The authors would like to acknowledge Stanford Department of Orthopaedic Surgery and Stanford Coulter Translational Seed Grant for funding. J.H.L. would like to thank National Science Foundation Graduate Fellowship and DARE Doctoral Fellowship for support.

Alle Experimente wurden in Übereinstimmung mit der Stanford University Menschliche Probanden Richtlinien durchgeführt und genehmigt Institutional Review Board-Protokoll. 1. Articular Chondrozyten Isolation <ol><li> Erhalten Knorpel von Gewebe während der Knieendoprothetik verworfen und sezieren wie zuvor 8 beschrieben. </li><li> Visuell wählen Sie die medialen oder lateralen Femurkondylen Knorpelproben für glatte Oberflächen und machen Schnitt an der Oberfläche des Knorpels durch ein Skalpell, um 4-5 mm Knorpelbiopsien, ohne den subchondralen Knochen zu entfernen. </li><li> Isolieren Chondrozyten aus der extrazellulären Matrix durch enzymatische Dissoziation des Gewebes mit O / N Behandlung mit gereinigter Collagenase. Verwendung 1: 1-Verhältnis von Collagenase II (125 U / ml) und Collagenase IV (160 U / ml) in Chondrozyten Wachstumsmedien bei 37 ° C. </li><li> Am folgenden Tag, filtern das verdaute Gewebe, das die dissoziierten Zellen durch eine 70mm Porengröße Nylon-Mesh. Zweimal Waschen mit 20 ml DMEM / F12 und zentrifugiert bei 600 g für 5 min. </li><li> Plate Die isolierten Chondrozyten in einer Dichte von 1 x 10 6 Zellen / 60 mm Kulturschale nach Resuspension in DMEM / F12 mit 10% fötalem Rinderserum (FBS), 25 ug / ml Ascorbat, 2 mM L-Glutamin, antimikrobielle Mittel (100 U / ml Penicillin, 100 ug / ml Streptomycin und 0,25 ug / ml Fungizon). Halten die Platten bei 37 ° C und der Primär Chondrozytenkulturen als hochdichten Monoschicht aufrechtzuerhalten vor der Verkapselung in biomimetischen Hydrogelen. </li></ol> 2. Biomimetic Hydrogel Fabrication Hinweis: Dieses Verfahren ermöglicht die Synthese eines biomimetischen Hydrogel Poly (ethylenglykoldiacrylat) (PEGDA, MW 5000 g / mol), Chondroitinsulfat-Methacrylat (CS-MA) in DPBS. Die Zugabe von Photoinitiator ermöglicht photoaktivierten Vernetzung. Die endgültige Hydrogel-Zusammensetzung enthält 7% wiegent / Volumen (w / v) PEGDA, 3% w / v des CS-MA und 0,05% w / v des Photoinitiators. <ol><li> Synthetisieren CS-MA durch Funktionalisierung CS Polymers mit Methacrylatgruppen. <ol><li> Vorbereitung gepufferte Lösung von 50 mM 2-Morpholinoethansulfonsäure (MES) und 0,5 M Natriumchlorid (NaCl) durch Auflösen von 1,952 g MES und 5,84 g NaCl in 200 ml entionisiertem Wasser (DiH 2 O). Aufzulösen 5 g Chondroitinsulfat-Natriumsalz in der gepufferten Lösung. </li><li> Hinzufügen von 0,532 g (4,62 mmol) N-Hydroxysuccinimid (NHS) und 1.771 g (9.24 mmol) 1-Ethyl-3- (3-dimethylaminopropyl) carbodiimid-Hydrochlorid (EDC) (Molverhältnis von NHS-EDC = 1: 2) die Lösung und rühren für 5 min. </li><li> Hinzufügen 0,765 g (4,62 mmol) 2-Aminoethyl-Methacrylat (AEMA) (Molverhältnis von NHS: EDC: AEMA = 1: 2: 1) zu der Lösung und Aufrechterhaltung der Reaktion bei RT für 24 h. </li><li> Reinigt das Gemisch durch Dialyse gegen deionisiertes Wasser (DIH 2 O) für 4 Tage unter Verwendung eines Dialyseschlauches (12-14 kDa MWCO). </li><li> Filtern Sie die purified Lösung durch ein 0,22 um-Filter und setzen Sie die offenen Röhrchen, die die Lösung in dem Exsikkator und Vakuum O / N. Stellen Sie sicher, dass alle Rohre werden vor Licht geschützt. Bewahren Sie die gelösten Polymers bei -20 ° C vor Licht und Feuchtigkeit geschützt. </li></ol></li></ol> 3. Zell Encapsulation <ol><li> Autoklaven werden die PCR Film und zylindrische Stäbe (zum Ausstanzen der zellbeladene Hydrogele 3D) und zu sterilisieren die maßgeschneiderte zylindrischen Gelform durch Eintauchen in 0,2 um gefiltert 70% Ethanol in Gewebekultur Haube unter UV-Licht. </li><li> Verschließen Sie den Boden des Gels Form mit autoklaviert PCR Folie ohne Luftblasen oder Lücken, um Lecks zu verhindern. Halten Sie es in einer sterilen 150 mm Platte. </li><li> Der Tag vor der Verkapselung von Zellen in biomimetischen Hydrogel, distanzieren die High-Density-Chondrozyten-Monoschicht mit O / N-Behandlung in Collagenase II und Collagenase IV-Lösung. Verwenden 125 U / ml Collagenase II und 160 U / ml Kollagenase VI jeweils in Chondrozyten Nährmedienbei 37 ° C. </li><li> Nehmen Sie einen 50-ml-Röhrchen und geben Sie 5% Gewicht / Volumen (w / v) Poly (Ethylenglykoldiacrylat) (PEGDA, MW = 5000 g / mol), 3% w / v Chondroitinsulfat-Methacrylat (CS-MA), und 0,05% w / v-Photoinitiator in DPBS. Das Röhrchen vortexen, um die Lösung zu mischen und halten Sie sie beiseite. Schützen Sie den Schlauch vor Licht. </li><li> Sammeln Sie die dissoziierten Zellen in einem 50 ml Falcon-Röhrchen, zählen und Zentrifuge bei 460 xg für 5 min. Saugen Sie alle Medien aus den Zellen Rohr und fügen Sie die obigen gemischten Gel-Material zu resuspendieren Zellen in einer Dichte von 15 x 10 6 Zellen / ml. Mischen Sie es 30 Mal gründlich unter Vermeidung von Luftblasen-Bildung. </li><li> Pipette 72 ul der Zellsuspension-Hydrogel oder Hydrogel allein in die maßgeschneiderte zylindrischen Gelform an und beeinflussen die Gelierung durch Bestrahlung mit UV-Licht (365 nm Wellenlänge) bei 3 mW / m 2 für 5 min. Bereiten Sie eine Hydrogel-Lösung ohne Zellinhalt. Verwenden Sie diese Konstrukte als Negativkontrollen