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-transparent scaffolds prevent light from penetrating without significant distortion15. To partly overcome these problems we herewith establish a method for cell assessment on/in knitted titanium made scaffolds using various fluorophores.
In order to enable knitting followed by folding of the titanium wires, the material comes into contact with strong corrosive and toxic substances (lubricant, mordant, electro-polishing solution), which might alter the biocompatibility of the scaffold if traces remain in/on the scaffold. With the help of a developed indirect-fluorescence protocol (Protocol 5) we could visualize the scaffold structure. Furthermore, scaffold characteristics, e.g., the material thickness, the individual pore size and shape, or the connective density, were analyzed using ImageJ. A higher magnified epifluorescence microscopic image allowed visualizing impurities in the scaffold as well as on the scaffold surface. Figure 3b thus represents the confirmatory result of the successfully developed cleaning protocol. The principle of this indirect staining protocol can be easily reproduced to other non-transparent scaffolds, taking into consideration the individual scaffold properties, e.g., pore size and corresponding diffusion which affects the incubation time. Also, auto-fluorescence of the scaffold and microscopic settings affects the choice of fluorophores used. The online tool fluorescence spectral viewer can help to choose the adequate fluorophores.
Cell-metal interaction has been examined indirectly by analyzing the adherence pattern of cells on the scaffold. Protocol 6 describes the methodology of how cells can be monitored in vitro if plated on non-transparent scaffolds in culture systems by using a GFP transfection strategy. Based upon preliminary results of in vitro assays, it has been predicted that the surface properties such as composition, micro-topography and roughness16 might play an important role in establishing adherence and spreading of target cells. Titanium being a biomaterial thus might be acting as a substrate template to provide base for cell attachment (refer Figure 4).
Implant surface topography has been reported to influence cell behavior16. In the present study, we analyzed the cell growth/spreading and viability using fluorescence staining techniques in combination with quantitative viability measurements. Analysis revealed subtle variation in cell percent viability and spreading depending on the scaffold material. However, cell functionality assessed quantitatively by resazurin conversion assay (mitochondrial activity), was not significantly affected by the scaffold material. The measurement of the mitochondrial activity by resazurin conversion has the advantage that it is not cell toxic and thus can be performed repeatedly over a long culture period. Special care has to be taken when washing off residual resazurin working solution, so to not accumulate background signal (false positive results). Despite these advantages, the resazurin conversion assay will not give any information on the cell spreading on the scaffold. The GFP infected cells hence can be tracked over a long culture period (GFP signal remained constant for over 14 days), thereby enabling to visualize specific growth pattern on the scaffold surface. Visualization of cells deeper in the scaffold is still limited by the scaffold material and thus will require dissection of the implant. The combination of these two techniques has the major advantage that it can be easily transferred to other cell types, considering that incubation time might have to be adapted to the cell type of interest. However, care has to be taken when transferring this method to other non-transparent scaffolds, e.g., collagen based scaffolds which generally exhibit a strong green auto-fluorescence17. In this case other fluorescent tags might be used. The combination of above stated two methods therefore has several advantages (see Table 2).
Scaffold characteristics, e.g., pore size might trap cells inside the scaffold. If not enough nutrients are supplied, these cells might die and secrete proteases that affect cell viability of the surrounding cells/tissue. We were able to adapt a fluorescent based staining protocol that is able to visualize live and dead cells in and on the knitted titanium scaffold. Similar to the indirect fluorescence staining protocol, the principle of this staining protocol can be easily transferred to other non-transparent scaffolds. While doing so, the individual scaffold properties, e.g., pore size and corresponding diffusion as well as possible auto-fluorescence, microscopic setting have to be taken into consideration as they might affect the incubation time and the choice of fluorophores used. Here, again the online tool fluorescence spectral viewer can help to choose the adequate fluorophores.
In summary, in vitro results indicate that the proposed knitted titanium nucleus implant model has a biological profile. Initial attachment of mesenchymal stromal precursor cells (SCP-1 cells) on this material suggests that this titanium alloy implant material is biocompatible. Although we have not analyzed association of different topographical parameters, scaffold surface modification might enhance cell adherence proliferation as well as differentiation18. Optimum compatibility between scaffold and cells raise the probability of better implant integration into the surrounding tissue and so improving in vivo longevity after the treatment19. Using the above stated test setup opens up the possibility to measure and visualize improvements in the biological performance of the scaffold induced by surface modifications. The preference of scaffolds with bioactive surface over unmodified implant designs suggests the better performance20 in terms of osteo-chondrogenic integration. This study is further enhanced by other reports on the knitted titanium implant where its mechanical property and bioactivity have been reported 6,7.

