このプロジェクトは、ドイツ研究協会（DFG、HE 4578/3-1）によって賄われていた。

A.軟骨生検（手術室、非滅菌準備室の手順1-5） <ol><li>適切に用量薬することができるように、手術後の体重を監視するために、ウサギ（ニュージーランドホワイトウサギ、女性、3.5〜4.0キロの体重、生後6ヶ月）の最終重量の制御を行う。 </li><li> 10 mg / kgをプロポフォールの静脈内注射によりウサギに麻酔を誘導する。 </li><li>挿管後、静脈内に1.5 mg / kg体重/分プロポフォール及び0.05 mg / kgでフェンタニルで麻酔を維持します。カプノグラフィ、パルスオキシメトリと脈拍数を用いて麻酔を監視する。 </li><li>電気クリッパー、真空毛皮でログオン操作する膝を剃る。 </li><li>徹底的に剃っているd膝を消毒、滅菌ドレッシングで​​ウサギの残りの部分をカバーしています。 </li><li>膝蓋骨を触診し、膝蓋骨の内側に皮膚切開を行います。 </li><li>無菌条件下で内側parapatellar関節切開による膝関節を開きます。どんな小さなSUを切断回避しようperficial血管。 </li><li>横方向に膝蓋骨を置換。 </li><li>任意の異常や巨視的軟骨病変に対する膝関節を点検。 </li><li>慎重に滅菌メス（ 図1）と滑 ​​車骨大腿の外に小さな軟骨片（2-3 mm）の皮をむく。 </li><li>使用直後の体外培養右を続行する完全培地（DMEM + 10％ウシ胎児血清（FCS）+ 1％ペニシリン/ストレプトマイシン（ペニシリン/ストレプトマイシン））で50 mlチューブにこれらの軟骨生検を配置。 </li><li>欠陥をきれいにし、滅菌生理食塩水でそれらを洗い流してください。 </li><li>滑車の溝内に膝蓋骨の位置を変更。 </li><li>シングルボタンの縫合（4-0 VICRYL）、継続皮膚縫合（4-0モノクリル）と層における創傷閉鎖。吸収性縫合糸の素材を使用しています。 </li><li>最後に、水蒸気透過性ドレッシングスプレーで傷を封印。 </li></ol> 体外培養における B. <ol><li>生検Cを処理無菌層流の組織培養フード内操作後できるだけ早くartilageはピース。 </li><li>室温で3分間、500×gで遠心分離し、後続とのPBS中の滅菌収穫軟骨2倍を洗ってください。 </li><li> 30分間1ミリメートル3、50ミリリットルファルコンへの転送部分で軟骨をカットし、10ミリリットルトリプシン-EDTA（0.25％）および10 mlのPBSで30分間シェーカーでそれらを消化</li><li> 30分後、完全培地を添加することにより、トリプシン処理を停止する。その後、無血清培地（DMEM + 1％ペニシリン/ストレプトマイシン）のコラゲナーゼ（0.21 U / mg）を、12時間のために別のために軟骨の部分を横に振る。 </li><li>室温で3分間、170×gで得られた溶液を遠心分離。 </li><li> 5ミリリットル完全培地中で細胞ペレットを再懸濁します。 </li><li>シード5ミリリットル完全培地で25cm 2の組織培養フラスコに上の孤立した軟骨細胞と37℃加湿組織培養インキュベーター中で保つ℃、5％CO 2。 </li><li> 80％の密度に軟骨細胞を成長させる。 </li><li>細胞fを放し3分間0.05％トリプシン-EDTAにさらす前に、PBS 2倍で洗浄することによりROMフラスコ。軟骨は切り離したら、10μlの完全培地の添加によりトリプシン処理を停止します。 </li><li>完全培地中300 XG（3分）とペレットを再懸濁しでサスペンションを遠心分離。 </li><li>種子組織培養フラスコ中の細胞（75cm 2の ）と1:3（〜5,000細胞/ cm 2）ごとに5 日目または80％コンフルエント時で分割。 </li><li>トリパンブルー染色により細胞数と生存率を決定します。 </li></ol>マトリックスのC.の細胞播種<ol><li>上述したように、注入洗浄および剥離細胞に先立っ。細胞数を実行し、1.5 mlのマイクロチューブに5×10 4個の細胞を移す。その後、室温で5分間、500×gで遠心。 </li><li>行列の完全な飽和状態のために15μlの完全培地中でペレットを再懸濁します。 </li><li>無菌の生検によって滑車欠陥の正確な寸法に重層コラーゲン足場をカットパンチ。 </li><li>適切な大きさの組織培養皿（ 例えば、24ウェルプレート）に、プレース行列。 </li><li>マトリックスの多孔質、細胞接着面への種子の細胞。これは離れてマトリックスから細胞遊走を阻止。 </li><li>軟骨細胞の完全な遵守を可能にするために1時間インキュベーター内でさらに細胞培養培地なしで細胞播種行列をしておきます。 </li><li>新鮮な完全培地で培養容器（ 例えば 、24ウェルプレート）に続いて、場所細胞-マトリックス構築物。強力な遵守が期待されるにもかかわらず行列オフ任意の細胞を洗い流すないように注意してください。 </li><li>現在、細胞 - マトリックス構築物は、再移植のための手術室に取り出すことができる。 </li></ol> D.マトリックス支援自家軟骨細胞移植<ol><li> （A. 1-8）上記のように反対側の膝にparapatellar関節切開を行います。 </li><li>無菌の空気のoperatinと滑車溝に2つの孤立した軟骨欠損を作成Gパワードリル（直径3.6ミリメートル）。 </li><li>欠陥をきれいにし、滅菌生理食塩水で洗い流してください。 </li><li>注目はいつでも骨髄腔を開くために支払われるべきではない。必要であれば、出血に対する電気焼灼と欠陥の底部をシールします。 </li><li>多孔質の側面下に（ 図2）で培養軟骨細胞を播種行列を植え込む。その後、ドリル穴に圧入し、周囲の表面を洗い流す。 </li><li>安全な固定を保証するためにフィブリン糊を少し（TissucolデュオS）を移植した行列を封印。 </li><li>数回凝固した後、滑車溝内膝蓋骨を再配置し、膝関節への動きの完全な範囲を適用する。 </li><li>もう一度横膝蓋骨を置換し、不安定な固定のいずれかのイベントやサインを確認してください。行列は場所ではまだ開催されている必要があります。 </li><li>再び膝蓋骨を交換して、上記のようにドレッシング層とスプレーで創傷閉鎖して操作を終了します。 </li></ol>