für die Zukunft biochemische und mechanische Prüfungen. Measure der Intensität der UV-Licht mit einer UV-Intensität Meter. So passen Intensität, ändern Sie den Abstand zwischen der UV-Lichtquelle und dem Hydrogel Gerüst während der Gelierung. HINWEIS: Da das Hydrogel CS wird die azelluläre Beitrag der biochemischen GAG-Gehalt der zellbeladenen Hydrogele abgezogen, um nur die Zell GAG darzustellen. </li><li> Verwenden Sie ein Skalpell, um den Film für leicht entfernen abgeschnitten und ziehen Sie sie vorsichtig. Mit Hilfe der zylindrischen Stangen, Push-out des Gels in einer 6-Well-Platte mit 5 ml sterilem DPBS abzuwaschen nicht polymerisierten Gel und lose Zellen. </li><li> Kultur die Zellbeladene Hydrogele in 24-Well-Platten mit 1,5 ml Knorpelzellwachstumsmedien in jedes Well. Verwenden Sie eine Einweg-Spatel, um die gewaschenen Hydrogele in den Brunnen zu übertragen. </li><li> Beurteilen Sie die Lebensfähigkeit der Zellen durch Live tot-Färbung von 24 Stunden nach der Einkapselung mit einem Lebensfähigkeit / Zytotoxizität Kit. Kultur die Zellbeladene Hydrogele und die leeren Hydrogele für 3-6 Wochen zu ändern, um frische Chondrozyten growth Medien alle 2 Tage vor der Ernte für Analysen. </li></ol> 4. RNA-Extraktion und Genexpressionsanalysen <ol><li> Die Medien sorgfältig absaugen und legte sterilem PBS 1X auf dem Cell-beladenen als auch die leeren Steuer Hydrogele. </li><li> Mit Hilfe eines sterilen Spatels Übertragung jedes Hydrogel in ein sauberes 1,5-ml-Eppendorf-Röhrchen und füge 250 ul Tri-Reagenz in jedes Röhrchen. Folgen Sie den Anweisungen des Herstellers, um RNA zu isolieren. </li><li> Nach RNA-Isolierung und Quantifizierung Verwendung 1 ug es von jeder Probe und führen Sie eine umgekehrte Transkription in cDNA unter Verwendung des High Capacity cDNA umge Transcription Kit. </li><li> Für die quantitative PCR verwenden TaqMan Gene Expression Arrays zur Untersuchung der Expression von Ihren Genen von Interesse. Normalisieren die Genexpression intern auf GAPDH. </li><li> Für die PCR-Bedingungen sind eine 2-minütige Inkubation bei 50 ° C zur vorherigen Amplicons mit Uracil-DNA-Glycosylase inaktivieren, gefolgt von einer 1060; min Inkubation bei 95 ° C, um die Taq-Polymerase zu aktivieren. Führen dann vierzig Zyklen PCR, bestehend aus 15 Sekunden bei 95 ° C und 1 min bei 60 ° C. </li></ol> 5. Biochemische Analysen <ol><li> Für jede Zelle-Hydrogel-Konstrukte zu quantifizieren DNA und sulfatiertes Glykosaminoglykan (SGAG) Produktion wie folgt. </li><li> Die Medien sorgfältig absaugen und legte sterilem PBS 1X auf dem Cell-beladenen als auch die leeren Steuer Hydrogele. Wiegen Sie eine leere Hülse und legte das Hydrogel im Inneren nach dem Blotten auf Tissue-Papier und das Gewicht aufgezeichnet oder Nassgewicht der Hydrogele. </li><li> Gefriert jedes Hydrogel bei -20 ° C in einem getrennten 1,5 ml Eppendorf-Röhrchen zur enzymatischen Verdauung und späteren Verwendung ein Aliquot der verdauten Produkt Picogreen Assay (für DNA) und Dimethyl-Methylenblau-Test (GAG) laufen. </li><li> Bereiten Sie die papainase Lösung für die enzymatische Verdauung der Hydrogele. <ol><li> Bereiten Sie zuerst die PBE-Puffer durch Auswiegen 7,1 g Natrium-phosphate zweibasigen (Na 2 HPO 4) und 1,6 g Ethylendiamintetraessigsäure-Dinatriumsalz (EDTA-Na 2). Lösen sich in 500 ml dH 2 O, der pH-Wert auf 6,5 eingestellt und filtriert die gereinigte Lösung durch ein 0,22-mm-Filter. </li><li> Aufzulösen 0,035 g L-Cystein in 20 ml PBE Puffer. Die Lösung wird filtriert, und 100 ul steriles Enzym Papain. </li></ol></li><li> Wenn Sie bereit sind, um die Tests durchzuführen, nehmen Sie die Schläuche von -20 ° C und 300 ul der bereit Papain Lösung der eingefrorenen Hydrogele. Crush das Gel mit einem Mörser und Stößel und mischen Sie es gut mit dem Motor Stößel. </li><li> Bringen Sie das Volumen auf 500 ul mit zusätzlichen Papain-Lösung. Führen Sie das gleiche Verfahren für die Steuer Gele. Inkubieren bei 60ºC für 16 Stunden 3,9. Die Lösung, die verdaut Hydrogel kann daher für die DNA- und GAG Quantifizierung verwendet. Messen Sie den DNA-Gehalt unter Verwendung des Picogreen Assay mit Lambda-Phagen-DNA als Standard folgende Hersteller protocol. </li><li> Quantifizierung der sulfatierten-GAG-Gehalt unter Verwendung des 1,9-Dimethylmethylen blau (DMMB) Farbstoffbindungsassay mit Hai Chondroitinsulfat als Standard 10. Bestimmen Sie die GAG-Gehalt durch Division der Menge an GAG für den entsprechenden Nassgewicht. </li></ol> 6. Mechanische Prüfung <ol><li> Führen einaxialen Druckversuche unter Verwendung eines Instron 5944-Testsystem mit einem 10 N Wägezelle ausgestattet. <ol><li> Nach der gewünschten Tagen der in vitro-Kultur, führen Sie den Druckversuch auf dem Cell-Hydrogel-Gerüst und die azellulären Steuer Hydrogele. </li><li> Tauchen Sie die Proben in einem PBS-Bad bei RT und Komprimieren in einer Rate von 1% Dehnung / sec zu einer maximalen Dehnung von 15% 11,12 da die physiologische Belastung von Knorpelgewebe unter Beladungszustand erfahren hat, wurde berichtet, dass 10-20% betragen 13,14. </li></ol></li><li> Erstellen Sie Stress gegen Dehnungskurven und Kurvenanpassung mit einem Polynom dritter Ordnung Gleichung. Bestimmen Sie die comprEssiv Tangentenmodul aus der Kurvengleichung bei Dehnungswerte von 15%. </li></ol>

