Name: matrix-assisted autologous chondrocyte transplantation for remodeling and repair of chondral defects in a rabbit model
Uploaded: 2013-05-21T12:00:00
Description: Watch this Scientific Journal Video about Matrix-assisted Autologous Chondrocyte Transplantation for Remodeling and Repair of Chondral Defects in a Rabbit Model at JoVE.com

Questo progetto è stato finanziato dalla German Research Association (DFG, SE 4578/3-1).

A. Cartilagine Biopsia (Chirurgia in camera; passaggi 1-5 nella camera di preparazione non sterile) <ol><li> Eseguire un controllo finale peso del coniglio (New Zealand White coniglio, femmina, 3,5-4,0 kg di peso corporeo, 6 mesi) per essere in grado di dose droghe correttamente e controllare il peso conseguente alla chirurgia. </li><li> Indurre l&#39;anestesia per il coniglio da una iniezione endovenosa di 10 mg / kg di propofol. </li><li> Dopo l&#39;intubazione, mantenere l&#39;anestesia con 1,5 mg / kg / min propof...

<ol>
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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dependence+of+autologous+chondrocyte+transplantation+on+varying+cellular+passage,+yield+and+culture+duration.">The dependence of autologous chondrocyte transplantation on varying cellular passage, yield and culture duration.</a> Biomaterials. 32, 5810-5818 (2011).</li><li>Frohlich, M., Malicev, E., Gorensek, M., Knezevic, M., Kregar Velikonja, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+rabbit+auricular+chondrocyte+isolation+and+growth+parameters+in+cell+culture.">Evaluation of rabbit auricular chondrocyte isolation and growth parameters in cell culture.</a> Cell Biol. Int. 31, 620-625 (2007).</li><li>Willers, C., Chen, J., Wood, D., Xu, J., Zheng, M. 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J. Radiol. 57, 24-31 (2006).</li><li>Benya, P. D., Shaffer, 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=Dedifferentiated+chondrocytes+reexpress+the+differentiated+collagen+phenotype+when+cultured+in+agarose+gels.">Dedifferentiated chondrocytes reexpress the differentiated collagen phenotype when cultured in agarose gels.</a> Cell. 30, 215-224 (1982).</li><li>Rudert, M., Hirschmann, F., Wirth, 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=Growth+behavior+of+chondrocytes+on+various+biomaterials.">Growth behavior of chondrocytes on various biomaterials.</a> Orthopade. 28, 68-75 (1999).</li><li>Hsu, S. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+biodegradable+polyesters+modified+by+type+II+collagen+and+Arg-Gly-Asp+as+Tissue+Engineering+scaffolding+materials+for+cartilage+regeneration.">Evaluation of biodegradable polyesters modified by type II collagen and Arg-Gly-Asp as Tissue Engineering scaffolding materials for cartilage regeneration.</a> Artificial Organs. 30, 42-55 (2006).</li><li>Brun, P., Cortivo, R., Zavan, B., Vecchiato, N., Abatangelo, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro++reconstructed+tissues+on+hyaluronan-based+temporary+scaffolding.">In vitro reconstructed tissues on hyaluronan-based temporary scaffolding.</a> J. Mater. Sci. Mater. Med. 10, 683-688 (1999).</li><li>Domm, C., Fay, J., Schunke, M., Kurz, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redifferentiation+of+dedifferentiated+joint+cartilage+cells+in+alginate+culture.+Effect+of+intermittent+hydrostatic+pressure+and+low+oxygen+partial+pressure.">Redifferentiation of dedifferentiated joint cartilage cells in alginate culture. Effect of intermittent hydrostatic pressure and low oxygen partial pressure.</a> Orthopade. 29, 91-99 (2000).</li><li>Kimura, T., Yasui, N., Ohsawa, S., Ono, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chondrocytes+embedded+in+collagen+gels+maintain+cartilage+phenotype+during+long-term+cultures.">Chondrocytes embedded in collagen gels maintain cartilage phenotype during long-term cultures.</a> Clin. Orthop. Relat. Res. , 231-239 (1984).</li><li>Kon, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Second-generation+autologous+chondrocyte+implantation:+results+in+patients+older+than+40+years.">Second-generation autologous chondrocyte implantation: results in patients older than 40 years.</a> Am. J. Sports Med. 39, 1668-1675 (2011).</li><li>Gavenis, K., Schmidt-Rohlfing, B., Mueller-Rath, R., Andereya, S., Schneider, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+comparison+of+six+different+matrix+systems+for+the+cultivation+of+human+chondrocytes.">In vitro comparison of six different matrix systems for the cultivation of human chondrocytes.</a> In Vitro Cell Dev. Biol. Anim. 42, 159-167 (2006).</li><li>Niemeyer, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristic+complications+after+autologous+chondrocyte+implantation+for+cartilage+defects+of+the+knee+joint.">Characteristic complications after autologous chondrocyte implantation for cartilage defects of the knee joint.</a> Am. J. Sports Med. 36, 2091-2099 (2008).</li><li>Tay, L. X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+outcomes+of+alginate-embedded+allogenic+mesenchymal+stem+cells+versus+autologous+chondrocytes+for+the+repair+of+focal+articular+cartilage+defects+in+a+rabbit+model.">Treatment outcomes of alginate-embedded allogenic mesenchymal stem cells versus autologous chondrocytes for the repair of focal articular cartilage defects in a rabbit model.</a> The American Journal of Sports Medicine. 40, 83-90 (2012).</li><li>Brittberg, M., Nilsson, A., Lindahl, A., Ohlsson, C., Peterson, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rabbit+articular+cartilage+defects+treated+with+autologous+cultured+chondrocytes.">Rabbit articular cartilage defects treated with autologous cultured chondrocytes.</a> Clin. Orthop. Relat. 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Il protocollo presentato fornisce un consolidato 9,12,13 e facilmente riproducibile tecnica per isolare i condrociti autologhi per la successiva proliferazione e re-impianto in difetti della cartilagine creato artificialmente in ginocchio coniglio. L&#39;uso di condrociti autologhi per il rimodellamento e la riparazione di lesioni della cartilagine articolare è già in uso clinico fornire risultati soddisfacenti a lungo termine 6. Problemi importanti come per esempio l...

