Ein. Isolation of Primary Chondrozyten Ernte 10-15 volle Dicke Knorpel Scheiben von den Gelenkflächen des tierischen Gelenken (zB das Mittelhandknochen-Phalangealgelenk von ausgewachsenem Skelett Kühen von einem lokalen Schlachthof gewonnen). <ol><li> Ort Knorpel Scheiben in einer 100 mm Petrischale und Inkubieren in 20 ml 0,5% Protease in Ham-F-12 (w / v) für 2 Stunden bei 37 ° C. Spülen Sie drei Mal in Ham-F-12 Kulturmedien und inkubieren mit 20 ml 0,15% Collagenase A in Hams F-12 Kulturmedien über Nacht bei 37 ° C. </li><li> Filtern der Zellsuspension durch ein 200 mesh-Sieb-Filter in einen sauberen Teller. Waschen Sie den Filter mit 5 ml Ham F-12 und zu dem Gericht. Übertragen der Zellsuspension in ein 50 ml konischen Röhrchen. Waschen Sie die Petrischale mit 10 ml Ham F-12 und zu dem konischen Rohr. </li><li> Zentrifugieren bei 600-800 rcf für 6-8 min bei Raumtemperatur. </li><li> Überstand, wodurch nicht th störene Zellpellet und in 40 ml Ham-F-12 Kulturmedium erneut zu suspendieren. </li><li> Zentrifugieren bei 600-800 rcf für 6-8 min. </li><li> Wiederholen Schritte 1.4 und 1.5 noch zweimal mit insgesamt 3 mal. Vor dem Zentrifugieren zum dritten Mal, das Pellet über Agitation und erhalten eine 500 ul Aliquot zur Zellzählung. </li><li> Zählen lebensfähigen Zellen unter Verwendung des Trypan-Blau-Ausschluss-Methode 9 mit einem Hämozytometer und eines invertierten Lichtmikroskops. </li><li> Überstand und resuspendieren des Pellets in einem Volumen von Ham-F-12 Medium, um den gewünschten Einsaatdichte verdoppeln (z. B. 20 x 10 6 Zellen / ml für eine Endkonzentration von 10 x 10 6 Zellen / ml nach der Verkapselung). </li></ol> 2. Chondrozyten-Agarose Hydrogel Encapsulation <ol><li> Wärme 0,8 g autoklavierten Typ VII Agarose in 20 ml sterilem 1x PBS (pH 7,4) auf einer Heizplatte bei 120 ° C bis zur Auflösung. Einmal gelöst, die Hitze auf 60 ° C höhere Temperaturen als will beeinträchtigen die Lebensfähigkeit der Zellen. </li><li> In einem 50 ml konischen Röhrchen wird gründlich gemischt gleichen Volumina der Zellsuspension mit der doppelten gewünschten Konzentration aus Agarose (z. B. 4% Agarose Suspension für eine abschließende 2% Agarose Hydrogel). Vermeiden Sie die Erstellung von großen Blasen, wie sie die Lebensfähigkeit der Zellen beeinflussen können und bauen Lebensdauer. </li><li> Sorgfältig Aliquot von 10 ml der Chondrozyten-Agarose Mischung in eine 60 mm Petrischale Durchmesser, unter Vermeidung der Bildung großer Blasen. </li><li> Lassen Sie stehen für 30 min bei Raumtemperatur bis geliert. </li><li> Zu erhalten, wie viele Chondrozyten-Agarose Hydrogel Proben nach Bedarf (typischerweise 20-30) mit einem 4 mm Biopsiestanze. </li><li> Messung und Aufzeichnung der physikalischen Abmessungen (Durchmesser und Höhe) von repräsentativen Proben mit einem Mikrometer. Konstrukte sollte etwa 5 mm betragen in der Höhe, verwerfen Proben aus einem Bereich, wo der Probenhöhe Varianz größer als 2% erhalten. </li><li> Übertragen Proben zu einem neuen 60 mm Petrischale und Ergänzungmit 10 ml komplettem Medium (z. B. 20% fötalem Rinderserum in Ham-F-12 Kulturmedium mit 20 mM HEPES, 100 ug / ml Ascorbinsäure und 2x Antibiotika / Antimykotika). </li></ol> 3. Mechanische Stimulation und Radiomarkierung von Chondrozyten-Agarose Hydrogele <ol><li> Autoklaven werden die metallischen Komponenten des Kompressions-rig. Sterilisieren Sie die Kunststoffteile mit 70% Ethanol (über Nacht) und UV-Licht (mindestens 15 min). </li><li> Konstrukte Umfallen, mit Pinzette Platz 12 sterile Plastik Halteringe (mit einem Innendurchmesser größer als der Durchmesser Konstrukt) (n = 6, Proben und Kontrollen) innerhalb der Vertiefungen einer 24-Well-Kulturplatte zu halten. </li><li> Mit einer Pinzette, transferieren es vorsichtig die Chondrozyten-Agarose Hydrogele aus der Petrischale auf den 24-Well-Platte, die Sicherung jedes in einem Haltering. </li><li> Übertragen 400 ul komplette Medien (zB 20% FBS in Ham-F-12 Kulturmedium mit 20 mM HEPES, 100 