Name: 3d hydrogel scaffolds for articular chondrocyte culture and cartilage generation
Uploaded: 2015-10-07T14:00:00.000+00:00
Duration: 12 min 37 s
Description: Watch this Scientific Journal Video about 3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation at JoVE.com

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><tbody><tr><td>juvenile chondrocytes (Clonetics Normal Human Chondrocyte Cell System )</td><td>Lonza</td><td>CC-2550</td><td></td></tr><tr><td>adult chondrocytes (Clonetics 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&amp;nbsp;</td><td>2959</td><td></td></tr><tr><td>sodium chloride&amp;nbsp;</td><td>Sigma</td><td>S9888</td><td></td></tr><tr><td>chondroitin sulfate sodium salt&amp;nbsp;</td><td>Sigma</td><td>C9819</td><td></td></tr><tr><td>N-hydroxysuccinimide&amp;nbsp;</td><td>Sigma</td><td>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>516155</td><td></td></tr><tr><td>dialysis tubing</td><td>Spectrum Laboratories&amp;nbsp;</td><td>132700</td><td></td></tr><tr><td>Collagenase 2</td><td>Worthington Biochemical&amp;nbsp;</td><td>LS004177</td><td></td></tr><tr><td>Collagenase 4</td><td>Worthington Biochemical&amp;nbsp;</td><td>LS004189</td><td></td></tr><tr><td>DMEM/F12 media</td><td>HyClone, Thermo Scientific&amp;nbsp;</td><td>SH3002301&amp;nbsp;</td><td></td></tr><tr><td>live/dead assay</td><td>Life Technologies</td><td>&lt;a href=&quot;https://www.lifetechnologies.com/order/catalog/product/L3224&quot;&gt;L3224&lt;/a&gt;</td><td></td></tr><tr><td>Tri reagent</td><td>Life Technologies</td><td>AM9738</td><td></td></tr><tr><td>Quant-iT PicoGreen dsDNA Assay Kit</td><td>Invitrogen</td><td>&lt;a href=&quot;https://www.lifetechnologies.com/order/catalog/product/P11496&quot;&gt;P11496&lt;/a&gt;</td><td></td></tr><tr><td>Sodium phosphate dibasic&amp;nbsp;</td><td>Sigma</td><td>S3264</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid disodium salt&amp;nbsp;</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&amp;nbsp;</td><td>Sigma</td><td>341088</td><td></td></tr><tr><td>Instruments</td><td></td><td></td><td></td></tr><tr><td>UV light equipment - XX-15LW Bench Lamp, 365 nm</td><td>UVP</td><td>95-0042-07</td><td></td></tr><tr><td>Instron 5944 testing system&amp;nbsp;</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

19.9K 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

Preparation of Chitosan-based Injectable Hydrogels and Its Application in 3D Cell Culture

The protocol presents a facile, efficient, and versatile method to prepare chitosan-based hydrogels using dynamic imine chemistry. The hydrogel is prepared by mixing solutions of glycol chitosan with a synthesized benzaldehyde terminated polymer gelator, and hydrogels are efficiently obtained in several minutes at room temperature. By varying ratios between glycol chitosan, polymer gelator, and water contents, versatile hydrogels with different gelation times and stiffness are obtained. When damaged, the hydrogel can recover its appearances and modulus, due to the reversibility of the dynamic imine bonds as crosslinkages. This self-healable property enables the hydrogel to be injectable since it can be self-healed from squeezed pieces to an integral bulk hydrogel after the injection process. The hydrogel is also multi-responsive to many bio-active stimuli due to different equilibration statuses of the dynamic imine bonds. This hydrogel was confirmed as bio-compatible, and L929 mouse fibroblast cells were embedded following standard procedures and the cell proliferation was easily assessed by a 3D cell cultivation process. The hydrogel can offer an adjustable platform for different research where a physiological mimic of a 3D environment for cells is profited. Along with its multi-responsive, self-healable, and injectable properties, the hydrogels can potentially be applied as multiple carriers for drugs and cells in future bio-medical applications.

Here we describe a facile preparation of chitosan-based injectable hydrogels using dynamic imine chemistry. Methods to adjust the hydrogel&#8217;s mechanical strength and its application in 3D cell culture are presented.

Vorbereitung der Chitosan-basierten injizierbaren Hydrogele und deren Anwendung in 3D Zellkultur

The protocol presents a facile, efficient, and versatile method to prepare chitosan-based hydrogels using dynamic imine chemistry. The hydrogel is ...