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.

<table><tbody><tr><td>6/24/48 well plates, T25/ T75 culture flask</td><td>Greiner Bio-One GmbH</td><td>*</td><td></td></tr><tr><td>* 24 well plates</td><td>Greiner Bio-One GmbH</td><td>CELLSTAR 662 160</td><td></td></tr><tr><td>* 48 well plates</td><td>Corning Incorporated USA</td><td>3548</td><td></td></tr><tr><td>* 6 well plates</td><td>Falcon</td><td>353046</td><td></td></tr><tr><td>* T25</td><td>Greiner Bio-One GmbH</td><td>690 175</td><td></td></tr><tr><td>* T75</td><td>Greiner Bio-One GmbH</td><td>658 175</td><td></td></tr><tr><td>Acetic acid, purum &amp;ge; 99.0%</td><td>Carl Roth</td><td>3738.4</td><td></td></tr><tr><td>Acetone</td><td>Carl Roth</td><td>5025.1</td><td></td></tr><tr><td>Axioplan-2&amp;nbsp;</td><td>Carl Zeiss, Germany</td><td></td><td></td></tr><tr><td>Biological safety cabinets</td><td>Thermo Scientific</td><td>safe 2020</td><td></td></tr><tr><td>Calcein acetoxymethyl ester (calcein AM)</td><td>Sigma</td><td>17783</td><td></td></tr><tr><td>Cell Culture Incubtator</td><td>Binder, Tuttlingen, Germany</td><td>9040-0078</td><td></td></tr><tr><td>Filter unit (0.22 &amp;micro;m)</td><td>Millipore, IRL</td><td>SLGP033RS</td><td></td></tr><tr><td>Centrifuges 5810 R And 5417 R</td><td>Thermo Fisher Scientific, NY</td><td>Megafuge 40R</td><td></td></tr><tr><td>Dimethylsulfoxid (DMSO)</td><td>Carl Roth</td><td>4720.2</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s PBS without Ca &amp;amp; Mg</td><td>Sigma</td><td>H15-002</td><td></td></tr><tr><td>Ethanol 99%</td><td>&amp;nbsp;SAV liquid prod. GmBH</td><td>475956</td><td></td></tr><tr><td>Ethidium homodimer</td><td>Sigma</td><td>46043</td><td></td></tr><tr><td>EVOS Fluorescence imaging system</td><td>Life technologies</td><td>AMF4300</td><td></td></tr><tr><td>Fetal Bovine Serum (FCS)</td><td>Gibco</td><td>10270-106</td><td></td></tr><tr><td>Hemocytometer</td><td>Hausser Scientific, PA, USA</td><td></td><td></td></tr><tr><td>Hoechst 33342</td><td>Sigma</td><td>14533-100MG</td><td></td></tr><tr><td>Knitted titanium nucleus implant</td><td>Buck co &amp;amp; KG,Germany</td><td></td><td></td></tr><tr><td>MEM Alpha Modification with Glutamine w/o nucleoside</td><td>Sigma</td><td>E15-832</td><td></td></tr><tr><td>Omega microplate Reader</td><td>BMG Labtech,Germany</td><td>FLUOstar Omega</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Sigma</td><td>P11-010</td><td></td></tr><tr><td>Resazurin sodium salt</td><td>Sigma</td><td>199303-1G</td><td></td></tr><tr><td>Sulforhodamine B sodium salt</td><td>Sigma</td><td>S1402-1G</td><td></td></tr><tr><td>Test tube rotator</td><td>Labinco B.V.,The Netherlands</td><td>Model LD-76</td><td></td></tr><tr><td>TRIS (hydroxymethyl) aminomethan</td><td>Carl Roth</td><td>AE15.1</td><td></td></tr><tr><td>Triton</td><td>Carl Roth</td><td>3051.2</td><td></td></tr><tr><td>Trypan Blue 0.5%</td><td>Carl Roth</td><td>CN76.1</td><td></td></tr><tr><td>Trypsin/EDTA</td><td>Sigma</td><td>L11-004</td><td></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 substance load on the scaffold. Figure 3 shows the efficiency of established cleaning protocol.
The success of implants used for arthroplasty treatment is determined by events that takes place at the cell-material interface. Figure 4 shows the cells attached on the scaffold after 24 hr of plating, as described in the protocol section 6. A significant transfection efficiency of SCP-1 cells was observed as we could image the growth pattern of mesenchymal stromal precursor cells on the scaffold (refer Figure 2). Direct visualization confirms the biocompatibility of the scaffold and also depicts the adherence pattern on the scaffold surface (Figure 4). Fluorescence staining can be done further to examine cell interaction with and spreading on the scaffold surface.
Fluorophores were successfully applied in order to examine cell death and proliferation over a period of time on the scaffold. Live-dead-staining images exemplify how staining can successfully be done on the scaffold to confirm the percent viability of cells over a period of time. Figure 6 shows blue nuclear staining (Hoechst 3342) in all cells, red fluorescently labelled (ethidium homodimer) dead cells, and green labelling for incorporation of calcein-AM as viability marker. Calcein AM is converted to calcein which exhibits a bright green fluorescence in the presence of calcium ions in the cytoplasm of the cells. Hoechst 33342 is cell wall permeable and intercalates into the cellular DNA. This way all cells will show blue nuclei (refer Figure 6). Ethidium homodimer is not cell wall permeable, thus it will only intercalate into the DNA of dead cells. This way, dead cells will show red nuclei. Furthermore, cell viability and fold increase in cell number on scaffold over a week was quantified by resazurin conversion assay and represented graphically (Figure 7).