ug / ml Ascorbinsäure und2x Antibiotika / Antimykotika) zu jedem Probenbehälter. </li><li> Montieren Sie die sterilisiert Kompression rig (Abbildung 1) und befestigen Sie die Platten mit Stellschrauben. Bringen Sie die Kompression rig auf die 24-well Platte. </li><li> Lösen und wieder sichern die Stellschrauben Platte-Hydrogel-Kontaktlinsen (spannungslosen Zustand) zu etablieren. </li><li> Legen Sie die zusammengebauten Kompression rig in eine Mach-1 Mikromechanische Testing System. Sichere rig auf die vertikale Stufe über Fixierstift und einrasten mit Stellschraube. Entfernen Sie den Abstand jig. </li><li> Anwenden dynamischen Druckbelastung an gewünschten Amplitude (z. B. 10% Dehnung Amplitude auf der ursprünglichen Höhe der Proben basierend), Frequenz (zB 1 Hz) und Dauer oder Anzahl der Zyklen (zB Zeitintervallen bis zu 60 min). </li><li> Nach Abschluss der mechanischen Stimulation, ersetzen Sie den Abstand jig, lösen Sie die Fixierstift Stellschraube und entfernen Sie die Kompression rig und Kultur Platte aus dem Mach-1 Mikromechanische Testing System. </li&gt; <li> Radiomarkierung Zell-Hydrogel-Konstrukte durch Ergänzung der Medien mit 5 uCi gewünschte Isotop (zB [3 H]-Prolin-Protein-Label für Chondrozyten-spezifische Kollagen-Synthese und [35 S]-Schwefel für Proteoglykan-Synthese). </li><li> Inkubieren der radiomarkierten Konstrukten für 24 h bei 37 ° C, 5% CO 2 10. </li><li> Ernten Sie die radioaktiv markierten Hydrogel Konstrukten und spülen 3 mal in 1x PBS (pH 7,4) auf eine nicht rechtsfähige Isotop entfernen. Ausgewählte Proben unter Verwendung von Papain (40 ug / ml in 20 mM Ammoniumacetat, 1 mM ethyldiaminetetraacetic Säure und 2 mM Dithiothreitol bei 65 ° C für 72 h). </li><li> Assay Digest für DNA-Gehalt mit dem PicoGreen Farbstoff Assay 11 und eingebauten Isotops Aktivität, normiert auf DNA-Gehalt von β-Flüssig-Szintillationszählung 12,13 unmittelbar folgenden Papainverdau. </li></ol> Hinweis: Der Kauf und die Entsorgung von radioaktiven Materialien (Abfälle Isotope und Artikeldie möglicherweise in Kontakt radioaktiver Stoffe gekommen sind) müssen den einschlägigen institutionellen (und / oder staatlichen) Vorschriften und Verfahren für die sichere Handhabung und Entsorgung von radioaktiven Substanzen. 4. Repräsentative Ergebnisse Bovine Gelenkchondrozytenprobe-Agarose Hydrogele Konstrukte (2% Agarose mit 10 x 10 6 Zellen / ml eingekapselten Zellen) wurden mechanisch mit einer Amplitude von 10% Stauchung bei einer Frequenz von 1 Hz für 20 bis 60 min (oder 1.200 bis 3.600 Zyklen angeregt ) und im Hinblick auf DNA-Gehalt und extrazellulären Matrix-Synthese durch Einbau Radioisotop. Und extrazellulären Matrix-DNA-Synthese wurde in einer Dosis-abhängigen Weise beeinflusst. DNA-Gehalt zeigte eine signifikante Abnahme von 35% als Ergebnis von 20 oder 30 min der Stimulation (p &lt;0,01), während 60 min Stimulation zeigte keinen Effekt (Abb. 2). Knorpel-spezifische Kollagen und Proteoglykan-Synthese (bestimmt durch [3 H]-Prolin und[35 S]-Schwefel Einarbeitung jeweils) zeigten signifikante Anstiege von etwa 60% in Reaktion auf 20 bis 30 min der Stimulation (p &lt;0,01), 60 min mit der Stimulation aufweist keinen beobachtbaren Effekt (Abb. 3). <img src="/files/ftp_upload/4229/4229fig1.jpg" alt="Abbildung 1" /> . Abbildung 1 Schematische Darstellung des custom-built dynamische Kompression rig zur Stimulierung Chondrozyten-Agarose Konstrukte. Links: Assembled rig. Rechts: Explosionszeichnung zeigt Einzelkomponenten. <img src="/files/ftp_upload/4229/4229fig2.jpg" alt="Abbildung 2" /> Abbildung 2. Veränderungen in der DNA-Gehalt der Chondrozyten-Agarose Hydrogele mechanisch unter einer 10% Stauchung Amplitude bei 1 Hz für 20 bis 60 min (Mittelwert ± SEM, n = 6/Gruppe) stimuliert. Abbildung 3. Veränderungen der extrazellulären Matrixsynthese (Kollagen und Proteoglykane) der Chondrozyten-Agarose Hydrogele mechanisch unter 10% Stauchung Amplitude bei 1 Hz für 20 bis 60 min (Mittelwert ± SEM, n = 6/Gruppe) stimuliert.