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The authors have nothing to disclose.

Wie in diesem Protokoll angegeben, sind 3D-Hydrogele in der Lage, von Chondrozyten Phänotyp in Kultur zu halten, die Vermeidung der Prozess der Zell Entdifferenzierung in Faserknorpel-Zellen in der Regel mit Monolayer-Kulturen 15 angetroffen. Außerdem Langzeitkulturen der chondrocytes- Hydrogel Konstrukt zeigte eine günstige Umgebung, die die innere Zellfunktionen mit dem Alter und Erkrankung assoziiert beibehält. Die Verwendung eines 3D biomimetischen Hydrogel hat mehrere Vorteile. Erstens ist die Aufnahme von Chondroitinsulfat (CS), eine wichtige Komponente im Gelenkknorpel gefunden, aktivieren Zellen, um die Hydrogelmatrix durch Sekretion von Chondroitinase abbauen und legte sich neu synthetisierte Knorpelextrazellulären Matrix 5, 16. Darüber hinaus CS hat sich gezeigt, In den entzündungshemmenden Eigenschaften im arthritischen Gelenk haben. Die biomimetischen Hydrogel kann auch als Gerüstmaterial für Zellabgabe bei der Knorpelreparatur verwendet werden, und können chemisch modifiziert seinum bessere Gewebe-Biomaterial Integration 17,18 zu erleichtern. Die Verwendung der PEG-CS Hydrogele ermöglicht Langzeitkulturen von Chondrozyten und die Bewertung der biochemischen und mechanischen Eigenschaften. Hier zeigen wir, wie diese Plattform kann nützlich für die vergleichende Analysen der verschiedenen Quellen von differenzierten Chondrozyten, um die optimale Zelltyp für die Knorpeltechnik zu definieren. Interessant ist, dass Chondrozyten in Hydrogele gekapselt lebensfähig bleiben und sich vermehren nach ihrem inneren Fähigkeiten. Hydrogelzusammensetzung Träger, in der Tat, das Wachstum von gesunden jugendlichen und erwachsenen Chondrozyten, wie in Abbildung 2 dargestellt. Die Zusammensetzung und Struktur der beschriebenen Hydrogele fördert auch die Knorpelgewebebildung, wie durch die Abscheidung einer funktionelle extrazelluläre Matrix durch Glycosaminoglycan beurteilt (GAG deutet ) Quantifizierung. Ein zusätzlicher Vorteil ist, dass die Chondrozyten-Hydrogel-Konstruktekönnen für die mechanischen Eigenschaften des neu gebildeten Knorpelgewebe zu bewerten. Beachten Sie, dass der einaxialen Drucktest sollte auf der azellulären Hydrogel zum Vergleich durchgeführt werden. Die Hydrogele in der Tat, eine Eigensteifigkeit auf Grund der Steifigkeit der CS-Einheiten. Einaxialen Druckverformung von 5-20% (bei ​​einer Verformungsgeschwindigkeit 1% / s) kann für die mechanische Prüfung von Knorpelgewebe 11,12, da die physiologische Belastung von Knorpelgewebe unter Beladungszustand erlebt anzuwenden wurde berichtet, dass 10 bis 20 sein 13,14%. Die Reaktion der beiden Zellbeladenen und azellulären Hydrogele mechanische Prüfung wurde bei der Kultur Endpunkt ausgewertet. In dem beschriebenen Beispiel über beobachteten wir eine vergleichbare Steifigkeit der Konstrukte, die bei Erwachsenen und Jugendlichen Chondrozyten in Gegensatz zu der geringeren Steifigkeit der Konstrukte enthaltenden OA Chondrozyten. Solche mechanischen Eigenschaften der zell Hydrogel Konstrukt erlauben die Beurteilung der funktionellen Eigenschaften desgebildete Gewebe geben eine eingehende Analyse der Zellreifung Fähigkeit. Abschließend kann die Verwendung der 3D biomimetischen Hydrogele, um das Potential verschiedener Chondrozytenpopulation studieren, um Knorpelgewebe erzeugen weithin angewandt werden. Neben den hier beschriebenen in vitro-Studien können in vivo Transplantation der Zellen beladene Konstrukten vorgesehen, um die Zellreifung und Regenerationspotential im physiologischen Bereich zu untersuchen. Weitere Modifikationen des Hydrogels Plattform mit zusätzlichen biomimetischen Faktoren können ebenfalls in Betracht gezogen werden, um Chondrozytenproliferation und Reifung zu optimieren.