<img alt="Figure 1" src="/files/ftp_upload/54537/54537fig1.jpg" /> 
Figure 1: Cell counting with a Hemocytometer. (A) Setup of a chamber assembly. (B) Illustration of counting chambers; 4 x 4 counting chamber is used for a cell count. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54537/54537fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54537/54537fig2.jpg" /> 
Figure 2: GFP transfection efficiency. SCP1 cells exhibit a strong green fluorescence indicating positive ad-GFP- transfection efficiency. Scale bar = 1,000 &#181;m, 4X magnification. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54537/54537fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54537/54537fig3.jpg" /> 
Figure 3: Sulforhodamine B staining negative images capture. (A) Scaffold before cleaning. Arrow indicates the presence of toxic/corrosive substances on scaffold. (B) Scaffold after the cleaning protocol. Scale bar = 1,000 &#181;m, 4X magnification. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54537/54537fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54537/54537fig4.jpg" /> 
Figure 4: Adherence pattern of SCP1 cells on scaffold. GFP signal indicates cell adherence and growth pattern on the surface of the knitted titanium scaffold. Scale bar = 1,000 &#181;m, 4X magnification. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54537/54537fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/54537/54537fig6.jpg" /> 
Figure 6: Co-fluorescence staining of cells on scaffold. (A) The Hoechst nuclear staining (blue) and (B) the Calcein-AM cytoplasmic staining (green). (C) Arrow indicates the presence of dead cell due to uptake of ethidium homodimer-1 stain (red). (D) shows the merged image. Scale bar = 1,000 &#181;m, 4X magnification. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54537/54537fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" src="/files/ftp_upload/54537/54537fig7.jpg" /> 
Figure 7: Resazurin conversion assay. (A) Biochemical reduction reaction of redox dye (resazurin) into an end product (resorufin) which emits fluorescence and undergoes colorimetric changes. (B) Mitochondrial activity was measured when cells were plated on 0.75 mg/cm&#179; density scaffold. Data was collected using fluorescence based measuring instrument. Fluorescence intensity at 590 nm (y-axis) at defined time points (x-axis) is depicted as a result of quantitative cell viability measurement (Ex = 540 nm, Em = 590 nm). The statistical significance was determined using Two way ANOVA and standard error of the mean (SEM) is shown as an error bars. Considering the scaffold physical properties, e.g., pore size and mechanical properties (e.g., damping feature) together, 0.75 mg/cm&#179; density scaffold was used for biocompatibility characterization. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54537/54537fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Media components</td>
			<td>Concentration</td>
		</tr>
		<tr>
			<td>Basal &#945;MEM media (%)</td>
			<td>90</td>
		</tr>
		<tr>
			<td>Serum (%)</td>
			<td>10</td>
		</tr>
		<tr>
			<td>Pen/Strep (%)</td>
			<td>1</td>
		</tr>
	</tbody>
</table>
Table 1: Cell culture (&#945;MEM) Media composition.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Problem</td>
			<td>Cause</td>
			<td>Solution</td>
		</tr>
		<tr>
			<td>Cell viability imaging on Non-transparent scaffold</td>
			<td>Scaffold prevents light from penetrating without distortion</td>
			<td>Use fluorophore based imaging technique for cell assessment.</td>
		</tr>
		<tr>
			<td>Interference of fluorescence intensity</td>
			<td>Auto fluorescence, background signal</td>
			<td>Pay attention to the fluorophores and use appropriate based on particular scaffold properties.</td>
		</tr>
		<tr>
			<td>Cell viability assessment on scaffold over a culture period</td>
			<td>Repeated measurements over a long time</td>
			<td>Transfect the cells with ad-GFP-virus particles.</td>
		</tr>
		<tr>
			<td>Cell functionality assessment on non-transparent scaffold</td>
			<td>Fluorescence imaging technique allows only cell spreading pattern analysis.</td>
			<td>Perform resazurin conversion assay (quantitative cell viability measurement) in a combination with imaging technique.</td>
		</tr>
	</tbody>
</table>
Table 2: Summary table: troubleshooting the cell viability imaging on non-transparent scaffold.