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<html> Das beschriebene Verfahren für das Auftragen kontrollierter mechanische Reize auf Zellen seeded Agarose Hydrogele ermöglicht die direkte Untersuchung der Auswirkungen von dynamischen Druckkräfte auf Chondrozytenmetabolismus. Die Verwendung des kundenspezifischen Prüfstand in Verbindung mit den Halteringen versehen seitliche Beschränkung für die Konstrukte, um mögliche Probleme der Probe Kippen zu vermeiden. Die Verwendung von dead-gewichtete Belastung Platten durch Mengen Crews gesichert gewährleistet den direkten Kontakt mit Konstrukten trotz möglicher Unterschiede in der Probe Höhe. Radiomarkieren post-Stimulation zu messen zelluläre Biosynthese vermindert die Gefahr einer Kontamination, so dass für einen einzigen Prüfstand für mehrere Anwendungen von dynamischen mechanische Reize verwendet werden. Dieses Verfahren kann leicht angepasst werden, um die Wirkung verschiedener mechanischer Belastung Modi (zB 1,4 Scher-, Zug 1,2) zu untersuchen oder zu erläutern spezifische Mechanotransduktion Signalwege verantwortlich. ove_content &quot;&gt; Die repräsentativen Ergebnisse dargestellt, dass eine geringe Menge an Zelltod von etwa 35% können durch die Anwendung der dynamische Stimulation, mit längeren Anwendungen der mechanische Stimulation nicht beeinflussen Konstrukt Zellularität 15-17. Biosynthese von extrazellulären Matrix Makromoleküle, bestimmt durch Radioisotop Einarbeitung veranschaulicht eine Dauer abhängigen Reaktion auf dynamische Kompression. Anwendungen der dynamischen Druckbelastung während eines Zeitraums von 20 bis 30 min resultierte ein maximaler anabolische Reaktion mit längeren Laufzeiten auftreten, um eine Desensibilisierung der auferlegten Effekt mechanische Reize auszulösen. Cellular Desensibilisierung gegen mechanische Beladung zuvor 1,2,5,13 stellte aber es gab wenig Untersuchung Charakterisierung des zeitabhängigen Natur dieser Phänomene. Diese Informationen verwendet werden, um die optimalen Bedingungen zu bestimmen Stimulation werden, um die Ansammlung von extrazellulärer Matrix mit der Maximierung Bau under wiederholt, oder langfristige, Anwendungen mechanischer Stimulation.