<ol>
	<li>Albrecht, C., 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=Gene+expression+and+cell+differentiation+in+matrix-associated+chondrocyte+transplantation+grafts:+a+comparative+study.">Gene expression and cell differentiation in matrix-associated chondrocyte transplantation grafts: a comparative study.</a> Osteoarthritis Cartilage. 19, 1219-1227 (2011).</li><li>Pridie, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+of+resurfacing+osteoarthritic+knee+joints.">A method of resurfacing osteoarthritic knee joints.</a> J. Bone Joint Surg. Br. 41, 618-619 (1959).</li><li>Johnson, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arthroscopic+abrasion+arthroplasty+historical+and+pathologic+perspective:+present+status.">Arthroscopic abrasion arthroplasty historical and pathologic perspective: present status.</a> Arthroscopy. 2, 54-69 (1986).</li><li>Steadman, J. R., Rodkey, W. G., Singelton, S. B., Briggs, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfracture+technique+for+full-thickness+chondral+defects:+technique+and+clinical+result.">Microfracture technique for full-thickness chondral defects: technique and clinical result.</a> Operat. Tech. Orthop. 7, 300-304 (1997).</li><li>Brittberg, 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=Treatment+of+deep+cartilage+defects+in+the+knee+with+autologous+chondrocyte+transplantation.">Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation.</a> N. Engl. J. Med. 331, 889-895 (1994).</li><li>Peterson, L., Vasiliadis, H. S., Brittberg, M., Lindahl, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autologous+chondrocyte+implantation:+a+long-term+follow-up.">Autologous chondrocyte implantation: a long-term follow-up.</a> Am. J. Sports Med. 38, 1117-1124 (2010).</li><li>Nehrer, S., 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=Chondrocyte-seeded+collagen+matrices+implanted+in+a+chondral+defect+in+a+canine+model.">Chondrocyte-seeded collagen matrices implanted in a chondral defect in a canine model.</a> Biomaterials. 19, 2313-2328 (1998).</li><li>Frenkel, S. R., Toolan, B., Menche, D., Pitman, M. I., Pachence, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chondrocyte+transplantation+using+a+collagen+bilayer+matrix+for+cartilage+repair.">Chondrocyte transplantation using a collagen bilayer matrix for cartilage repair.</a> J. Bone. Joint Surg. Br. 79, 831-836 (1997).</li><li>Salzmann, G. 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. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autologous+chondrocyte+implantation+with+collagen+bioscaffold+for+the+treatment+of+osteochondral+defects+in+rabbits.">Autologous chondrocyte implantation with collagen bioscaffold for the treatment of osteochondral defects in rabbits.</a> Tissue Eng. 11, 1065-1076 (2005).</li><li>Vogt, S., 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+influence+of+the+stable+expression+of+BMP2+in+fibrin+clots+on+the+remodelling+and+repair+of+osteochondral+defects.">The influence of the stable expression of BMP2 in fibrin clots on the remodelling and repair of osteochondral defects.</a> Biomaterials. 30, 2385-2392 (2009).</li><li>Ueblacker, 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=In+vivo+analysis+of+retroviral+gene+transfer+to+chondrocytes+within+collagen+scaffolds+for+the+treatment+of+osteochondral+defects.">In vivo analysis of retroviral gene transfer to chondrocytes within collagen scaffolds for the treatment of osteochondral defects.</a> Biomaterials. 28, 4480-4487 (2007).</li><li>Marlovits, S., Zeller, P., Singer, P., Resinger, C., Vecsei, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cartilage+repair:+generations+of+autologous+chondrocyte+transplantation.">Cartilage repair: generations of autologous chondrocyte transplantation.</a> Eur. 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. Res. , 270-283 (1996).</li></ol>