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

Universidad San Sebastian

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

Postproduction Processing of Electrospun Fibres for Tissue Engineering

Electrospinning is a commonly used and versatile method to produce scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo undergo biaxial distension to varying extents such as skin, bladder, pelvic floor and even the hard palate as children grow. In producing scaffolds for these purposes there is a need to develop scaffolds of appropriate biomechanical properties (whether achieved without or with cells) and which are sterile for clinical use. The focus of this paper is not how to establish basic electrospinning parameters (as there is extensive literature on electrospinning) but on how to modify spun scaffolds post production to make them fit for tissue engineering purposes - here thickness, mechanical properties and sterilisation (required for clinical use) are considered and we also describe how cells can be cultured on scaffolds and subjected to biaxial strain to condition them for specific applications.
Electrospinning tends to produce thin sheets; as the electrospinning collector becomes coated with insulating fibres it becomes a poor conductor such that fibres no longer deposit on it. Hence we describe approaches to produce thicker structures by heat or vapour annealing increasing the strength of scaffolds but not necessarily the elasticity. Sequential spinning of scaffolds of different polymers to achieve complex scaffolds is also described. Sterilisation methodologies can adversely affect strength and elasticity of scaffolds. We compare three methods for their effects on the biomechanical properties on electrospun scaffolds of poly lactic-co-glycolic acid (PLGA).
Imaging of cells on scaffolds and assessment of production of extracellular matrix (ECM) proteins by cells on scaffolds is described. Culturing cells on scaffolds in vitro can improve scaffold strength and elasticity but the tissue engineering literature shows that cells often fail to produce appropriate ECM when cultured under static conditions. There are few commercial systems available that allow one to culture cells on scaffolds under dynamic conditioning regimes - one example is the Bose Electroforce 3100 which can be used to exert a conditioning programme on cells in scaffolds held using mechanical grips within a media filled chamber.4 An approach to a budget cell culture bioreactor for controlled distortion in 2 dimensions is described. We show that cells can be induced to produce elastin under these conditions. Finally assessment of the biomechanical properties of processed scaffolds cultured with or without cells is described.