<table border="1"><tbody><tr><td> Name des Reagens oder Ausrüstung </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare (optional) </td></tr><tr><td> Ham-F-12 </td><td> Thermo Fisher Scientific </td><td> SH3001002 </td><td></td></tr><tr><td> Collagenase A </td><td> Sigma Aldrich Ltd </td><td> C0130 </td><td></td></tr><tr><td> Protease </td><td> Sigma Aldrich Ltd </td><td> P5147 </td><td></td></tr><tr><td> Fötales Rinderserum </td><td> Sigma Aldrich Ltd </td><td> F1051 </td><td></td></tr><tr><td> Ascorbat </td><td> Sigma Aldrich Ltd </td><td> A4034 </td><td></td></tr><tr><td> Antibiotika / Antimykotika </td><td> Sigma Aldrich Ltd </td><td> A5955 </td><td></td></tr><tr><td> HEPES </td><td> Bioshop Canada Ltd </td><td> HEP001 </td><td></td></tr><tr><td> Trypanblau </td><td> Sigma Aldrich Ltd </td><td> 93595 </td><td></td></tr><tr><td> Reichert Hell-Line Hämazytometer </td><td> Hausser Scientific </td><td> 1490 </td><td></td></tr><tr><td> Quant-iT PicoGreen </td><td> Invitrogen </td><td> P7589 </td><td></td></tr><tr><td> Papain aus Papaya Latex </td><td> Sigma Aldrich Ltd </td><td> P3125 </td><td></td></tr><tr><td> Ammoniumacetat </td><td> Sigma Aldrich Ltd </td><td> A1542 </td><td></td></tr><tr><td> Ethyldiaminetetraacetic Säure </td><td> Sigma Aldrich Ltd </td><td> E9884 </td><td></td></tr><tr><td> DL-Dithiothreitol </td><td> Sigma Aldrich Ltd </td><td> 43819 </td><td></td></tr><tr><td> Agarose mit niedrigem Schmelzpunkt, Typ VII </td><td> Sigma Aldrich Ltd </td><td> A9045 </td><td></td></tr> &lt;tr&gt; <td> Mesh-Screen (200) Filter </td><td> Sigma Aldrich Ltd </td><td> S4145 </td><td></td></tr><tr><td> Mach-1 Mikromechanische Tester </td><td> Biomomentum Inc. </td><td> V500cs </td><td></td></tr><tr><td> Compression Loading Jig </td><td> Custom-built </td><td></td><td> Ein Produkt ähnlich könnte Biomomentum Inc. geliefert werden </td></tr><tr><td> Falcon 24 Well Zellkulturplatten </td><td> Thermo Fisher Scientific </td><td> B353047 </td><td></td></tr><tr><td> β-Flüssigkeits-Szintillationszähler </td><td> Beckman Coulter </td><td> LS6500 </td><td></td></tr><tr><td> [3H] Proline </td><td> Perkin-Elmer </td><td> NET323005MC </td><td></td></tr><tr><td> [35S] Sulfur </td><td> Perkin-Elmer </td><td> NEX041005MC </td><td></td></tr></tbody></table>