With its limited self-repair potential, human articular cartilage undergoes frequent irreversible damages. Extensive efforts are currently focused on the development of efficient cell-based approaches for treatment of articular cartilage injuries. The success of these cell-based therapies is highly dependent on the selection of an optimal cell source and the maintenance of its regenerative potential. Chondrocytes are a common cell source for cartilage repair, but they are limited in supply and can de-differentiate during in vitro expansion in 2D monolayer culture thereby limiting their generation of hyaline cartilage 1. 
The aim of this protocol is to establish a 3-dimensional hydrogel platform for an in vitro comparative study of human chondrocytes from different ages and disease state. Unlike conventional two-dimensional (2D) culture, three-dimensional (3D) hydrogels allow chondrocytes to maintain their morphology and phenotype and provides a physiologically relevant environment enabling chondrocytes to produce cartilage tissue 2,3. In addition to providing a 3D physical structure for chondrocyte culture, hydrogels mimic the function of native cartilage extracellular matrix (ECM). Specifically, the inclusion of chondroitin sulfate methacrylate provides a potential reservoir for secreted paracrine factors 4 and enables cell-mediated degradation and matrix turnover 5. Although many 3D hydrogel culture systems have been utilized widely in various studies including agarose and alginate gels, we have used a biomimetic 3D culture system that has some distinct advantages for chondrocyte culture. Chondroitin sulfate (CS) is an abundant component in articular cartilage and the PEG-CS hydrogels have been shown to maintain and even enhance chondrogenic phenotype and facilitate cell-mediated matrix degradation and turnover 2,5. In addition, the mechanical properties of the hydrogel scaffold can be easily modulated by changing concentration of PEG and hence can be utilized to further enhance the regeneration potential of chondrocytes or a related cell type 6,7. PEG/CSMA is also biocompatible and hence has the potential for a direct clinical application in cartilage defects for example. The limitation for this system is its complexity and the use of photopolymerization that can potentially affect cell viability as compared to simpler systems like agarose, however the advantages for the chondrocyte culture outweigh the potential limitations.
The 3D hydrogel culture is compatible with conventional assay for evaluation of cell phenotype (gene expression, protein immunostaining) and functional outcome (quantification of cartilage matrix production, mechanical testing). This favorable 3D environment was tested to compare the tissue regeneration potential of human chondrocytes from three different aged populations in long-term 3D cultures. 
The outcomes were evaluated via both phenotypic and functional assays. Juvenile, adult and OA chondrocytes showed differential responses in the 3D biomimetic hydrogel culture. After 3 and 6 weeks, chondrogenic gene expression was upregulated in juvenile and adult chondrocytes but was downregulated in OA chondrocytes. Deposition of cartilage tissue components including aggrecan, type II collagen, and glycosaminoglycan (GAG) was high for juvenile and adult chondrocytes but not for OA chondrocytes. The compressive moduli of the resulting cartilage constructs also exhibited similar trends. In conclusion, both juvenile and adult chondrocytes exhibited chondrogenic and cartilage matrix disposition up to 6 weeks of 3D culture in hydrogels. In contrast, osteoarthritic chondrocytes revealed a loss of cartilage phenotype and minimal ability to generate robust cartilage.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>juvenile chondrocytes (Clonetics&#8482; Normal Human Chondrocyte Cell System )</td>
			<td>Lonza</td>
			<td>CC-2550</td>
			<td></td>
		</tr>
		<tr>
			<td>adult chondrocytes (Clonetics&#8482; Normal Human Chondrocyte Cell System)</td>
			<td>Lonza</td>
			<td>CC-2550</td>
			<td></td>
		</tr>
		<tr>
			<td>poly(ethylene glycol diacrylate)</td>
			<td>Laysan Bio</td>
			<td>ACRL-PEG-ACRL-1000-1g</td>
			<td></td>
		</tr>
		<tr>
			<td>2-morpholinoethanesulfonic acid</td>
			<td>Sigma</td>
			<td>M5287</td>
			<td></td>
		</tr>
		<tr>
			<td>photoinitiator</td>
			<td>Irgacure&#160;</td>
			<td align="right">2959</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium chloride&#160;</td>
			<td>Sigma</td>
			<td>S9888</td>
			<td></td>
		</tr>
		<tr>
			<td>chondroitin sulfate sodium salt&#160;</td>
			<td>Sigma</td>
			<td>C9819</td>
			<td></td>
		</tr>
		<tr>
			<td>N-hydroxysuccinimide&#160;</td>
			<td>Sigma</td>
			<td align="right">130672</td>
			<td></td>
		</tr>
		<tr>
			<td>1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride</td>
			<td>Sigma</td>
			<td>E1769</td>
			<td></td>
		</tr>
		<tr>
			<td>2-aminoethyl methacrylate</td>
			<td>Sigma</td>
			<td align="right">516155</td>
			<td></td>
		</tr>
		<tr>
			<td>dialysis tubing</td>
			<td>Spectrum Laboratories&#160;</td>
			<td align="right">132700</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase 2</td>
			<td>Worthington Biochemical&#160;</td>
			<td>LS004177</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase 4</td>
			<td>Worthington Biochemical&#160;</td>
			<td>LS004189</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12 media</td>
			<td>HyClone, Thermo Scientific&#160;</td>
			<td>SH3002301&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>live/dead assay</td>
			<td>Life Technologies</td>
			<td><a href="https://www.lifetechnologies.com/order/catalog/product/L3224">L3224</a></td>
			<td></td>
		</tr>
		<tr>
			<td>Tri reagent</td>
			<td>Life Technologies</td>
			<td>AM9738</td>
			<td></td>
		</tr>
		<tr>
			<td>Quant-iT&#8482; PicoGreen&#174; dsDNA Assay Kit</td>
			<td>Invitrogen</td>
			<td><a href="https://www.lifetechnologies.com/order/catalog/product/P11496">P11496</a></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic&#160;</td>
			<td>Sigma</td>
			<td>S3264</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid disodium salt&#160;</td>
			<td>Sigma</td>
			<td>E5134</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Cysteine</td>
			<td>Sigma</td>
			<td>C1276</td>
			<td></td>
		</tr>
		<tr>
			<td>1,9-dimethylmethylene blue&#160;</td>
			<td>Sigma</td>
			<td align="right">341088</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Instruments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>UV light equipment - XX-15LW Bench Lamp, 365nm</td>
			<td>UVP</td>
			<td>95-0042-07</td>
			<td></td>
		</tr>
		<tr>
			<td>Instron 5944 testing system&#160;</td>
			<td>Instron Corporation</td>
			<td>E5940</td>
			<td></td>
		</tr>
	</tbody>
</table>