Electrospun scaffolds can be processed post production for tissue engineering applications. Here we describe methods for spinning complex scaffolds (by consecutive spinning), for making thicker scaffolds (by multi-layering using heat or vapour annealing), for achieving sterility (aseptic production or sterilisation post production) and for achieving appropriate biomechanical properties.

Electrospinning is a commonly used and versatile method to produce  scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo ...

Multi-Scale Modification of Metallic Implants With Pore Gradients, Polyelectrolytes and Their Indirect Monitoring In vivo

Metallic implants, especially titanium implants, are widely used in clinical applications. Tissue in-growth and integration to these implants in the tissues are important parameters for successful clinical outcomes. In order to improve tissue integration, porous metallic implants have being developed. Open porosity of metallic foams is very advantageous, since the pore areas can be functionalized without compromising the mechanical properties of the whole structure. Here we describe such modifications using porous titanium implants based on titanium microbeads. By using inherent physical properties such as hydrophobicity of titanium, it is possible to obtain hydrophobic pore gradients within microbead based metallic implants and at the same time to have a basement membrane mimic based on hydrophilic, natural polymers. 3D pore gradients are formed by synthetic polymers such as Poly-L-lactic acid (PLLA) by freeze-extraction method. 2D nanofibrillar surfaces are formed by using collagen/alginate followed by a crosslinking step with a natural crosslinker (genipin). This nanofibrillar film was built up by layer by layer (LbL) deposition method of the two oppositely charged molecules, collagen and alginate. Finally, an implant where different areas can accommodate different cell types, as this is necessary for many multicellular tissues, can be obtained. By, this way cellular movement in different directions by different cell types can be controlled. Such a system is described for the specific case of trachea regeneration, but it can be modified for other target organs. Analysis of cell migration and the possible methods for creating different pore gradients are elaborated. The next step in the analysis of such implants is their characterization after implantation. However, histological analysis of metallic implants is a long and cumbersome process, thus for monitoring host reaction to metallic implants in vivo an alternative method based on monitoring CGA and different blood proteins is also described. These methods can be used for developing in vitro custom-made migration and colonization tests and also be used for analysis of functionalized metallic implants in vivo without histology.

Multi-Scale Modification of Metallic Implants With Pore Gradients, Polyelectrolytes and Their Indirect Monitoring In vivo

In this video, we will demonstrate modification techniques for porous metallic implants to improve their functionality and to control cell migration. Techniques include development of pore gradients to control cell movement in 3D and production of basement membrane mimics to control cell movement in 2-D. Also, a HPLC-based method for monitoring implant integration in-vivo via analysis of blood proteins is described.

Metallic implants, especially titanium implants, are widely used in  clinical applications. Tissue in-growth and integration to these implants in  the ...

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to evaluate new strategies that aim at improving vascularization, reliable methods are required to make the in-growth of new blood vessels into bio-artificial scaffolds visible and quantify the results. Over the past couple of years, our group has introduced a full skin defect model that enables the direct visualization of blood vessels by transillumination and provides the possibility of quantification through digital segmentation. In this model, one surgically creates full skin defects in the back of mice and replaces them with the material tested. Molecules or cells of interest can also be incorporated in such materials to study their potential effect. After an observation time of one&#8217;s own choice, materials are explanted for evaluation. Bilateral wounds provide the possibility of making internal comparisons that minimize artifacts among individuals as well as of decreasing the number of animals needed for the study. In comparison to other approaches, our method offers a simple, reliable and cost effective analysis. We have implemented this model as a routine tool to perform high-resolution screening when testing vascularization of different biomaterials and bio-activation approaches.

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Vascularization is key to approaches in successful tissue engineering. Therefore, reliable technologies are required to evaluate the development of vascular networks in tissue-constructs. Here we present a simple and cost-effective method to visualize and quantify vascularization in vivo.

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to ...