著者らは、開示することは何もありません。

提示プロトコルは、ウサギのひざで人工的に作成された軟骨欠損へのその後の増殖および再移植のため自家軟骨細胞を単離する9,12,13を設立し、簡単に再現可能な技術を提供する。関節軟骨損傷のリモデリングおよび修復のための自家軟骨細胞の使用は満足な長期的結果を提供すること6臨床既に使用されている。 骨膜肥大や石灰化などのような主...

<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

関節軟骨は自己再生1限られた容量を有するので、関節軟骨の欠陥が重大な健康問題と考えられている。未処理の軟骨病変は進行中の痛みにつながる、否定的に生活の質に影響を与えると変形性関節症のためにやすく。過去数十年の間に、いくつかの外科技術は、このような病変を治療するために開発されてきた。しかし、今まで、硝子関節軟骨を有する欠陥を覆う又は十分な長期の回復2-4を提供するという点で完全な修復を達成することは不可能であった。したがって、関節軟骨の損傷は、そのような組織工学などの再生技術のための主な目標のまま。多くの場合、繊維状または線維軟骨組織の形成をもたらす他の外科技術とは対照的に、組織工学は、完全に移植された細胞の軟骨産生能を使用して、元の関節軟骨複合体の構造および特性を回復することを目的とする。 Recent発展は再生軟骨の治療のための有望な可能性を開いた。 全層軟骨や骨軟骨病変の治療のための最初のセルベースのアプローチは、臨床自家軟骨細胞移植（ACI）5を開拓ラース·ピーターソンとマットBrittbergによって1994年に行われた。今日では、技術は臨床的にも、10〜20年後に移植6良好な臨床結果を維持し、膝の大硝子軟骨欠損の治療のために十分に確立されています。近年では、自家軟骨細胞の移植は、急速な進行を受けた。その後、細胞を植え替えされた人工的な三次元コラーゲンマトリックスの使用は、ますます人気7-9となった。 MACTが2つの外科的処置を含む：軟骨細胞を収集するために、まず、軟骨生検は、tの非体重を支える軟骨領域から行う必要がある彼は膝関節を。その後、軟骨細胞は、抽出、精製し、 インビトロで十分な細胞数に拡張されている。軟骨細胞は、その後、三次元マトリックス上に播種され、その後、再移植することができる。組織工学インプラントを調製する場合、増殖速度および分化能力が成功した組織再生10にとって極めて重要である。細胞担体としての三次元マトリクスの使用は、これらの細胞の特性11を支持すると考えられる。 以下のプロトコルは、3Dマトリックス（ 軟骨·ジッド 、Geistlichバイオマテリアル、Wollhusen、スイス）の上に軟骨生検、in vitroでのそれらの増殖とその播種から軟骨細胞を単離するための手法を要約し、デモンストレーションを行います。最終的に、ウサギの膝関節の人工的に作成された軟骨細胞の欠陥にマトリクス構築物の注入について説明する。この技術は、実験的な設定として使用することができる軟骨修復のさらなる実験のために。

記載外科技術は、人工軟骨欠損部に自家軟骨細胞の正常な分離と移植を可能にします。実験装置は、周囲の軟骨にインプラント統合の成功をもたらした。 in vivoでの 12週間後、軟骨欠損は、インプラント（ 図4）にせん断応力及び損傷を低減均一で無傷の表面で修復組織によって充填した。また、インプラントのない肥大や石灰化が見られなかった。?...