Forschung

Chemie

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and regenerative medicine. With digital control&#160;cells, scaffolds, and growth factors can be precisely deposited to the desired two-dimensional (2D) and three-dimensional (3D) locations rapidly. Therefore, this technology is an ideal approach to fabricate tissues mimicking their native anatomic structures. In order to engineer cartilage with native zonal organization, extracellular matrix composition (ECM), and mechanical properties, we developed a bioprinting platform using a commercial inkjet printer with simultaneous photopolymerization capable for 3D cartilage tissue engineering. Human chondrocytes suspended in poly(ethylene glycol) diacrylate (PEGDA) were printed for 3D neocartilage construction via layer-by-layer assembly. The printed cells were fixed at their original deposited positions, supported by the surrounding scaffold in simultaneous photopolymerization. The mechanical properties of the printed tissue were similar to the native cartilage. Compared to conventional tissue fabrication, which requires longer UV exposure, the viability of the printed cells with simultaneous photopolymerization was significantly higher. Printed neocartilage demonstrated excellent glycosaminoglycan (GAG) and collagen type II production, which was consistent with gene expression. Therefore, this platform is ideal for accurate cell distribution and arrangement for anatomic tissue engineering.

The methods described in this paper show how to convert a commercial inkjet printer into a bioprinter with simultaneous UV polymerization. The printer is capable of constructing 3D tissue structure with cells and biomaterials. The study demonstrated here constructed a 3D neocartilage.

Menschliches Knorpelgewebe Herstellung unter Verwendung von dreidimensionalen Inkjet-Drucktechnologie

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and ...

Biotechnik

Layered Alginate Constructs: A Platform for Co-culture of Heterogeneous Cell Populations

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential mechanosensing characteristics. Engineering and analysis of these tissue types remain a challenge. Layered hydrogel constructs provide an opportunity for investigating the interactions among multiple cell populations within single constructs. Alginate hydrogels are both biocompatible and allow for easy isolation of cells after experimentation. Here, we describe a method for the development of small sized dual layered alginate hydrogel discs. This process maintains high cell viability of human mesenchymal stem cells during the formation process and these layered discs can withstand unconfined cyclic compression, commonly used for stimulation of hMSCs undergoing chondrogenesis. These layered constructs can potentially be scaled up to include additional levels, and also be used to segregate cell populations initially after layering. This dual layer alginate hydrogel culture platform can be used for many different applications including engineering and analysis of cells of load bearing tissues and co-cultures of other cell types.

Engineering and analysis of load bearing tissues with heterogeneous cell populations are still a challenge. Here, we describe a method for creating bi-layered alginate hydrogel discs as a platform for co-culture of diverse cell populations within one construct.

Layered Alginate Konstrukten: Eine Plattform für Co-Kultur von heterogenen Zellpopulationen

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential ...

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 ...

Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an environment that closely mimics the structural features of non-polarized tissue. Furthermore, the particular intrinsic nanofiber structure of the scaffold makes it transparent to visual light, which allows for easy visualization of the sample under microscopy. This advantage was largely used to study cell migration, organization, proliferation, and differentiation and thus any development of their particular cellular function by staining with specific dyes or probes. Furthermore, in this work, we describe the good performance of this system to easily study the redifferentiation of expanded human articular chondrocytes into cartilaginous tissue. Cells were encapsulated into self-assembling peptide scaffolds and cultured under specific conditions to promote chondrogenesis. Three-dimensional cultures showed good viability during the 4 weeks of the experiment. As expected, samples cultured with chondrogenic inducers (compared to non-induced controls) stained strongly positive for toluidine blue (which stains glycosaminoglycans (GAGs) that are highly present in cartilage extracellular matrix) and expressed specific molecular markers, including collagen type I, II and X, according to Western Blot analysis. This protocol is easy to perform and can be used at research laboratories, industries and for educational purposes in laboratory courses.

Here, we present a protocol to obtain 3D-culture systems in self-assembling peptide scaffolds to promote the differentiation of dedifferentiated human articular chondrocytes into cartilage-like tissue.

Kultivierung von Säugerzellen in dreidimensionale Peptid Gerüste

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an ...