<table border="1">
	<tbody>
		<tr>
			<td>Name of reagent/equipment</td>
			<td>Company</td>
			<td>Catalogue Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Biochrom AG</td>
			<td>F 0415</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase A</td>
			<td>Roche</td>
			<td>10 103 586 001</td>
			<td>0.21 U/mg</td>
		</tr>
		<tr>
			<td>Fetal calf serum (FCS)</td>
			<td>PAN Biotech GmbH</td>
			<td>3702-P103009</td>
			<td></td>
		</tr>
		<tr>
			<td>Propofol</td>
			<td>Fresenius Kabi</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Biochrom AG</td>
			<td>A 2213</td>
			<td>10,000 U/ml/10,000 &mu;g/ml</td>
		</tr>
		<tr>
			<td>PBS Dulbecco (1X)</td>
			<td>Biochrom AG</td>
			<td>L1815</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol (70%)</td>
			<td>Merck KgaA</td>
			<td>410230</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA 0.25 %/0.02 %</td>
			<td>Biochrom AG</td>
			<td>L2163</td>
			<td>in PBS w/o Ca2+, Mg2+</td>
		</tr>
		<tr>
			<td>Fentanyl</td>
			<td>Delta Select GmBH</td>
			<td>1819340</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl solution (0.9%)</td>
			<td>Bbraun</td>
			<td>8333A193</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture dishes 100 mm/150 mm</td>
			<td>TPP AG</td>
			<td>93100/93150</td>
			<td>Growth area 60.1 mm2/147.8 mm2</td>
		</tr>
		<tr>
			<td>Tissue culture flasks 25/75 mm2</td>
			<td>TPP AG</td>
			<td>90025/90075</td>
			<td>25 mm2, 75 mm2</td>
		</tr>
		<tr>
			<td>Centrifuge Tubes (50 ml)</td>
			<td>TPP AG</td>
			<td>91050</td>
			<td>Gamma-sterilized</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>Brand GmbH+Co KG</td>
			<td>717810</td>
			<td>Neubauer</td>
		</tr>
		<tr>
			<td>Trypan Blue Solution 0.4%</td>
			<td>Sigma-Aldrich</td>
			<td>L8154</td>
			<td></td>
		</tr>
		<tr>
			<td>Spray dressing (OpSite)</td>
			<td>Smith&amp;Nephew</td>
			<td>66004978</td>
			<td>Permeable for water vapor</td>
		</tr>
		<tr>
			<td>Chondro-Gide&Ograve;</td>
			<td>Geistlich Pharma AG</td>
			<td>30915.5</td>
			<td></td>
		</tr>
		<tr>
			<td>Biopsy Punch</td>
			<td>pfm medical ag</td>
			<td>48351</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissucol Duo S</td>
			<td>Baxter</td>
			<td>3419627</td>
			<td>0.5 ml</td>
		</tr>
	</tbody>
</table>

matrix-assisted autologous chondrocyte transplantation for remodeling and repair of chondral defects in a rabbit model