3d hydrogel scaffolds for articular chondrocyte culture and cartilage generation

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer cartilage tissue offer a promising solution to repair articular cartilage. To select the optimal cell source for tissue repair, it is important to develop an appropriate culture platform to systematically examine the biological and biomechanical differences in the tissue-engineered cartilage by different cell sources. Here we applied a three-dimensional (3D) biomimetic hydrogel culture platform to systematically examine cartilage regeneration potential of juvenile, adult, and osteoarthritic (OA) chondrocytes. The 3D biomimetic hydrogel consisted of synthetic component poly(ethylene glycol) and bioactive component chondroitin sulfate, which provides a physiologically relevant microenvironment for in vitro culture of chondrocytes. In addition, the scaffold may be potentially used for cell delivery for cartilage repair in vivo. Cartilage tissue engineered in the scaffold can be evaluated using quantitative gene expression, immunofluorescence staining, biochemical assays, and mechanical testing. Utilizing these outcomes, we were able to characterize the differential regenerative potential of chondrocytes of varying age, both at the gene expression level and in the biochemical and biomechanical properties of the engineered cartilage tissue. The 3D culture model could be applied to investigate the molecular and functional differences among chondrocytes and progenitor cells from different stages of normal or aberrant development.

Bioaktive Hydrogele, die PEG und CS-Einheiten (Abbildung 1) stellen eine ideale Plattform für Kultur und Reifung von menschlichen Chondrozyten 2,3,5,7. Chondrozyten aus unterschiedlichem Alter und Krankheitszustände können mit dem beschriebenen Verfahren kultiviert und auf ihren Phänotyp, der Genexpression und biochemische und mechanische Eigenschaften des erzeugten Knorpelgewebe zu analysieren. Drei Chondrozyten-Proben, juvenile- 6 Monate, Erwachsenen- 34 Jahre und osteoarthritic- 74 Jahre, wurden nach 3 Wochen Kultur in 3D biomimetische Hydrogele geerntet. Genexpressionsanalysen der normalen Chondrozyten, sowohl jugendlichen und erwachsenen, zeigten einen Anstieg der Expression der Chondrozyten-Genen COL2A1 und Col6a1. Im Gegensatz dazu zeigten erkrankten Chondrozyten eine dramatische Abnahme der COL2A1, während die Expression von Col6a1 (Abbildung 2), die einen Verlust von chondrogenen Phänotyp, obwohl sie in einem günstigen biomimetischen Umgebung kultiviert. Die CS-PEG Hydrogele auch als ein dynamisches Umfeld für die Chondrozyten zur Proliferation zu dienen, verdauen die bereits bestehenden Matrix von PEG und CS und legen ihre perizellulären und der extrazellulären Matrix, die Hauptbestandteile des reifen Knorpelgewebe. Angesichts dieser Möglichkeiten, Chondrozyten Expansion nach 3 Wochen in vitro-Kultur kann durch Quantifizierung der DNA mit dem Picogreen Farbstoff geschätzt werden. Vergleichsanalyse der drei Gruppen von Zellen zeigen, dass die Zelldichte von jugendlichen und erwachsenen Bevölkerung war unverändert, während OA Chondrozyten zeigte einen dramatischen Abfall im Vergleich zu Tag 1 der Kultur. Ein Verlust von DNA-Gehalt wurde auch für OA, aber keine anderen Chondrozyten Eingabe mögliche Zelltod während der Langzeit-Kultur (Abbildung 3) beobachtet. Das sezernierte Matrix wurde als sulfatiertes GAG-Gehalt von DMMB Farbstoffbindungstest am Endpunkt Stufe nach 3 Wochen der Kulturen quantifiziert. GAG-Gehalt wird durch das Feuchtgewicht des Hydrogels als recor normalisiertded vor dem enzymatischen Abbau und dem azellulären Beitrag subtrahiert. Wie in 4 gezeigt, abgeschieden Chondrozyten eine ähnliche signifikante Menge an GAG während 3 Wochen Kultur in 3D Hydrogelen. Die Anwesenheit eines physikalischen Gerüst ermöglicht die Auswertung der biomechanischen Eigenschaften der Proben durch einaxialen Druckversuch 2,3. Während azellulären Hydrogele werden einer Abnahme der Druckmoduln gehalten zellbeladene Konstrukte die Druckmodule nach 3 Wochen in Kultur (Abbildung 4). Die phänotypische, hier beschriebenen biochemischen und biomechanischen Analysen sind daher nützlich für die Bewertung und das Verständnis des Potenzials der verschiedenen Knorpelzellpopulationen für Engineering Knorpel. <img alt="Abbildung 1" src="/files/ftp_upload/53085/53085fig1.jpg" /> Abbildung 1. PEG-CS biomimetische Hydrogele für 3D-Chondrozyten-cultur. Chondrozyten werden in einer Mischung, die Poly (ethylenglykoldiacrylat) (PEGDA) und Chondroitinsulfat-Methacrylat (CS-MA) und in die maßgeschneiderte zylindrischen Gelform gegossen resuspendiert. Nach der UV-Belichtung, verfestigte Gele werden aus den Formen gesammelt und die Lebensfähigkeit der Zellen wird durch Live-tot-Färbung von 24 Stunden nach der Einkapselung bewertet. <a href="https://www.jove.com/files/ftp_upload/53085/53085fig1large.jpg" target="_blank">Bitte klicken Sie hier, um eine größere Version dieser Figur zu sehen.</a> <img alt="Figur 2" src="/files/ftp_upload/53085/53085fig2.jpg" /> Abbildung 2. Die Expression von Genen chondrogene in humanen Chondrozyten in 3D biomimetische Hydrogele kultiviert. Quantitative Genexpressionsknorpelmarker COL2A1 und Col6a1 von jugendlichen, erwachsenen und OA Chondrozyten nach 3 Wochen der Kultur in 3D biomimetische Hydrogele. Werte werden auf Genexpressionsebene an Tag 1. Fehler b normiertars stellen Mittelwert ± Standardabweichung. p * &lt;0,05, wie durch einen zweiseitigen Student t-Test bestimmt. Geändert von Smeriglio et al. 2 <a href="https://www.jove.com/files/ftp_upload/53085/53085fig2large.jpg" target="_blank">Klicken Sie hier, um eine größere Version dieser Figur zu sehen.</a> <img alt="Figur 3" src="/files/ftp_upload/53085/53085fig3.jpg" /> Abbildung 3. DNA-Gehalt in humanen Chondrozyten in 3D biomimetische Hydrogele kultiviert. DNA-Quantifizierung durch Picogreen Assay in juvenile, Erwachsenen-und OA Chondrozyten nach 3 Wochen der Kultur in 3D biomimetische Hydrogele. Werte werden auf DNA-Ebene an Tag 1 normiert <a href="https://www.jove.com/files/ftp_upload/53085/53085fig3large.jpg" target="_blank">Bitte klicken Sie hier, um eine größere Version dieser Figur zu sehen.</a> <img alt="Figur 4" src="/files/ftp_upload/53085/53085fig4.jpg" /> &lt;br /&gt; Abbildung 4. GAG-Gehalt Messung und einaxialen Druckversuch der menschlichen Knorpelzellen an Tag 1 und nach 3 Wochen der Kultur in 3D biomimetische Hydrogele. (A) GAG Quantifizierung durch DMMB Assay in Chondrozyten an Tag 1 und nach 3 Wochen der Kultur in 3D biomimetische Hydrogele. Die Werte sind normiert auf Gewicht (ww) benetzen und ausgedrückt als &amp; mgr; g / g. (B) Druckmodul (kPa) von azellulären und zellbeladene Hydrogele am Tag 1 und nach 3 Wochen Kultur in 3D biomimetische Hydrogele. <a href="https://www.jove.com/files/ftp_upload/53085/53085fig4large.jpg" target="_blank">Bitte klicken Sie hier, um zu sehen eine größere Version dieser Figur.</a>

