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 av primära ledkondrocyter Skörd 10-15 fulla skivor tjocklek brosk från artikulära ytorna av animaliska fogar (t.ex. metakarpala-falangala leden moget skelett kor erhållna från en lokal Abbatoir). <ol><li> Placera brosk segment i en 100 mm petriskål och inkubera i 20 ml 0,5% proteas i Hams F-12 (vikt / volym) under 2 h vid 37 ° C. Skölj tre gånger i Hams F-12 odlingsmedium och inkubera med 20 ml 0,15% kollagenas A i Hams F-12 odlingsmedium över natten vid 37...

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	<li>Grodzinsky, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cartilage+tissue+remodeling+in+response+to+mechanical+forces.">Cartilage tissue remodeling in response to mechanical forces.</a> Annual Review of Biomedical Engineering. 2, 691-713 (2000).</li><li>Kuettner, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemistry+of+articular+cartilage+in+health+and+disease.">Biochemistry of articular cartilage in health and disease.</a> Clinical Biochemistry. 25, 155-163 (1992).</li><li>Neu, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+interface+of+functional+biotribology+and+regenerative+medicine+in+synovial+joints.">The interface of functional biotribology and regenerative medicine in synovial joints.</a> Tissue Engineering Part B: Reviews. 14, 235-247 (2008).</li><li>Demarteau, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+compression+of+cartilage+constructs+engineered+from+expanded+human+articular+chondrocytes.">Dynamic compression of cartilage constructs engineered from expanded human articular chondrocytes.</a> Biochemical and Biophysical Research Communications. 310, 580-588 (2003).</li><li>Waldman, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+intermittent+compressive+stimulation+improves+the+composition+and+mechanical+properties+of+tissue-engineered+cartilage.">Long-term intermittent compressive stimulation improves the composition and mechanical properties of tissue-engineered cartilage.</a> Tissue Engineering. 10, 1323-1331 (2004).</li><li>Hunter, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+compression+of+chondrocyte-seeded+fibrin+gels:+effects+on+matrix+accumulation+and+mechanical+stiffness.">Dynamic compression of chondrocyte-seeded fibrin gels: effects on matrix accumulation and mechanical stiffness.</a> Osteoarthritis and Cartilage. 12, 117-130 (2004).</li><li>Buschmann, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+compression+modulates+matrix+biosynthesis+in+chondrocyte/agarose+culture.">Mechanical compression modulates matrix biosynthesis in chondrocyte/agarose culture.</a> Journal of Cell Science. 108 (Pt 4), 1497-1508 (1995).</li><li>Quinn, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+compression+alters+proteoglycan+deposition+and+matrix+deformation+around+individual+cells+in+cartilage+explants.">Mechanical compression alters proteoglycan deposition and matrix deformation around individual cells in cartilage explants.</a> Journal of Cell Science. 111 (Pt 5), 573-583 (1998).</li><li>Kuettner, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+cartilage+matrix+by+mammalian+chondrocytes+in+vitro.+I.+Isolation,+culture+characteristics,+and+morphology.">Synthesis of cartilage matrix by mammalian chondrocytes in vitro. I. Isolation, culture characteristics, and morphology.</a> The Journal of Cell Biology. 93, 743-750 (1982).</li><li>Lee, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+loading+of+chondrocytes+embedded+in+3D+constructs:+in+vitro+methods+for+assessment+of+morphological+and+metabolic+response+to+compressive+strain.">Mechanical loading of chondrocytes embedded in 3D constructs: in vitro methods for assessment of morphological and metabolic response to compressive strain.</a> Methods in Molecular Medicine. 100, 307-324 (2004).</li><li>McGowan, K. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+quantification+of+DNA+in+human+articular+and+septal+cartilage+using+PicoGreen+and+Hoechst+33258.">Biochemical quantification of DNA in human articular and septal cartilage using PicoGreen and Hoechst 33258.</a> Osteoarthritis and Cartilage. 10, 580-587 (2002).</li><li>Fan, J. C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+intermittent+static+biaxial+tensile+strains+on+tissue+engineered+cartilage.">The effect of intermittent static biaxial tensile strains on tissue engineered cartilage.</a> Annals of Biomedical Engineering. 38, 1672-1682 (2010).</li><li>Kaupp, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+vibrations+increase+the+proliferation+of+articular+chondrocytes+in+high-density+culture.">Mechanical vibrations increase the proliferation of articular chondrocytes in high-density culture.</a> Proceedings of the Institution of Mechanical Engineers, Part H: Journal of Engineering in Medicine. 222, 695-703 (2008).</li><li>Waldman, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+intermittent+shear+deformation+improves+the+quality+of+cartilaginous+tissue+formed+in+vitro.">Long-term intermittent shear deformation improves the quality of cartilaginous tissue formed in vitro.</a> Journal of Orthopaedic Research. 21, 590-596 (2003).</li><li>Waldman, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+single+application+of+cyclic+loading+can+accelerate+matrix+deposition+and+enhance+the+properties+of+tissue-engineered+cartilage.">A single application of cyclic loading can accelerate matrix deposition and enhance the properties of tissue-engineered cartilage.</a> Osteoarthritis and Cartilage. 14, 323-330 (2006).</li><li>Kisiday, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+dynamic+compressive+loading+on+chondrocyte+biosynthesis+in+self-assembling+peptide+scaffolds.">Effects of dynamic compressive loading on chondrocyte biosynthesis in self-assembling peptide scaffolds.</a> Journal of Biomechanics. 37, 595-604 (2004).</li><li>Chowdhury, T. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+regulation+of+chondrocyte+metabolism+in+agarose+constructs+subjected+to+dynamic+compression.">Temporal regulation of chondrocyte metabolism in agarose constructs subjected to dynamic compression.</a> Archives of Biochemistry and Biophysics. 417, 105-111 (2003).</li></ol>