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

The development of Tissue Engineered Vessels (TEVs) is advanced by the ability to routinely and effectively implant TEVs (4-5 mm in diameter) into a large animal model. A step by-step protocol for inter-positional placement of the TEV and real-time digital assessment of the TEV and native carotid arteries is described here. In vivo monitoring is made possible by the implantation of flow probes, catheters and ultrasonic crystals (capable of recording dynamic diameter changes of implanted TEVs and native carotid arteries) at the time of surgery. Once implanted, researchers can calculate arterial blood flow patterns, invasive blood pressure and artery diameter yielding parameters such as pulse wave velocity, augmentation index, pulse pressures and compliance. Data acquisition is accomplished using a single computer program for analysis throughout the duration of the experiment. Such invaluable data provides insight into TEV matrix remodeling, its resemblance to native/sham controls and overall TEV performance in vivo.

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

A step-by-step protocol for the inter-positional placement of Tissue Engineered Vessels (TEVs) into the carotid artery of a sheep using end-to-end anastomosis and real-time digital assessment in vivo until animal sacrifice.

The development of Tissue Engineered Vessels (TEVs) is advanced by the ability to routinely and effectively implant TEVs (4-5 mm in diameter) into a large ...

Three-Dimensional In Vitro Biomimetic Model of Neuroblastoma Using Collagen-Based Scaffolds

Neuroblastoma is the most common extracranial solid tumor in children, accounting for 15% of overall pediatric cancer deaths. The native tumor tissue is a complex three-dimensional (3D) microenvironment involving layers of cancerous and non-cancerous cells surrounded by an extracellular matrix (ECM). The ECM provides physical and biological support and contributes to disease progression, patient prognosis, and therapeutic response.
This paper describes a protocol for assembling a 3D scaffold-based system to mimic the neuroblastoma microenvironment using neuroblastoma cell lines and collagen-based scaffolds. The scaffolds are supplemented with either nanohydroxyapatite (nHA) or glycosaminoglycans (GAGs), naturally found at high concentrations in the bone and bone marrow, the most common metastatic sites of neuroblastoma. The 3D porous structure of these scaffolds allows neuroblastoma cell attachment, proliferation and migration, and the formation of cell clusters. In this 3D matrix, the cell response to therapeutics is more reflective of the in vivo situation.
The scaffold-based culture system can maintain higher cell densities than conventional two-dimensional (2D) cell culture. Therefore, optimization protocols for initial seeding cell numbers are dependent on the desired experimental timeframes. The model is monitored by assessing cell growth via DNA quantification, cell viability via metabolic assays, and cell distribution within the scaffolds via histological staining.
This model&#39;s applications include the assessment of gene and protein expression profiles as well as cytotoxicity testing using conventional drugs and miRNAs. The 3D culture system allows for the precise manipulation of cell and ECM components, creating an environment more physiologically similar to native tumor tissue. Therefore, this 3D in vitro model will advance the understanding of the disease pathogenesis and improve the correlation between results obtained in vitro, in vivo in&#160;animal models, and human subjects.

Three-Dimensional In Vitro Biomimetic Model of Neuroblastoma Using Collagen-Based Scaffolds

This paper lists the steps required to seed neuroblastoma cell lines on previously described three-dimensional collagen-based scaffolds, maintain cell growth for a predetermined timeframe, and retrieve scaffolds for several cell growth and cell behavior analyses and downstream applications, adaptable to satisfy a range of experimental aims.

Neuroblastoma is the most common extracranial solid tumor in children, accounting for 15% of overall pediatric cancer deaths. The native tumor tissue is a ...

Cancer Research

Distinctive Capillary Action by Micro-channels in Bone-like Templates can Enhance Recruitment of Cells for Restoration of Large Bony Defect