mechanical stimulation of chondrocyte-agarose hydrogels

Gelenkknorpel leidet unter einer begrenzten Kapazität, wenn Reparatur durch mechanische Insult oder abgebaute durch Krankheiten, wie Osteoarthritis beschädigt. Um diesem Mangel abzuhelfen, haben mehrere medizinische Eingriffe entwickelt. Eine solche Methode ist, um den beschädigten Bereich mit Tissue-Engineering Knorpel wieder auftauchen, aber die gezüchteten Gewebe in der Regel fehlen die biochemischen Eigenschaften und Haltbarkeit von nativen Knorpel Frage der langfristigen Überlebensfähigkeit. Dies beschränkt die Anwendung von Knorpelgewebe Engineering zur Reparatur von kleinen fokalen Fehlern, die sich auf das umgebende Gewebe, um die implantierte Material zu schützen. Um die Eigenschaften des entwickelten Gewebe zu verbessern, ist eine mechanische Stimulation eine beliebte Methode verwendet, um die Synthese von Knorpel extrazellulären Matrix sowie die resultierenden mechanischen Eigenschaften des gezüchteten Gewebe zu verbessern. Mechanische Stimulation gilt Kräfte auf das Gewebe konstruiert analog zu den erfahrensten in vivo. Diesbasiert auf der Prämisse, dass die mechanischen Umgebung, teilweise, reguliert die Entwicklung und Wartung von nativem Gewebe 1,2 basiert. Die am häufigsten angewandte Form der mechanischen Stimulation im Knorpel Tissue engineering ist Dynamikkompression bei physiologischem Stämme von etwa 5-20% bei einer Frequenz von 1 Hz 1,3. Mehrere Studien haben die Auswirkungen der dynamischen Kompression untersucht und haben gezeigt, dass es einen positiven Effekt auf Chondrozytenmetabolismus und Biosynthese haben, letztendlich auch die funktionellen Eigenschaften des entwickelten Gewebe 4-8. In diesem Papier zeigen wir die Methode zur mechanischen Stimulierung Chondrozyten-Agarose Hydrogel Konstrukten unter dynamischer Kompression und Analyse der Veränderungen in der Biosynthese durch biochemische und radioaktiven Assays. Dieses Verfahren kann auch ohne weiteres modifiziert werden, um alle möglicherweise induzierte Veränderungen in zelluläre Reaktion als Folge der mechanischen Reizen zu beurteilen.

Watch this Scientific Journal Video about Mechanical Stimulation of Chondrocyte-agarose Hydrogels at JoVE.com

Mechanical Stimulation of Chondrocyte-agarose Hydrogels

Die Biosynthese von knorpeligen extrazellulären Matrix durch Chondrocyten können durch Einwirkung mechanischer Reize beeinflußt werden. Diese Methode beschreibt die Technik der Anwendung dynamisch Druckspannungen zu Chondrozyten in 3D-Konstrukte und die Auswertung der induzierten Veränderungen in Chondrozytenmetabolismus verkapselt.

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 ...

mechanical-stimulation-of-chondrocyte-agarose-hydrogels

Nanyang Technological University

Research

JoVE Journal

Biology

11.6K Aufrufe.  Queen's University. Die Biosynthese von knorpeligen extrazellulären Matrix durch Chondrocyten können durch Einwirkung mechanischer Reize beeinflußt werden. Diese Methode beschreibt die Technik der Anwendung dynamisch Druckspannungen zu Chondrozyten in 3D-Konstrukte und die Auswertung der induzierten Veränderungen in Chondrozytenmetabolismus verkapselt.

Serie: Mechanical Stimulation of Chondrocyte-agarose Hydrogels

Chondrogenic Differentiation Induction of Adipose-derived Stem Cells by Centrifugal Gravity

Impaired cartilage cannot heal naturally. Currently, the most advanced therapy for defects in cartilage is the transplantation of chondrocytes differentiated from stem cells using cytokines. Unfortunately, cytokine-induced chondrogenic differentiation is costly, time-consuming, and associated with a high risk of contamination during in vitro differentiation. However, biomechanical stimuli also serve as crucial regulatory factors for chondrogenesis. For example, mechanical stress can induce chondrogenic differentiation of stem cells, suggesting a potential therapeutic approach for the repair of impaired cartilage. In this study, we demonstrated that centrifugal gravity (CG, 2,400 &#215; g), a mechanical stress easily applied by centrifugation, induced the upregulation of sex determining region Y (SRY)-box 9 (SOX9) in adipose-derived stem cells (ASCs), causing them to express chondrogenic phenotypes. The centrifuged ASCs expressed higher levels of chondrogenic differentiation markers, such as aggrecan (ACAN), collagen type 2 alpha 1 (COL2A1), and collagen type 1 (COL1), but lower levels of collagen type 10 (COL10), a marker of hypertrophic chondrocytes. In addition, chondrogenic aggregate formation, a prerequisite for chondrogenesis, was observed in centrifuged ASCs.

