Projektet er delvist finansieret af Zentrales Innovationsprogramm Mittelstand (ZIM) des Bundesministeriums für Wirtschaft und Energie -KF3010902AJ4. Gebyret publikation er omfattet af BG traume hospital Tübingen, Tyskland.

BEMÆRK: immortaliseret human mesenchymal stromale precursorceller (SCP-1 celler) blev anvendt til forsøgene. SCP-1 celler blev leveret af professor Matthias Schieker 12. 1. Udvidelse af SCP-1-celler <ol><li> Forud for arbejdet med SCP-1 celler, korrekt rengøre arbejdsområdet (betegnet biosikkerhed kabinet I) med 70% ethanol (v / v) iført handsker. </li><li> I den rensede biosikkerhed kabinet forberede et passende volumen cellekulturmedium ved at blande de nødvendige kompon...

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	<li>Bridwell, K. H., Anderson, P. A., Boden, S. D., Vaccaro, A. R., Wang, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What's+new+in+spine+surgery.">What's new in spine surgery.</a> J Bone Joint Surg Am. 95, 1144-1150 (2013).</li><li>Adams, M. 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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. 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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. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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>

Stilladset overflade spiller en vigtig rolle i dets interaktion med omgivende væv in vivo for derved at bestemme implantater funktionel holdbarhed. Således er biokompatibilitet af stilladset undersøgt ved in vitro-assays ved anvendelse af celler (SCP1 cellelinie), når udpladet på stilladser. Mikroskopiteknikker, som fungerer godt med tynde og optisk transparente stilladser er dårligt egnet til uigennemsigtige stilladser at studere biokompatibilitet. Dette er hovedsage...

Kroniske rygsmerter er en multifaktoriel sygdom. Interessen for en minimalt invasiv løsning for den degenerative disc sygdom behandling er vokset siden 1950&#39;erne. Indtil i dag, multi-segmental fusion af rygsøjlen er den mest udbredte behandling. Da denne fremgangsmåde ofte fører til begrænsninger i mobiliteten af det påvirkede segment 1,2, udforskning af artroplastik æra blev en bred interesse. Væsentlige fremskridt i alt disk udskiftning og kerne udskiftning er blevet et godt alternativ til behandling af kroniske rygsmerter 1. Trods de enorme fremskridt, har ingen af ​​de metoder blevet klinisk evalueret. De mindre stive nucleus implantater udgør et lovende alternativ til total udskiftning af bremseskive, forudsat at fibrøse ring er intakt 3,4. Imidlertid er de for tiden foreliggende nucleus implantater på markedet ofte forbundet med komplikationer som ændringer i hvirvellegemet, dislokation, lodrette højde tab af skiven og than mangler nødvendige tilhørende mekanisk stivhed 5. For at overvinde de nuværende ulemper, har en hidtil ukendt kerne implantat lavet af strikkede titanium ledninger blevet udviklet 6. På grund af den unikke strikkede struktur, har denne nyudviklede stillads vist fornemme biomekaniske egenskaber, f.eks dæmpning funktion, porestørrelse, lastning kapacitet og pålidelighed 7. Med sigte på at teste biokompatibilitet af denne hidtil ukendte kerne implantat, afbildet alvorlige begrænsninger i de (optiske) analyseteknikker tilskrives den uigennemsigtige natur af implantatet. For at teste biokompatibilitet, celle-metal interaktion spiller en fremtrædende rolle 8-10. En interaktion mellem cellerne og stilladset er nødvendig for stabilisering og dermed for bedre implantat integration i værtssystemet. Dog kan en stigende indvækst dybde ændre de mekaniske egenskaber af stilladset. Med sigte på at undersøtigate om stilladset overflade tilvejebringer en base for cellevedhæftning, proliferation og differentiering, eller om metallet påvirker cellelevedygtighed, er det vigtigt at foretage fejlfinding af fælles velkendt problem med billeddannelse celler på / i ikke-transparente og uigennemsigtige stilladser. For at overvinde denne begrænsning flere fluorescerende blev baserede teknikker udforsket. Virksomheder giver en bred vifte af fluoroforer at visualisere levende celler, cellulære rum, eller endda specifikke cellulære stater 11. Fluoroforer for dette eksperiment blev valgt ved hjælp af online-værktøjet spektral seeren med henblik på bedst passer til vores fluorescerende mikroskop. Den udviklede strategi for analysen af ​​den vedhængende celler adfærd på / i uigennemsigtige strikket titan stillads omfatter følgende: 1) fluorescerende (grønt fluorescerende protein / GFP) mærkning af de osteochondro-progenitorceller for at tillade sporing af cellerne på stillads, 2) måling af levedygtigheden (mitochondrial aktivitet) af cellerne, og 3) visualisering celle-celle- og celle-materiale interaktioner inden stilladset. Proceduren har den fordel, at det let kan overføres til andre adhærente celler og andre ikke-transparent eller uigennemsigtig stillads. Endvidere kan levedygtighed og indvækst mønster overvåges over flere dage, således den kan bruges med begrænsede mængder af stillads materiale eller celler. Den foreliggende undersøgelse viser den vellykkede anvendelse af vores aktuelle protokol til måling af cellelevedygtighed og visualisere i-vækstmønster osteochondro-progenitorceller på / i uigennemsigtige strikket titan stillads. Desuden kan de udviklede protokoller anvendes for at bestemme stilladset urenheder og kontrollere rengøring protokoller.