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

ウサギの膝関節内の軟骨欠損の治療のための実験技術が記載されている。マトリックス上に播種自家軟骨細胞の移植は、満足の長期的な結果を提供する関節軟骨病変の改造や修理のために広く受け入れ方法です。マトリックス支援自家軟骨細胞移植（MACT）が標準化され、臨床的に確立された注入方法を提供しています。

ウサギモデルにおける軟骨欠陥の改造と修理のためのマトリックス支援自家軟骨細胞移植

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

matrix-assisted-autologous-chondrocyte-transplantation-for-remodeling

Research

JoVE Journal

Medicine

14.3K ビュー.  Klinikum rechts der Isar der Technischen Universität München. ウサギの膝関節内の軟骨欠損の治療のための実験技術が記載されている。マトリックス上に播種自家軟骨細胞の移植は、満足の長期的な結果を提供する関節軟骨病変の改造や修理のために広く受け入れ方法です。マトリックス支援自家軟骨細胞移植（MACT）が標準化され、臨床的に確立された注入方法を提供しています。

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

A Mouse Model of in Utero Transplantation

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus. For example, in utero transplantation (IUT) of hematopoietic stem cells has been used to successfully treat patients with severe combined immunodeficiency.1,2 In several other conditions, however, IUT has been attempted without success.3 Given these mixed results, the availability of an efficient non-human model to study the biological sequelae of stem cell transplantation and gene therapy is critical to advance this field. We and others have used the mouse model of IUT to study factors affecting successful engraftment of in utero transplanted hematopoietic stem cells in both wild-type mice4-7 and those with genetic diseases.8,9 The fetal environment also offers considerable advantages for the success of in utero gene therapy. For example, the delivery of adenoviral10, adeno-associated viral10, retroviral11, and lentiviral vectors12,13 into the fetus has resulted in the transduction of multiple organs distant from the site of injection with long-term gene expression. in utero gene therapy may therefore be considered as a possible treatment strategy for single gene disorders such as muscular dystrophy or cystic fibrosis. Another potential advantage of IUT is the ability to induce immune tolerance to a specific antigen. As seen in mice with hemophilia, the introduction of Factor IX early in development results in tolerance to this protein.14 
In addition to its use in investigating potential human therapies, the mouse model of IUT can be a powerful tool to study basic questions in developmental and stem cell biology. For example, one can deliver various small molecules to induce or inhibit specific gene expression at defined gestational stages and manipulate developmental pathways. The impact of these alterations can be assessed at various timepoints after the initial transplantation. Furthermore, one can transplant pluripotent or lineage specific progenitor cells into the fetal environment to study stem cell differentiation in a non-irradiated and unperturbed host environment.
The mouse model of IUT has already provided numerous insights within the fields of immunology, and developmental and stem cell biology. In this video-based protocol, we describe a step-by-step approach to performing IUT in mouse fetuses and outline the critical steps and potential pitfalls of this technique.

A Mouse Model of in Utero Transplantation

The mouse model of in utero transplantation is a versatile tool that can be used to study the potential clinical applications of stem cell transplantation and gene therapy in the fetus. In this protocol, we present a general approach to performing this technique

のマウスモデル子宮内で移植

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus.  For example, in ...