Watch this Scientific Journal Video about 3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation at JoVE.com

3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation

Cartilage repair represents an unmet medical challenge and cell-based approaches to engineer human articular cartilage are a promising solution. Here, we describe three-dimensional (3D) biomimetic hydrogels as an ideal tool for the expansion and maturation of human articular chondrocytes.

3D-Hydrogel Scaffolds für Articular Chondrozyten-Kultur und Knorpel-Generation

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer ...

3d-hydrogel-scaffolds-for-articular-chondrocyte-culture-cartilage

Research

JoVE Journal

Bioengineering

19.8K Aufrufe.  Stanford University. Cartilage repair represents an unmet medical challenge and cell-based approaches to engineer human articular cartilage are a promising solution. Here, we describe three-dimensional (3D) biomimetic hydrogels as an ideal tool for the expansion and maturation of human articular chondrocytes. 

Serie: 3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation

A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells within a tissue, however, are typically encased within a closely packed tissue mass in which cells establish intimate connections with many near-neighbors and with extracellular matrix components. Accordingly, the chemical milieu and physical forces experienced by cells within a 3D tissue are fundamentally different than those experienced by cells grown in monolayer culture. This has been shown to markedly impact cellular morphology and signaling. Several methods have been devised to generate 3D cell cultures including encapsulation of cells in collagen gels1or in biomaterial scaffolds2. Such methods, while useful, do not recapitulate the intimate direct cell-cell adhesion architecture found in normal tissues. Rather, they more closely approximate culture systems in which single cells are loosely dispersed within a 3D meshwork of ECM products. Here, we describe a simple method in which cells are placed in hanging drop culture and incubated under physiological conditions until they form true 3D spheroids in which cells are in direct contact with each other and with extracellular matrix components. The method requires no specialized equipment and can be adapted to include addition of any biological agent in very small quantities that may be of interest in elucidating effects on cell-cell or cell-ECM interaction. The method can also be used to co-culture two (or more) different cell populations so as to elucidate the role of cell-cell or cell-ECM interactions in specifying spatial relationships between cells. Cell-cell cohesion and cell-ECM adhesion are the cornerstones of studies of embryonic development, tumor-stromal cell interaction in malignant invasion, wound healing, and for applications to tissue engineering. This simple method will provide a means of generating tissue-like cellular aggregates for measurement of biomechanical properties or for molecular and biochemical analysis in a physiologically relevant model.

We describe a simple, rapid method of generating 3D tissue-like spheroids and their potential application to quantify differences in cell-cell interactions.

A Simple hängenden Tropfen Cell Culture Protokoll zur Generierung von 3D-Sphäroide

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells ...