<table border="0" cellpadding="0" cellspacing="0" width="623">
	<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>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:275px;">* 48 well plates</td>
			<td style="width:205px;">Corning Incorporated USA</td>
			<td style="width:144px;">3548</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:275px;">* 6 well plates</td>
			<td style="width:205px;">Falcon</td>
			<td style="width:144px;">353046</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:275px;">* T25</td>
			<td style="width:205px;">Greiner Bio-One GmbH</td>
			<td style="width:144px;">690 175</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:275px;">* T75</td>
			<td style="width:205px;">Greiner Bio-One GmbH</td>
			<td style="width:144px;">658 175</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:275px;">Acetic acid, purum &#8805; 99,0 %</td>
			<td style="width:205px;">Carl Roth</td>
			<td style="width:144px;">3738.4</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:275px;">Acetone</td>
			<td style="width:205px;">Carl Roth</td>
			<td style="width:144px;">5025.1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Axioplan-2&#160;</td>
			<td style="width:205px;">Carl Zeiss, Germany</td>
			<td style="width:144px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Biological safety cabinets</td>
			<td style="width:205px;">Thermo Scientific</td>
			<td style="width:144px;">safe 2020</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:275px;">Calcein acetoxymethyl ester (calcein AM)</td>
			<td style="width:205px;">Sigma</td>
			<td style="width:144px;">17783</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:275px;">Cell Culture Incubtator</td>
			<td style="width:205px;">Binder, Tuttlingen, Germany</td>
			<td style="width:144px;">9040-0078</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:275px;">Filter unit (0.22&#181;m)</td>
			<td style="width:205px;">Millipore, IRL</td>
			<td style="width:144px;">SLGP033RS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Centrifuges 5810 R And 5417 R</td>
			<td style="width:205px;">Thermo Fisher Scientific, NY</td>
			<td style="width:144px;">Megafuge 40R</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:275px;">Dimethylsulfoxid (DMSO)</td>
			<td style="width:205px;">Carl Roth</td>
			<td style="width:144px;">4720.2</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:275px;">Dulbecco&#8217;s PBS without Ca &#38; Mg</td>
			<td style="width:205px;">Sigma</td>
			<td style="width:144px;">H15-002</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:275px;">Ethanol 99 %</td>
			<td style="width:205px;">&#160;SAV liquid prod. GmBH</td>
			<td style="width:144px;">475956</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:275px;">Ethidium homodimer</td>
			<td style="width:205px;">Sigma</td>
			<td style="width:144px;">46043</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">EVOS Fluorescence imaging system</td>
			<td style="width:205px;">Life technologies</td>
			<td style="width:144px;">AMF4300</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:275px;">Fetal Bovine Serum (FCS)</td>
			<td style="width:205px;">Gibco</td>
			<td style="width:144px;">10270-106</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Hemocytometer</td>
			<td style="width:205px;">Hausser Scientific, PA, USA</td>
			<td style="width:144px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:275px;">Hoechst 33342</td>
			<td style="width:205px;">Sigma</td>
			<td style="width:144px;">14533-100MG</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Knitted titanium nucleus implant</td>
			<td style="width:205px;">Buck co &#38; KG,Germany</td>
			<td style="width:144px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:275px;">MEM Alpha Modification with Glutamine w/o nucleoside</td>
			<td style="width:205px;">Sigma</td>
			<td style="width:144px;">E15-832</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Omega microplate Reader</td>
			<td style="width:205px;">BMG Labtech,Germany</td>
			<td style="width:144px;">FLUOstar Omega</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:275px;">Penicillin/Streptomycin</td>
			<td style="width:205px;">Sigma</td>
			<td style="width:144px;">P11-010</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:275px;">Resazurin sodium salt</td>
			<td style="width:205px;">Sigma</td>
			<td style="width:144px;">199303-1G</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:275px;">Sulforhodamine B sodium salt</td>
			<td style="width:205px;">Sigma</td>
			<td style="width:144px;">S1402-1G</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Test tube rotator</td>
			<td style="width:205px;">Labinco B.V.,The Netherlands</td>
			<td style="width:144px;">Model LD-76</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:275px;">TRIS (hydroxymethyl) aminomethan</td>
			<td style="width:205px;">Carl Roth</td>
			<td style="width:144px;">AE15.1</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:275px;">Triton</td>
			<td style="width:205px;">Carl Roth</td>
			<td style="width:144px;">3051.2</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:275px;">Trypan Blue 0.5 %</td>
			<td style="width:205px;">Carl Roth</td>
			<td style="width:144px;">CN76.1</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:275px;">Trypsin/EDTA</td>
			<td style="width:205px;">Sigma</td>
			<td style="width:144px;">L11-004</td>
		</tr>
	</tbody>
</table>