<html> Den beskrivna metoden för att applicera kontrollerade mekaniska stimuli till cell-seedade agaros hydrogeler möjliggör direkt utredning om effekterna av dynamiska tryckkrafter på kondrocyt ämnesomsättning. Användningen av den specialanpassade provningsanordningens i samband med de kvarhållande ringarna som sidled hinder för konstruktionerna för att undvika eventuella problem med prov tipp. Användningen av dead-viktade lastning plattorna säkrade genom uppsättningar besättningar säkerställer direktkon...

<table border="1"><tbody><tr><td> Namn på reagens eller utrustning </td><td> Företag </td><td> Katalognummer </td><td> Kommentarer (tillval) </td></tr><tr><td> Hams F-12 </td><td> Thermo Fisher Scientific </td><td> SH3001002 </td><td></td></tr><tr><td> Kollagenas En </td><td> Sigma Aldrich Ltd </td><td> C0130 </td><td></td></tr><tr><td> Proteas </td><td> Sigma Aldrich Ltd </td><td> P5147 </td><td></td></tr><tr><td> Fetalt bovint serum </td><td> Sigma Aldrich Ltd </td><td> F1051 </td><td></td></tr><tr><td> Askorbat </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> Trypanblått </td><td> Sigma Aldrich Ltd </td><td> 93.595 </td><td></td></tr><tr><td> Reichert Bright-Line Hemacytometer </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 från 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> Agaros med låg smältpunkt, typ VII </td><td> Sigma Aldrich Ltd </td><td> A9045 </td><td></td></tr> &lt;tr&gt; <td> Mesh (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> Komprimering Laddar Jig </td><td> Specialbyggda </td><td></td><td> Liknande produkt kan levereras av Biomomentum Inc. </td></tr><tr><td> Falcon 24 brunnars odlingsplatta </td><td> Thermo Fisher Scientific </td><td> B353047 </td><td></td></tr><tr><td> β-vätskescintillationsräknare </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] Svavel </td><td> Perkin-Elmer </td><td> NEX041005MC </td><td></td></tr></tbody></table>

mechanical stimulation of chondrocyte-agarose hydrogels

Ledbrosk lider av en begränsad reparation kapacitet när den skadats av mekanisk förolämpning eller bryts ned av sjukdom, såsom artros. För att råda bot på denna brist har flera medicinska åtgärder utvecklats. En sådan metod är att dyka upp igen det skadade området med vävnadstekniska brosk, men det vävnadstekniska vanligtvis saknar de biokemiska egenskaper och hållbarhet hos infödda brosk, ifrågasätta sin långsiktiga överlevnadsförmåga. Detta begränsar tillämpningen av broskvävnad engineering till reparation av små fokala defekter, att förlita sig på den omgivande vävnaden för att skydda det implanterade materialet. För att förbättra egenskaperna hos den utvecklade vävnaden, är mekanisk stimulering en populär metod som används för att öka syntesen av brosk extracellulär matris liksom de resulterande mekaniska egenskaperna hos den manipulerade vävnaden. Mekanisk stimulering tillämpar krafter till vävnaden konstruerar analoga med dem upplevt in vivo. Dettaär baserad på antagandet att den mekaniska miljön, delvis reglerar utveckling och underhåll av nativ vävnad 1,2. Den mest allmänt vedertagen form av mekanisk stimulering i brosk vävnadsteknik är dynamisk kompression vid fysiologiska stammar av cirka 5-20% vid en frekvens av 1 Hz 1,3. Flera studier har undersökt effekterna av dynamisk kompression och har visat att det har en positiv effekt på kondrocyt metabolism och biosyntes i slutändan påverkar de funktionella egenskaperna hos den utvecklade vävnaden 4-8. I detta dokument visar vi metoden att mekaniskt stimulera kondrocyt-agaros hydrogel konstruktioner under dynamisk kompression och analysera förändringar i biosyntes genom biokemiska och radioisotoper analyser. Denna metod kan också lätt modifieras för att bedöma eventuella potentiellt inducerade förändringar i cellulär respons som ett resultat av mekaniska stimuli.

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

Mechanical Stimulation of Chondrocyte-agarose Hydrogels

Biosyntesen av brosk extracellulär matris genom kondrocyter kan påverkas av applicering av mekaniska stimuli. Denna metod beskriver tekniken att tillämpa dynamiska komprimerande stammar till kondrocyter inkapslade i 3D konstruktioner och utvärdering av inducerade förändringar i kondrocyt ämnesomsättning.

Mekanisk stimulering av kondrocyt-agaros 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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