tissue. This paper describes the decellularization process in detail and provides suggestions to use these scaffolds in in vitro settings.

Osteochondral defects lack sufficient intrinsic repair capacity to regenerate functionally sound bone and cartilage tissue. To this extent, cartilage ...

Imaging Cell Viability on Non-transparent Scaffolds — Using the Example of a Novel Knitted Titanium Implant

Summary

Explore More Videos

Chapters in this video

Density Gradient Multilayered Polymerization (DGMP): A Novel Technique for Creating Multi-compartment, Customizable Scaffolds for Tissue Engineering

Wide-field Fluorescent Microscopy and Fluorescent Imaging Flow Cytometry on a Cell-phone

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Cell Patterning on Photolithographically Defined Parylene-C: SiO₂ Substrates

A Novel Method for Localizing Reporter Fluorescent Beads Near the Cell Culture Surface for Traction Force Microscopy

Viability of Bioprinted Cellular Constructs Using a Three Dispenser Cartesian Printer

Selective Cell Elimination from Mixed 3D Culture Using a Near Infrared Photoimmunotherapy Technique

Neonatal Cardiac Scaffolds: Novel Matrices for Regenerative Studies

Determination of Zeta Potential via Nanoparticle Translocation Velocities through a Tunable Nanopore: Using DNA-modified Particles as an Example

Fabrication of Decellularized Cartilage-derived Matrix Scaffolds

Imaging Cell Viability on Non-transparent Scaffolds — Using the Example of a Novel Knitted Titanium Implant

Summary

Explore More Videos

Chapters in this video

Density Gradient Multilayered Polymerization (DGMP): A Novel Technique for Creating Multi-compartment, Customizable Scaffolds for Tissue Engineering

Wide-field Fluorescent Microscopy and Fluorescent Imaging Flow Cytometry on a Cell-phone

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Cell Patterning on Photolithographically Defined Parylene-C: SiO2 Substrates

A Novel Method for Localizing Reporter Fluorescent Beads Near the Cell Culture Surface for Traction Force Microscopy

Viability of Bioprinted Cellular Constructs Using a Three Dispenser Cartesian Printer

Selective Cell Elimination from Mixed 3D Culture Using a Near Infrared Photoimmunotherapy Technique

Neonatal Cardiac Scaffolds: Novel Matrices for Regenerative Studies

Determination of Zeta Potential via Nanoparticle Translocation Velocities through a Tunable Nanopore: Using DNA-modified Particles as an Example

Fabrication of Decellularized Cartilage-derived Matrix Scaffolds

Cell Patterning on Photolithographically Defined Parylene-C: SiO₂ Substrates