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

Intervertebral disc degeneration and disc herniation is one of the major causes of lower back pain. Depletion of extracellular matrix, culminating in nucleus pulposus (NP) extrusion leads to intervertebral disc destruction. Currently available surgical treatments reduce the pain but do not restore the mechanical functionality of the spine. In order to preserve mechanical features of the spine, total disc or nucleus replacement thus became a wide interest. However, this arthroplasty era is still in an immature state, since none of the existing products have been clinically evaluated.
This study intends to test the biocompatibility of a novel nucleus implant made of knitted titanium wires. Despite all mechanical advantages, the material has its limits for conventional optical analysis as the resulting implant is non-transparent. Here we present a strategy that describes in vitro visualization, tracking and viability testing of osteochondro-progenitor cells on the scaffold. This protocol can be used to visualize the efficiency of the cleaning protocol as well as to investigate the biocompatibility of these and other non-transparent scaffolds. Furthermore, this protocol can be used to show adherence pattern of cells as well as cell viability and proliferation rates on/in the scaffold. This in vitro biocompatibility testing assay provides a propitious tool to analyze cell-material interaction in non-transparent and opaque scaffolds.

Foreløbige resultater viste, at den beskrevne hidtil ukendte kerne implantat ikke kun har gode dæmpningsegenskaber funktioner, men også er biokompatibel med SCP-1-celler. Under produktionen af ​​implantatet, det kommer i kontakt med stærke ætsende og giftige stoffer (smøremiddel, bejdsemiddel, elektro-polering opløsning). Med hjælp af indirekte fluorescerende farvningsteknikker kunne vi visualisere resterende urenheder og følgelig optimere en rengøringsprotokol viser signif...

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

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

Vi præsenterer her en fluorofor baseret imaging teknik til at detektere cellelevedygtighed på et ikke-transparent titanium stillads samt at detektere glimt af stilladset urenheder. Denne protokol fejlfinding af den ulempe afbildning celle-celle eller celle-metal interaktioner på uigennemsigtige stilladser.

Imaging Cell levedygtighed på uigennemsigtige Stilladser - Brug af Eksempel på en roman Strikkede Titanium Implant

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

Research

JoVE Journal

Bioengineering

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

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

Densitetsgradient flerlags Polymerisation (DGMP): En ny teknik til at skabe Multi-rum, Customizable stilladser til Tissue Engineering

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

Wide-field Fluorescent Microscopy og Fluorescerende Imaging Flowcytometri på en Cell-telefon

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

Selvrapportering Stilladser 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 ...

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

Cell Patterning på fotolitografisk Defineret Parylene-C: SiO<sub&gt; 2.</sub&gt; Underlag

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

Viability of Bioprinted Cellular Constructs Using a Three Dispenser Cartesian Printer

Levedygtighed Bioprinted Cellular Konstruktioner Ved hjælp af en tre Dispenser kartesiske Printer

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

Selektiv Cell Elimination fra Mixed 3D Kultur Ved hjælp af en i nærheden af ​​infrarød Photoimmunotherapy Teknik

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

Neonatal Cardiac Stilladser: Nye Matricer til regenerativ 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

Bestemmelse af Zeta Potentielle via Nanopartikel Translokation Hastigheder gennem en Tunable nanopore: Brug af DNA-modificerede Partikler som eksempel

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

Fabrikation af Decellularized brusk-afledte Matrix stilladser

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

High-Dimensionality Flow Cytometry for Immune Function Analysis of Dissected Implant Tissues

Højdimensionalitetsflowcytometri til immunfunktionsanalyse af dissekeret implantatvæv

The success of implanting laboratory-grown tissue or a medical device in an individual is subject to the immune response of the recipient host. ...