Mechanical stress can induce the chondrogenic differentiation of stem cells, providing a potential therapeutic approach for the repair of impaired cartilage. We present a protocol to induce the chondrogenic differentiation of adipose-derived stem cells (ASCs) using centrifugal gravity (CG). CG-induced upregulation of SOX9 results in the development of chondrogenic phenotypes.

Chondrogene Differenzierung Induktion von Fettgewebe gewonnene Stammzellen durch Zentrifugalkraft Gravity

Impaired cartilage cannot heal naturally. Currently, the most advanced therapy for defects in cartilage is the transplantation of chondrocytes ...

Forschung

Entwicklungsbiologie

Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

We designed a loading device that is capable of applying uniaxial or biaxial mechanical strain to a tissue engineered biocomposites fabricated for transplantation. While the device primarily functions as a bioreactor that mimics the native mechanical strains, it is also outfitted with a load cell for providing force feedback or mechanical testing of the constructs. The device subjects engineered cartilage constructs to biaxial mechanical loading with great precision of loading dose (amplitude and frequency) and is compact enough to fit inside a standard tissue culture incubator. It loads samples directly in a tissue culture plate, and multiple plate sizes are compatible with the system. The device has been designed using components manufactured for precision-guided laser applications. Bi-axial loading is accomplished by two orthogonal stages. The stages have a 50 mm travel range and are driven independently by stepper motor actuators, controlled by a closed-loop stepper motor driver that features micro-stepping capabilities, enabling step sizes of less than 50 nm. A polysulfone loading platen is coupled to the bi-axial moving platform. Movements of the stages are controlled by Thor-labs Advanced Positioning Technology (APT) software. The stepper motor driver is used with the software to adjust load parameters of frequency and amplitude of both shear and compression independently and simultaneously. Positional feedback is provided by linear optical encoders that have a bidirectional repeatability of 0.1 &mu;m and a resolution of 20 nm, translating to a positional accuracy of less than 3 &mu;m over the full 50 mm of travel. These encoders provide the necessary position feedback to the drive electronics to ensure true nanopositioning capabilities. In order to provide the force feedback to detect contact and evaluate loading responses, a precision miniature load cell is positioned between the loading platen and the moving platform. The load cell has high accuracies of 0.15% to 0.25% full scale.

We designed a novel mechanical loading bioreactor that can apply uniaxial or biaxial mechanical strain to a cartilage biocomposite prior to transplantation into an articular cartilage defect.

Entwurf eines Biaxiale Mechanische Belastung Bioreaktor für Tissue Engineering

We  designed a loading device that is capable of applying uniaxial or biaxial  mechanical strain to a tissue engineered biocomposites fabricated for  ...

Biotechnik

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 ...

Chemie

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.

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 ...

Synthesis of Thermogelling Poly(N-isopropylacrylamide)-graft-chondroitin Sulfate Composites with Alginate Microparticles for Tissue Engineering

Injectable biomaterials are defined as implantable materials that can be introduced into the body as a liquid and solidify in situ. Such materials offer the clinical advantages of being implanted minimally invasively and easily forming space-filling solids in irregularly shaped defects. Injectable biomaterials have been widely investigated as scaffolds for tissue engineering. However, for the repair of certain load-bearing areas in the body, such as the intervertebral disc, scaffolds should possess adhesive properties. This will minimize the risk of dislocation during motion and ensure intimate contact with the surrounding tissue, providing adequate transmission of forces. Here, we describe the preparation and characterization of a scaffold composed of thermally sensitive poly(N-isopropylacrylamide)-graft-chondroitin sulfate (PNIPAAM-g-CS) and alginate microparticles. The PNIPAAm-g-CS copolymer forms a viscous solution in water at RT, into which alginate particles are suspended to enhance adhesion. Above the lower critical solution temperature (LCST), around 30 &#176;C, the copolymer forms a solid gel around the microparticles. We have adapted standard biomaterials characterization procedures to take into account the reversible phase transition of PNIPAAm-g-CS. Results indicate that the incorporation of 50 or 75 mg/ml&#160;alginate particles into 5% (w/v) PNIPAAm-g-CS solutions quadruple the adhesive tensile strength of PNIPAAm-gCS alone (p&#60;0.05). The incorporation of alginate microparticles also significantly increases swelling capacity of PNIPAAm-g-CS (p&#60;0.05), helping to maintain a space-filling gel within tissue defects. Finally, results of the in vitro toxicology assay kit, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) and Live/Dead viability assay indicate that the adhesive is capable of supporting the survival and proliferation of encapsulated Human Embryonic Kidney (HEK) 293 cells over 5 days.

