Name: mechanical stimulation of chondrocyte-agarose hydrogels
Uploaded: 2012-10-27T19:00:00
Description: Watch this Scientific Journal Video about Mechanical Stimulation of Chondrocyte-agarose Hydrogels at JoVE.com

1. Isolering af primære led-chondrocytter Høst 10-15 fuld tykkelse brusk skiver fra de artikulære overflader af animalske leddene (f.eks metacarpal-falankse led af fuldvoksne køer opnået fra en lokal abbatoir). <ol><li> Sted brusk skiver i en 100 mm petriskål og inkuberes i 20 ml 0,5% protease i Hams F-12 (w / v) i 2 timer ved 37 ° C. Skylles tre gange i Hams F-12 kulturmedier og inkuberes med 20 ml 0,15% collagenase A i Hams F-12 kulturmedier natten over ved 37 ° C. </li>...

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<html> Den beskrevne metode til påføring af kontrollerede mekaniske stimuli til celle-seedede agarose hydrogeler giver mulighed for direkte undersøgelse af virkningerne af dynamiske trykkræfter på chondrocyt stofskifte. Brugen af ​​custom-prøvningsapparaturet i forbindelse med de låseringe forudsat lateral begrænsning for konstruktionerne for at undgå potentielle problemer med prøve deponering. Brugen af ​​døde-vægtede loading plader sikret ved sæt besætninger sikrer direkte kontakt med konstruktion...

<table border="1"><tbody><tr><td> Navn på reagenset eller udstyr </td><td> Firma </td><td> Katalognummer </td><td> Kommentarer (valgfrit) </td></tr><tr><td> Hams 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> Føtalt bovint serum </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> Trypanblåt </td><td> Sigma Aldrich Ltd </td><td> 93.595 </td><td></td></tr><tr><td> Reichert Bright-Line Hæmacytometer </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 fra papayalatex </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 Acid </td><td> Sigma Aldrich Ltd </td><td> E9884 </td><td></td></tr><tr><td> DL-dithiothreitol </td><td> Sigma Aldrich Ltd </td><td> 43.819 </td><td></td></tr><tr><td> Agarose med lavt smeltepunkt, Type 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 mikromekanisk Tester </td><td> Biomomentum Inc. </td><td> V500cs </td><td></td></tr><tr><td> Compression Loading Jig </td><td> Kundetilpasset </td><td></td><td> Lignende produkt kunne levere Biomomentum Inc. </td></tr><tr><td> Falcon 24 brønds dyrkningsplade </td><td> Thermo Fisher Scientific </td><td> B353047 </td><td></td></tr><tr><td> β-væskescintillationstæller </td><td> Beckman Coulter </td><td> LS6500 </td><td></td></tr><tr><td> [3H] prolin </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

Ledbrusk lider af en begrænset reparationskapacitet beskadiget på grund af mekanisk defekt eller nedbrydes af sygdomme, såsom osteoarthritis. For at afhjælpe denne mangel, har flere medicinske indgreb blevet udviklet. En sådan metode er at dukke op igen det beskadigede område med væv-manipuleret brusk, men den konstruerede væv typisk mangler de biokemiske egenskaber og holdbarhed for indfødte brusk, spørgende sit langsigtede overlevelsesevne. Dette begrænser anvendelsen af ​​bruskvæv engineering til reparation af små fokale defekter med henvisning til det omgivende væv for at beskytte den implanterede materiale. At forbedre egenskaberne af den udviklede væv, mekanisk stimulering er en populær metode anvendes til at forstærke syntesen af ​​bruskagtig extracellulær matrix såvel som de resulterende mekaniske egenskaber af konstruerede væv. Mekanisk stimulering anvender kræfter til vævskonstruktioner analog med dem, der opleves in vivo. Detteer baseret på den forudsætning, at den mekaniske miljø, til dels regulerer udvikling og vedligeholdelse af nativt væv 1,2. De mest almindeligt anvendte form for mekanisk stimulation i bruskvæv engineering er dynamisk kompression ved fysiologiske stammer af ca 5-20% ved en frekvens på 1 Hz 1,3. Adskillige undersøgelser har undersøgt effekten af dynamisk kompression og har vist, at det har en positiv effekt på chondrocyt metabolisme og biosyntese, i sidste ende påvirker de funktionelle egenskaber af den udviklede væv 4-8. I dette dokument viser vi fremgangsmåden til mekanisk at stimulere chondrocyt-agarose hydrogel konstruktioner under dynamisk kompression og analysere ændringer i biosyntesen via biokemiske og radioisotop assays. Denne metode kan også let modificeres til at vurdere potentielt inducerede ændringer i cellulær reaktion som følge af mekaniske stimuli.

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

Mechanical Stimulation of Chondrocyte-agarose Hydrogels

Biosyntesen af ​​bruskagtig extracellulær matrix ved hjælp af chondrocytter kan påvirkes af anvendelse af mekaniske stimuli. Denne metode beskriver teknik for at anvende dynamiske trykstyrke stammer til chondrocytter indkapslet i 3D konstruktioner og evalueringen af ​​inducerede ændringer i chondrocyt stofskifte.

Mekanisk stimulering af chondrocyt-agarose Hydrogeler

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

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