A 3D System for Culturing Human Articular Chondrocytes in Synovial Fluid

Cartilage destruction is a central pathological feature of osteoarthritis, a leading cause of disability in the US. Cartilage in the adult does not regenerate very efficiently in vivo; and as a result, osteoarthritis leads to irreversible cartilage loss and is accompanied by chronic pain and immobility 1,2. Cartilage tissue engineering offers promising potential to regenerate and restore tissue function. This technology typically involves seeding chondrocytes into natural or synthetic scaffolds and culturing the resulting 3D construct in a balanced medium over a period of time with a goal of engineering a biochemically and biomechanically mature tissue that can be transplanted into a defect site in vivo 3-6. Achieving an optimal condition for chondrocyte growth and matrix deposition is essential for the success of cartilage tissue engineering. 
 In the native joint cavity, cartilage at the articular surface of the bone is bathed in synovial fluid. This clear and viscous fluid provides nutrients to the avascular articular cartilage and contains growth factors, cytokines and enzymes that are important for chondrocyte metabolism 7,8. Furthermore, synovial fluid facilitates low-friction movement between cartilaginous surfaces mainly through secreting two key components, hyaluronan and lubricin 9 10. In contrast, tissue engineered cartilage is most often cultured in artificial media. While these media are likely able to provide more defined conditions for studying chondrocyte metabolism, synovial fluid most accurately reflects the natural environment of which articular chondrocytes reside in.
 Indeed, synovial fluid has the advantage of being easy to obtain and store, and can often be regularly replenished by the body. Several groups have supplemented the culture medium with synovial fluid in growing human, bovine, rabbit and dog chondrocytes, but mostly used only low levels of synovial fluid (below 20&#37;) 11-25. While chicken, horse and human chondrocytes have been cultured in the medium with higher percentage of synovial fluid, these culture systems were two-dimensional 26-28. Here we present our method of culturing human articular chondrocytes in a 3D system with a high percentage of synovial fluid (up to 100&#37;) over a period of 21 days. In doing so, we overcame a major hurdle presented by the high viscosity of the synovial fluid. This system provides the possibility of studying human chondrocytes in synovial fluid in a 3D setting, which can be further combined with two other important factors (oxygen tension and mechanical loading) 29,30 that constitute the natural environment for cartilage to mimic the natural milieu for cartilage growth. Furthermore, This system may also be used for assaying synovial fluid activity on chondrocytes and provide a platform for developing cartilage regeneration technologies and therapeutic options for arthritis.

A 3D system of culturing human articular chondrocytes in high levels of synovial fluid is described. Synovial fluid reflects the most natural microenvironment for articular cartilage, and can be easily obtained and stored. This system thus can be used for studying cartilage regeneration and for screening therapeutics for treating arthritis.

Ein 3D-System für Züchtung menschlichen Chondrozyten in der Synovialflüssigkeit

Cartilage destruction is a central pathological feature of osteoarthritis, a leading cause of disability in the US. Cartilage in the adult does not ...

Biologie

Mechanical Stimulation of Chondrocyte-agarose Hydrogels

Articular cartilage suffers from a limited repair capacity when damaged by mechanical insult or degraded by disease, such as osteoarthritis. To remedy this deficiency, several medical interventions have been developed. One such method is to resurface the damaged area with tissue-engineered cartilage; however, the engineered tissue typically lacks the biochemical properties and durability of native cartilage, questioning its long-term survivability. This limits the application of cartilage tissue engineering to the repair of small focal defects, relying on the surrounding tissue to protect the implanted material. To improve the properties of the developed tissue, mechanical stimulation is a popular method utilized to enhance the synthesis of cartilaginous extracellular matrix as well as the resultant mechanical properties of the engineered tissue. Mechanical stimulation applies forces to the tissue constructs analogous to those experienced in vivo. This is based on the premise that the mechanical environment, in part, regulates the development and maintenance of native tissue1,2. The most commonly applied form of mechanical stimulation in cartilage tissue engineering is dynamic compression at physiologic strains of approximately 5-20% at a frequency of 1 Hz1,3. Several studies have investigated the effects of dynamic compression and have shown it to have a positive effect on chondrocyte metabolism and biosynthesis, ultimately affecting the functional properties of the developed tissue4-8. In this paper, we illustrate the method to mechanically stimulate chondrocyte-agarose hydrogel constructs under dynamic compression and analyze changes in biosynthesis through biochemical and radioisotope assays. This method can also be readily modified to assess any potentially induced changes in cellular response as a result of mechanical stimuli.

The biosynthesis of cartilaginous extracellular matrix by chondrocytes can be affected by application of mechanical stimuli. This method describes the technique of applying dynamic compressive strains to chondrocytes encapsulated in 3D constructs and the evaluation of induced changes in chondrocyte metabolism.

Mechanische Stimulation der Chondrozyten-Agarose Hydrogele

Articular cartilage suffers from a limited repair capacity when damaged by mechanical insult or degraded by disease, such as osteoarthritis. To remedy ...