Without an active, thriving cell population that is well-distributed and stably anchored to the inserted template, exceptional bone regeneration does not occur. With conventional templates, the absence of internal micro-channels results in the lack of cell infiltration, distribution, and inhabitance deep inside the templates. Hence, a highly porous and uniformly interconnected trabecular-bone-like template with micro-channels (biogenic microenvironment template; BMT) has been developed to address these obstacles. The novel BMT was created by innovative concepts (capillary action) and fabricated with a sponge-template coating technique. The BMT consists of several structural components: inter-connected primary-pores (300-400 &#181;m) that mimic pores in trabecular bone, micro-channels (25-70 &#181;m) within each trabecula, and nanopores (100-400 nm) on the surface to allow cells to anchor. Moreover, the BMT has been documented by mechanical test study to have similar mechanical strength properties to those of human trabecular bone (~3.8 MPa)12.
The BMT exhibited high absorption, retention, and habitation of cells throughout the bridge-shaped (&#928;) templates (3 cm height and 4 cm length). The cells that were initially seeded into one end of the templates immediately mobilized to the other end (10 cm distance) by capillary action of the BMT on the cell media. After 4 hr, the cells homogenously occupied the entire BMT and exhibited normal cellular behavior. The capillary action accounted for the infiltration of the cells suspended in the media and the distribution (active migration) throughout the BMT. Having observed these capabilities of the BMT, we project that BMTs will absorb bone marrow cells, growth factors, and nutrients from the periphery under physiological conditions.
The BMT may resolve current limitations via rapid infiltration, homogenous distribution and inhabitance of cells in large, volumetric templates to repair massive skeletal defects.

A step-by-step generic process to create a bone-like template with engineered micro-channels is presented. High absorption and retention capabilities of the template are demonstrated by capillary action via micro-channels.

Without an active, thriving cell population that is well-distributed and stably anchored to the inserted template, exceptional bone regeneration does not ...

Fabrication of Mechanically Tunable and Bioactive Metal Scaffolds for Biomedical Applications

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness and low bioactivity of metals. In this study, we have developed a new method towards fabricating a new type of bioactive and mechanically reliable porous metal scaffolds-densified porous Ti scaffolds. The method consists of two fabrication processes, 1) the fabrication of porous Ti scaffolds by dynamic freeze casting, and 2) coating and densification of the porous scaffolds. The dynamic freeze casting method to fabricate porous Ti scaffolds allowed the densification of porous scaffolds by minimizing the chemical contamination and structural defects. The densification process is distinctive for three reasons. First, the densification process is simple, because it requires a control of only one parameter (degree of densification). Second, it is effective, as it achieves mechanical enhancement and sustainable release of biomolecules from porous scaffolds. Third, it has broad applications, as it is also applicable to the fabrication of functionally graded porous scaffolds by spatially varied strain during densification.

Bioactive and mechanically reliable metal scaffolds have been fabricated through a method which consists of two processes, dynamic freeze casting for the fabrication of porous Ti, and coating and densification of the Ti scaffolds. The densification process is simple, effective and applicable to the fabrication of functionally graded scaffolds.

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness ...

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair

The reconstruction of critically sized bone defects remains a serious clinical problem because of poor angiogenesis within tissue-engineered scaffolds during repair, which gives rise to a lack of sufficient blood supply and causes necrosis of the new tissues. Rapid vascularization is a vital prerequisite for new tissue survival and integration with existing host tissue. The de novo generation of vasculature in scaffolds is one of the most important steps in making bone regeneration more efficient, allowing repairing tissue to grow into a scaffold. To tackle this problem, the genetic modification of a biomaterial scaffold is used to accelerate angiogenesis and osteogenesis. However, visualizing and tracking in vivo blood vessel formation in real-time and in three-dimensional (3D) scaffolds or new bone tissue is still an obstacle for bone tissue engineering. Multiphoton microscopy (MPM) is a novel bio-imaging modality that can acquire volumetric data from biological structures in a high-resolution and minimally-invasive manner. The objective of this study was to visualize angiogenesis with multiphoton microscopy in vivo in a genetically modified 3D-PLGA/nHAp scaffold for calvarial critical bone defect repair. PLGA/nHAp scaffolds were functionalized for the sustained delivery of a growth factor pdgf-b gene carrying lentiviral vectors (LV-pdgfb) in order to facilitate angiogenesis and to enhance bone regeneration. In a scaffold-implanted calvarial critical bone defect mouse model, the blood vessel areas (BVAs) in PHp scaffolds were significantly higher than in PH scaffolds. Additionally, the expression of pdgf-b and angiogenesis-related genes, vWF and VEGFR2, increased correspondingly. MicroCT analysis indicated that the new bone formation in the PHp group dramatically improved compared to the other groups. To our knowledge, this is the first time multiphoton microscopy was used in bone tissue-engineering to investigate angiogenesis in a 3D bio-degradable scaffold in vivo and in real-time.