Difetti della cartilagine articolare sono considerati un problema sanitario importante in quanto la cartilagine articolare ha una limitata capacità di auto-rigenerazione 1. Lesioni cartilaginee non trattate portano a dolore in corso, influenzare negativamente la qualità della vita e di predisporre per l&#39;artrosi. Durante gli ultimi decenni, diverse tecniche chirurgiche sono state sviluppate per il trattamento di tali lesioni. Tuttavia, finora non è stato possibile ottenere una riparazione completa in termini di copertura del difetto di cartilagine ialina articolare o di fornire recupero a lungo termine soddisfacenti 2-4. Pertanto, le lesioni della cartilagine articolare rimane un obiettivo primario per le tecniche rigenerative come Tissue Engineering. A differenza di altre tecniche chirurgiche, che spesso portano alla formazione di tessuto fibroso o fibrocartilagineo, Tissue Engineering mira a ripristinare completamente la struttura complessa e le proprietà della cartilagine articolare originale utilizzando il potenziale condrogenico delle cellule trapiantate. Ralcune recenti sviluppi hanno aperto possibilità promettenti per le terapie rigenerative della cartilagine. Il primo approccio basato cellulare per il trattamento di cartilagine a tutto spessore o lesioni osteocondrali è stata eseguita nel 1994 da Lars Peterson e Mats Brittberg pioniere clinico impianto di condrociti autologhi (ACI) 5. Oggi, la tecnica è clinicamente ben consolidata per il trattamento dei grandi difetti della cartilagine ialina del ginocchio, il mantenimento di buoni risultati clinici anche 10 o 20 anni dopo l&#39;impianto 6. Negli ultimi anni, l&#39;impianto di condrociti autologhi subito una progressione rapida. L&#39;uso di un tridimensionale collageno-matrice artificiale su cui le cellule vengono successivamente reimpiantate diventata sempre più popolare 7-9. MACT compone di due procedure chirurgiche: in primo luogo, al fine di raccogliere condrociti, una biopsia cartilagine deve essere eseguita da una zona di cartilagine non sostengono pesi tgiunto egli ginocchio. Poi, condrociti vengono estratti, purificati e ampliato per un numero di cellule sufficiente in vitro. I condrociti sono poi seminate su una matrice tridimensionale e possono essere successivamente reimpiantati. Quando si prepara un impianto tissutale, tasso di proliferazione e la capacità di differenziazione sono cruciali per una rigenerazione dei tessuti di successo 10. L&#39;uso di una matrice tridimensionale come vettore cella è pensato per supportare queste caratteristiche cellulari 11. Il seguente protocollo riassumerà e dimostrare una tecnica per l&#39;isolamento di condrociti da biopsie cartilagine, la loro proliferazione in vitro e loro semina su un 3D-matrice (Chondro-Gide, Geistlich Biomaterials, Wollhusen, Svizzera). Infine, l&#39;impianto degli cellula-matrice-costrutti in artificialmente creati difetti condrali del ginocchio di coniglio sarà descritto. Questa tecnica può essere utilizzata come ambiente sperimentaleper ulteriori esperimenti di riparazione della cartilagine.

La tecnica chirurgica descritta permette un isolamento di successo e l&#39;impianto di condrociti autologhi in un difetto condrale artificiale. Il setup sperimentale ha comportato una integrazione dell&#39;impianto nella cartilagine circostante. Dopo 12 settimane in vivo, il difetto condrale stato riempito da tessuto di riparazione con una superficie omogenea e intatta, che ha ridotto sforzo di taglio e danneggiamento dell&#39;impianto (Figura 4). Inoltre, nessuna i...

Watch this Scientific Journal Video about Matrix-assisted Autologous Chondrocyte Transplantation for Remodeling and Repair of Chondral Defects in a Rabbit Model at JoVE.com

Matrix-assisted Autologous Chondrocyte Transplantation for Remodeling and Repair of Chondral Defects in a Rabbit Model

Una tecnica sperimentale per il trattamento dei difetti condrali in ginocchio di coniglio è descritto. L&#39;impianto di condrociti autologhi seminate su una matrice è un metodo ben accettato per il rimodellamento e la riparazione di lesioni della cartilagine articolare che fornisca risultati soddisfacenti a lungo termine. Matrix-assistito trapianto autologo di condrociti (MACT) offre un metodo standardizzato di impianto e clinicamente stabilita.

Matrix-assistito trapianto autologo di condrociti per il rimodellamento e la riparazione dei difetti condrali in un modello di coniglio

Articular cartilage defects are considered a major health problem because articular cartilage has a limited capacity for self-regeneration 1. Untreated ...

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