Forschung

Biotechnik

Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells inside of 3D biomaterial scaffolds provides a way to accurately mimic these microenvironments, providing an advantage over traditional 2D culture methods using polystyrene as well as a method for engineering replacement tissues 2. While 2D tissue culture polystrene has been used for the majority of cell culture experiments, 3D biomaterial scaffolds can more closely replicate the microenvironments found in vivo by enabling more accurate establishment of cell polarity in the environment and possessing biochemical and mechanical properties similar to soft tissue.3 A variety of naturally derived and synthetic biomaterial scaffolds have been investigated as 3D environments for supporting stem cell growth. While synthetic scaffolds can be synthesized to have a greater range of mechanical and chemical properties and often have greater reproducibility, natural biomaterials are often composed of proteins and polysaccharides found in the extracelluar matrix and as a result contain binding sites for cell adhesion and readily support cell culture. Fibrin scaffolds, produced by polymerizing the protein fibrinogen obtained from plasma, have been widely investigated for a variety of tissue engineering applications both in vitro and in vivo 4. Such scaffolds can be modified using a variety of methods to incorporate controlled release systems for delivering therapeutic factors 5. Previous work has shown that such scaffolds can be used to successfully culture embryonic stem cells and this scaffold-based culture system can be used to screen the effects of various growth factors on the differentiation of the stem cells seeded inside 6,7.
 This protocol details the process of polymerizing fibrin scaffolds from fibrinogen solutions using the enzymatic activity of thrombin. The process takes 2 days to complete, including an overnight dialysis step for the fibrinogen solution to remove citrates that inhibit polymerization. These detailed methods rely on fibrinogen concentrations determined to be optimal for embryonic and induced pluripotent stem cell culture. Other groups have further investigated fibrin scaffolds for a wide range of cell types and applications - demonstrating the versatility of this approach 8-12.

This work details the preparation of 3D fibrin scaffolds for culturing and differentiating plutipotent stem cells. Such scaffolds can be used to screen the effects of various biological compounds on stem cell behavior as well as modified to contain drug delivery systems.

Erstellung von 3D-Fibrin-Gerüste für Stem Cell Culture Anwendungen

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells ...

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.

Selbstberichts Gerüste für 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 ...

Generation of ESC-derived Mouse Airway Epithelial Cells Using Decellularized Lung Scaffolds

Lung lineage differentiation requires integration of complex environmental cues that include growth factor signaling, cell-cell interactions and cell-matrix interactions. Due to this complexity, recapitulation of lung development in vitro to promote differentiation of stem cells to lung epithelial cells has been challenging. In this protocol, decellularized lung scaffolds are used to mimic the 3-dimensional environment of the lung and generate stem cell-derived airway epithelial cells. Mouse embryonic stem cell are first differentiated to the endoderm lineage using an embryoid body (EB) culture method with activin A. Endoderm cells are then seeded onto decellularized scaffolds and cultured at air-liquid interface for up to 21 days. This technique promotes differentiation of seeded cells to functional airway epithelial cells (ciliated cells, club cells, and basal cells) without additional growth factor supplementation. This culture setup is defined, serum-free, inexpensive, and reproducible. Although there is limited contamination from non-lung endoderm lineages in culture, this protocol only generates airway epithelial populations and does not give rise to alveolar epithelial cells. Airway epithelia generated with this protocol can be used to study cell-matrix interactions during lung organogenesis and for disease modeling or drug-discovery platforms of airway-related pathologies such as cystic fibrosis.

This protocol efficiently directs mouse embryonic stem cell-derived definitive endoderm to mature airway epithelial cells. This differentiation technique uses 3-dimensional decellularized lung scaffolds to direct lung lineage specification, in a defined, serum-free culture setting.

Die Erzeugung von ESC-derived Maus Airway epithelialen Zellen unter Verwendung von Dezellularisierte Lung Scaffolds

Lung lineage differentiation requires integration of complex environmental cues that include growth factor signaling, cell-cell interactions and ...

An Experimental and Finite Element Protocol to Investigate the Transport of Neutral and Charged Solutes across Articular Cartilage

Osteoarthritis (OA) is a debilitating disease that is associated with degeneration of articular cartilage and subchondral bone. Degeneration of articular cartilage impairs its load-bearing function substantially as it experiences tremendous chemical degradation, i.e. proteoglycan loss and collagen fibril disruption. One promising way to investigate chemical damage mechanisms during OA is to expose the cartilage specimens to an external solute and monitor the diffusion of the molecules. The degree of cartilage damage (i.e. concentration and configuration of essential macromolecules) is associated with collisional energy loss of external solutes while moving across articular cartilage creates different diffusion characteristics compared to healthy cartilage. In this study, we introduce a protocol, which consists of several steps and is based on previously developed experimental micro-Computed Tomography (micro-CT) and finite element modeling. The transport of charged and uncharged iodinated molecules is first recorded using micro-CT, which is followed by applying biphasic-solute and multiphasic finite element models to obtain diffusion coefficients and fixed charge densities across cartilage zones.

We propose a protocol to investigate the transport of charged and uncharged molecules across articular cartilage with the aid of recently developed experimental and numerical methods.

Eine experimentelle und Finite-Elemente-Protokoll den Transport von neutralen und geladenen gelösten Stoffen durch Gelenkknorpel zur Untersuchung

Osteoarthritis (OA) is a debilitating disease that is associated with degeneration of articular cartilage and subchondral bone. Degeneration of articular ...

3D Magnetic Stem Cell Aggregation and Bioreactor Maturation for Cartilage Regeneration

Cartilage engineering remains a challenge due to the difficulties in creating an in vitro functional implant similar to the native tissue. An approach recently explored for the development of autologous replacements involves the differentiation of stem cells into chondrocytes. To initiate this chondrogenesis, a degree of compaction of the stem cells is required; hence, we demonstrated the feasibility of magnetically condensing cells, both within thick scaffolds and scaffold-free, using miniaturized magnetic field sources as cell attractors. This magnetic approach was also used to guide aggregate fusion and to build scaffold-free, organized, three-dimensional (3D) tissues several millimeters in size. In addition to having an enhanced size, the tissue formed by magnetic-driven fusion presented a significant increase in the expression of collagen II, and a similar trend was observed for aggrecan expression. As the native cartilage was subjected to forces that influenced its 3D structure, dynamic maturation was also performed. A bioreactor that provides mechanical stimuli was used to culture the magnetically seeded scaffolds over a 21-day period. Bioreactor maturation largely improved chondrogenesis into the cellularized scaffolds; the extracellular matrix obtained under these conditions was rich in collagen II and aggrecan. This work outlines the innovative potential of magnetic condensation of labeled stem cells and dynamic maturation in a bioreactor for improved chondrogenic differentiation, both scaffold-free and within polysaccharide scaffolds.