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair

Here, we present a protocol to visualize blood vessel formation in vivo and in real-time in 3D scaffolds by multiphoton microscopy. Angiogenesis in genetically modified scaffolds was studied in a murine calvarial critical bone defect model. More new blood vessels were detected in the treatment group than in controls.

The reconstruction of critically sized bone defects remains a serious clinical problem because of poor angiogenesis within tissue-engineered scaffolds ...

Nondestructive Monitoring of Degradable Scaffold-Based Tissue-Engineered Blood Vessel Development Using Optical Coherence Tomography

Engineered vascular grafts with structural and mechanical properties similar to natural blood vessels are expected to meet the growing demand for arterial bypass. Characterization of the growth dynamics and remodeling process of degradable polymer scaffold-based tissue-engineered blood vessels (TEBVs) with pulsatile stimulation is crucial for vascular tissue engineering. Optical imaging techniques stand out as powerful tools for monitoring vascularization of engineered tissue enabling high-resolution imaging in real-time culture. This paper demonstrates a nondestructive and fast real-time imaging strategy to monitor the growth and remodeling of TEBVs in long-term culture by using optical coherence tomography (OCT). Geometric morphology is evaluated, including vascular remodeling process, wall thickness, and comparison of TEBV thickness in different culture time points and presence of pulsatile stimulation. Finally, OCT provides practical possibilities for real-time observation of the degradation of polymer in the reconstructing tissues under pulsatile stimulation or not and in each vessel segment, by compared with the assessment of polymer degradation using scanning electron microscopic(SEM) and polarized microscope.

A step by step protocol for nondestructive and long-period monitoring the process of vascular remodeling and scaffold degradation in real-time culture of biodegradable polymeric scaffold-based tissue-engineered blood vessels with pulsatile stimulation using optical coherence tomography is described here.

Engineered vascular grafts with structural and mechanical properties similar to natural blood vessels are expected to meet the growing demand for arterial ...

Core/shell Printing Scaffolds For Tissue Engineering Of Tubular Structures

Three-dimensional (3D) printing of core/shell filaments allows direct fabrication of channel structures with a stable shell that is cross-linked at the interface with a liquid core. The latter is removed post-printing, leaving behind a hollow tube. Integrating an additive manufacturing technique (like the one described here with tailor-made [bio]inks, which structurally and biochemically mimic the native extracellular matrix [ECM]) is an important step towards advanced tissue engineering. However, precise fabrication of well-defined structures requires tailored fabrication strategies optimized for the material in use. Therefore, it is sensible to begin with a set-up that is customizable, simple-to-use, and compatible with a broad spectrum of materials and applications. This work presents an easy-to-manufacture core/shell nozzle with luer-compatibility to explore core/shell printing of woodpile structures, tested with a well-defined, alginate-based scaffold material formulation.

Presented here is a simple-to-use, core/shell, three-dimensional bioprinting set-up for one-step fabrication of hollow scaffolds, suitable for tissue engineering of vascular and other tubular structures.

Three-dimensional (3D) printing of core/shell filaments allows direct fabrication of channel structures with a stable shell that is cross-linked at the ...

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

Summary

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Chapters in this video

Postproduction Processing of Electrospun Fibres for Tissue Engineering

Multi-Scale Modification of Metallic Implants With Pore Gradients, Polyelectrolytes and Their Indirect Monitoring In vivo

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

Three-Dimensional In Vitro Biomimetic Model of Neuroblastoma Using Collagen-Based Scaffolds

Distinctive Capillary Action by Micro-channels in Bone-like Templates can Enhance Recruitment of Cells for Restoration of Large Bony Defect

Fabrication of Mechanically Tunable and Bioactive Metal Scaffolds for Biomedical Applications

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair

Nondestructive Monitoring of Degradable Scaffold-Based Tissue-Engineered Blood Vessel Development Using Optical Coherence Tomography

Core/shell Printing Scaffolds For Tissue Engineering Of Tubular Structures