Synthesis of Thermogelling PolyN-isopropylacrylamide-graft-chondroitin Sulfate Composites with Alginate Microparticles for Tissue Engineering

An injectable tissue engineering scaffold composed of poly(N-isopropylacrylamide)-graft-chondroitin sulfate (PNIPAAm-g-CS)-containing alginate microparticles was prepared. The adhesive strength, swelling properties and in vitro biocompatibility are analyzed in this study. The characterization techniques developed here may be applicable to other thermogelling systems.

Synthese von thermogelierenden Poly (N-isopropylacrylamid) -Pfropf-Chondroitinsulfat Composites mit Alginate Mikroteilchen für Tissue Engineering

Injectable biomaterials are defined as implantable materials that can be introduced into the body as a liquid and solidify in situ. Such materials offer ...

Gradient Strain Chip for Stimulating Cellular Behaviors in Cell-laden Hydrogel

Artificial guidance for cellular alignment is a hot topic in the field of tissue engineering. Most of the previous research has investigated single strain-induced cellular alignment on a cell-laden hydrogel by using complex experimental processes and mass controlling systems, which are usually associated with contamination issues. Thus, in this article, we propose a simple approach to building a gradient static strain using a fluidic chip with a plastic PDMS cover and a UV transparent glass substrate for the stimulation of cellular behavior in a 3D hydrogel. Overloading photo-patternable cell prepolymer in the fluidic chamber can generate a convex curved PDMS membrane on the cover. After UV crosslinking, through a concentric circular micropattern under the curved PDMS membrane, and buffer washing, a microenvironment for investigating cell behaviors under a variety of gradient strains is self-established in a single fluidic chip, without external instruments. NIH3T3 cells were demonstrated after observing the change in the cellular alignment trend under geometry guidance, in cooperation with strain stimulation, which varied from 15 - 65% on hydrogels. After a 3-day incubation, the hydrogel geometry dominated the cell alignment under low compressive strain, where cells aligned along the hydrogel elongation direction under high compressive strain. Between these, the cells showed random alignment due to the dissipation of the radical guidance of hydrogel elongation and the geometry guidance of the patterned hydrogel.

This article introduces a simple approach to providing non-continuous gradient static strains on a concentric cell-laden hydrogel to regulate cell alignment for tissue engineering.

Gradient Stamm Chip für anregende zellulären Verhaltensweisen in Zelle beladenen Hydrogel

Artificial guidance for cellular alignment is a hot topic in the field of tissue engineering. Most of the previous research has investigated single ...

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 ...

A Microfluidic Platform for Stimulating Chondrocytes with Dynamic Compression

Mechanical stimuli are known to modulate biological functions of cells and tissues. Recent studies have suggested that compressive stress alters growth plate cartilage architecture and results in growth modulation of long bones of children. To determine the role of compressive stress in bone growth, we created a microfluidic device actuated by pneumatic pressure, to dynamically (or statically) compress growth plate chondrocytes embedded in alginate hydrogel cylinders. In this article, we describe detailed methods for fabricating and characterizing this device. The advantages of our protocol are: 1) Five different magnitudes of compressive stress can be generated on five technical replicates in a single platform, 2) It is easy to visualize cell morphology via a conventional light microscope, 3) Cells can be rapidly isolated from the device after compression to facilitate downstream assays, and 4) The platform can be applied to study mechanobiology of any cell type that can grow in hydrogels.

This article provides detailed methods for fabricating and characterizing a pneumatically actuating microfluidic device for chondrocyte compression.

Eine mikrofluidische Plattform zur Stimulierung von Chondrozyten mit dynamischer Kompression

Mechanical stimuli are known to modulate biological functions of cells and tissues. Recent studies have suggested that compressive stress alters growth ...

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

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 ...