Creation of a Knee Joint-on-a-Chip for Modeling Joint Diseases and Testing Drugs

The high prevalence of debilitating joint diseases like osteoarthritis (OA) poses a high socioeconomic burden. Currently, the available drugs that target joint disorders are mostly palliative. The unmet need for effective disease-modifying OA drugs (DMOADs) has been primarily caused by the absence of appropriate models for studying the disease mechanisms and testing potential DMOADs. Herein, we describe the establishment of a miniature synovial joint-mimicking microphysiological system (miniJoint) comprising adipose, fibrous, and osteochondral tissue components derived from human mesenchymal stem cells (MSCs). To obtain the three-dimensional (3D) microtissues, MSCs were encapsulated in photocrosslinkable methacrylated gelatin before or following differentiation. The cell-laden tissue constructs were then integrated into a 3D-printed bioreactor, forming the miniJoint. Separate flows of osteogenic, fibrogenic, and adipogenic media were introduced to maintain the respective tissue phenotypes. A commonly shared stream was perfused through the cartilage, synovial, and adipose tissues to enable tissue crosstalk. This flow pattern allows the induction of perturbations in one or more of the tissue components for mechanistic studies. Furthermore, potential DMOADs can be tested via either &#34;systemic administration&#34; through all the medium streams or &#34;intraarticular administration&#34; by adding the drugs to only the shared &#34;synovial fluid&#34;-simulating flow. Thus, the miniJoint can serve as a versatile in vitro platform for efficiently studying disease mechanisms and testing drugs in personalized medicine.

We provide detailed methods for generating four types of tissues from human mesenchymal stem cells, which are used to recapitulate the cartilage, bone, fat pad, and synovium in the human knee joint. These four tissues are integrated into a customized bioreactor and connected through microfluidics, thus generating a knee joint-on-a-chip.

Erstellung eines Kniegelenk-on-a-Chips zur Modellierung von Gelenkerkrankungen und zur Erprobung von Medikamenten

The high prevalence of debilitating joint diseases like osteoarthritis (OA) poses a high socioeconomic burden. Currently, the available drugs that target ...

Synthesis of Decellularized Cartilage Extracellular Matrix Hydrogels

Decellularized cartilage extracellular matrix (DC-ECM) hydrogels are promising biomaterials for tissue engineering and regenerative medicine due to their biocompatibility and ability to mimic natural tissue properties. This protocol aims to produce DC-ECM hydrogels that closely mimic the native ECM of cartilage tissue. The protocol involves a combination of physical and chemical disruption and enzymatic digestion to remove the cellular material while preserving the structure and composition of the ECM. The DC-ECM is cross-linked using a chemical agent to form a stable and biologically active hydrogel. The DC-ECM hydrogel has excellent biological activity, spatial structure, and biological induction function, as well as low immunogenicity. These characteristics are beneficial in promoting cell adhesion, proliferation, differentiation, and migration and for creating a superior microenvironment for cell growth. This protocol provides a valuable resource for researchers and clinicians in the field of tissue engineering. Biomimetic hydrogels can potentially enhance the development of effective tissue engineering strategies for cartilage repair and regeneration.

This paper introduces a new method for the synthesis of decellularized cartilage extracellular matrix (DC-ECM) hydrogels. DC-ECM hydrogels have excellent biocompatibility and provide a superior microenvironment for cell growth. Therefore, they can be ideal cell scaffolds and biological delivery systems.

Synthese von Hydrogelen aus dezellularisiertem Knorpel mit extrazellulärer Matrix

Decellularized cartilage extracellular matrix (DC-ECM) hydrogels are promising biomaterials for tissue engineering and regenerative medicine due to their ...

Generating Spheroids from Various Chondrocytes using Low-Adhesive Conditions under Gravity and Homemade Mini-Bioreactors

Cartilage repair in chronic joint diseases demands advanced cell-based therapies to regenerate damaged tissues effectively. This protocol provides a step-by-step method for differentiating induced pluripotent stem cells (iPSCs) into chondrocyte-based spheroids, supporting tissue engineering and cell therapy applications. The differentiation process is carefully structured to promote chondrogenic lineage commitment, beginning with iPSCs cultured in specific media that sequentially guide cells through critical stages of differentiation. Initially, iPSCs are expanded to reach optimal confluency before induction toward chondrogenic lineage using a series of defined media changes. By day 10, cells are transitioned to a chondrogenesis-promoting medium that enhances the formation of chondrocyte-like cells expressing key markers of mature chondrocytes. Further aggregation in 96-well agarose-coated plates leads to the formation of three-dimensional spheroids, which are then cultured in custom mini-bioreactors designed to simulate a microenvironment that encourages extracellular matrix (ECM) deposition. By enabling scalable production of chondrocyte spheroids that mimic native cartilage characteristics, this approach offers a promising, reproducible solution for developing cell-based treatments for cartilage defects, providing broad utility for clinical and research applications in musculoskeletal regenerative medicine.

This study describes a method for producing chondrocytic spheroids by aggregating cells into spheroids under low-adhesion conditions using gravity, followed by culturing the resulting spheroids in mini-bioreactors.

Erzeugung von Sphäroiden aus verschiedenen Chondrozyten unter Verwendung von niedrigadhäsiven Bedingungen unter Schwerkraft und selbstgemachten Mini-Bioreaktoren

Cartilage repair in chronic joint diseases demands advanced cell-based therapies to regenerate damaged tissues effectively. This protocol provides a ...