Chondrogenesis from stem cells requires fine tuning the culture conditions. Here, we present a magnetic approach for condensing cells, an essential step to initiate chondrogenesis. In addition, we show that dynamic maturation in a bioreactor applies mechanical stimulation to the cellular constructs and enhances cartilaginous extracellular matrix production.

3D Magnetic Stem Cell Aggregation und Bioreactor Ausreifung für Knorpelregeneration

Cartilage engineering remains a challenge due to the difficulties in creating an in vitro functional implant similar to the native tissue. An approach ...

Bioprinting of Cartilage and Skin Tissue Analogs Utilizing a Novel Passive Mixing Unit Technique for Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both cartilage and skin analogs were fabricated after bioink pre-cellularization utilizing a novel passive mixing unit technique. This technique was developed with the aim to simplify the steps involved in the mixing of a cell suspension into a highly viscous bioink. The resolution of filaments deposited through bioprinting necessitates the assurance of uniformity in cell distribution prior to printing to avoid the deposition of regions without cells or retention of large cell clumps that can clog the needle. We demonstrate the ability to rapidly blend a cell suspension with a bioink prior to bioprinting of both cartilage and skin analogs. Both tissue analogs could be cultured for up to 4 weeks. Histological analysis demonstrated both cell viability and deposition of tissue specific extracellular matrix (ECM) markers such as glycosaminoglycans (GAGs) and collagen I respectively.

Cartilage and skin analogs were bioprinted using a nanocellulose-alginate based bioink. The bioinks were cellularized prior to printing via a single step passive mixing unit. The constructs were demonstrated to be uniformly cellularized, have high viability, and exhibit favorable markers of differentiation.

Bioprinting von Knorpel und Haut-Gewebe-Analoga nutzen eine neuartige Passive Einheit Mischtechnik für Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both ...

Real-time Visualization and Analysis of Chondrocyte Injury Due to Mechanical Loading in Fully Intact Murine Cartilage Explants

Homeostasis of articular cartilage depends on the viability of resident cells (chondrocytes). Unfortunately, mechanical trauma can induce widespread chondrocyte death, potentially leading to irreversible breakdown of the joint and the onset of osteoarthritis. Additionally, maintenance of chondrocyte viability is important in osteochondral graft procedures for optimal surgical outcomes. We present a method to assess the spatial extent of cell injury/death on the articular surface of intact murine synovial joints after application of controlled mechanical loads or impacts. This method can be used in comparative studies to investigate the effects of different mechanical loading regimens, different environmental conditions or genetic manipulations, as well as different stages of cartilage degeneration on short- and/or long-term vulnerability of in situ articular chondrocytes. The goal of the protocol introduced in the manuscript is to assess the spatial extent of cell injury/death on the articular surface of murine synovial joints. Importantly, this method enables testing on fully intact cartilage without compromising native boundary conditions. Moreover, it allows for real-time visualization of vitally stained articular chondrocytes and single image-based analysis of cell injury induced by application of controlled static and impact loading regimens. Our representative results demonstrate that in healthy cartilage explants, the spatial extent of cell injury depends sensitively on load magnitude and impact intensity. Our method can be easily adapted to investigate the effects of different mechanical loading regimens, different environmental conditions or different genetic manipulations on the mechanical vulnerability of in situ articular chondrocytes.

We present a method to assess the spatial extent of cell injury/death on the articular surface of intact murine joints after application of controlled mechanical loads or impacts. This method can be used to investigate how osteoarthritis, genetic factors and/or different loading regimens affect the vulnerability of in situ chondrocytes.

Echtzeit-Visualisierung und Analyse der Knorpelzelltransplantation Verletzungen aufgrund der mechanischen Belastung in völlig intakt murinen Knorpel Explantaten

Homeostasis of articular cartilage depends on the viability of resident cells (chondrocytes). Unfortunately, mechanical trauma can induce widespread ...

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.

Herstellung von Decellularized Knorpel abgeleitet Matrix Gerüste

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

3D Printed Porous Cellulose Nanocomposite Hydrogel Scaffolds

This work demonstrates the use of three-dimensional (3D) printing to produce porous cubic scaffolds using cellulose nanocomposite hydrogel ink, with controlled pore structure and mechanical properties. Cellulose nanocrystals (CNCs, 69.62 wt%) based hydrogel ink with matrix (sodium alginate and gelatin) was developed and 3D printed into scaffolds with uniform and gradient pore structure (110-1,100 &#181;m). The scaffolds showed compression modulus in the range of 0.20-0.45 MPa when tested in simulated in vivo conditions (in distilled water at 37 &#176;C). The pore sizes and the compression modulus of the 3D scaffolds matched with the requirements needed for cartilage regeneration applications. This work demonstrates that the consistency of the ink can be controlled by the concentration of the precursors and porosity can be controlled by the 3D printing process and both of these factors in return defines the mechanical properties of the 3D printed porous hydrogel scaffold. This process method can therefore be used to fabricate structurally and compositionally customized scaffolds according to the specific needs of patients.&#160;

The three critical steps of this protocol are i) developing the right composition and consistency of the cellulose hydrogel ink, ii) 3D printing of scaffolds into various pore structures with good shape fidelity and dimensions and iii) demonstration of the mechanical properties in simulated body conditions for cartilage regeneration.

3D-Druck poröse Zellulose Nanocomposites Hydrogel Gerüste

This work demonstrates the use of three-dimensional (3D) printing to produce porous cubic scaffolds using cellulose nanocomposite hydrogel ink, with ...