医学

Development of Obliterative Bronchiolitis in a Murine Model of Orthotopic Lung Transplantation

Orthotopic lung transplantation in rats was first reported by Asimacopoulos and colleagues in 1971 1. Currently, this method is well accepted and standardized not only for the study of allo-rejection but also between syngeneic strains for examining mechanisms of ischemia-reperfusion injury after lung transplantation. Although the application of the rat and other large animal model 2 contributed significantly to the elucidation of these studies, the scope of those investigations is limited by the scarcity of knockout and transgenic rats. Due to no effective therapies for obliterative bronchiolitis, the leading cause of death in lung transplant patients, there has been an intensive search for pre-clinical models that replicate obliterative bronchiolitis. The tracheal allograft model is the most widely used and may reproduce some of the histopathologic features of obliterative bronchiolitis 3. However, the lack of an intact vasculature with no connection to the recipient's conducting airways, and incomplete pathologic features of obliterative bronchiolitis limit the utility of this model 4. Unlike transplantation of other solid organs, vascularized mouse lung transplants have only recently been reported by Okazaki and colleagues for the first time in 2007 5. Applying the basic principles of the rat lung transplant, our lab initiated the obliterative bronchiolitis model using minor histoincompatible antigen murine orthotopic single-left lung transplants which allows the further study of obliterative bronchiolitis immunopathogenesis6.

Obliterative bronchiolitis is the key impediment to the long-term survival of lung transplant recipients and the lack of a robust preclinical model precludes examining obliterative bronchiolitis immunopathogenesis. Unlike other solid organ transplants, vascularized mouse lung transplantation has only recently been developed. Here we show our independently developed obliterative bronchiolitis model after murine orthotopic single-lung transplantation.

同所性肺移植のマウスモデルにおける閉塞性細気管支炎の開発

Orthotopic lung transplantation in rats was first reported by Asimacopoulos and colleagues in 1971 1. Currently, this method is well accepted and ...

A Mouse Fetal Skin Model of Scarless Wound Repair

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs across species but is age dependent. The transition from a scarless to scarring phenotype occurs in the third trimester of pregnancy in humans and around embryonic day 18 (E18) in mice. However, this varies with the size of the wound with larger defects generating a scar at an earlier gestational age. The emergence of lineage tracing and other genetic tools in the mouse has opened promising new avenues for investigation of fetal scarless wound healing. However, given the inherently high rates of morbidity and premature uterine contraction associated with fetal surgery, investigations of fetal scarless wound healing in vivo require a precise and reproducible surgical model. Here we detail a reliable model of fetal scarless wound healing in the dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

During mammalian development, early gestational skin wounds heal without a scar. Here we detail a reliable and reproducible model of fetal scarless wound healing in the cutaneous dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

瘢痕創傷修復のマウス胎児皮膚モデル

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs ...

A Rat Carotid Balloon Injury Model to Test Anti-vascular Remodeling Therapeutics

The rat carotid balloon injury is a well-established surgical model that has been used to study arterial remodeling and vascular cell proliferation. It is also a valuable model system to test, and to evaluate therapeutics and drugs that negate maladaptive remodeling in the vessel. The injury, or barotrauma, in the vessel lumen caused by an inflated balloon via an inserted catheter induces subsequent neointimal growth, often leading to hyperplasia or thickening of the vessel wall that narrows, or obstructs the lumen. The method described here is sufficiently sensitive, and the results can be obtained in relatively short time (2 weeks after the surgery). The efficacy of the drug or therapeutic against the induced-remodeling can be evaluated either by the post-mortem pathological and histomorphological analysis, or by ultrasound sonography in live animals. In addition, this model system has also been used to determine the therapeutic window or the time course of the administered drug. These studies can leadto the development of a better administrative strategy and a better therapeutic outcome. The procedure described here provides a tool for translational studies that bring drug and therapeutic candidates from bench research to clinical applications.

The rat carotid balloon injury model described below allows researchers to evaluate drugs or therapeutics that negate injury-induced arterial hyperplasia. Detailed pre-surgical preparation, surgical procedure, and post-surgical cares of the animal are described.

ラット頸動脈バルーン損傷モデルは、抗血管リモデリングの治療薬をテストします

The rat carotid balloon injury is a well-established surgical model that has been used to study arterial remodeling and vascular cell proliferation. It is ...

Lentiviral Vector-mediated Gene Therapy of Hepatocytes Ex Vivo for Autologous Transplantation in Swine

Gene therapy is an ideal choice to cure many inborn errors of metabolism of the liver. Ex-vivo, lentiviral vectors have been used successfully in the treatment of many hematopoietic diseases in humans, as their use offers stable transgene expression due to the vector&#39;s ability to integrate into the host genome. This method demonstrates the application of ex vivo gene therapy of hepatocytes to a large animal model of hereditary tyrosinemia type I. This process consists of 1) isolation of primary hepatocytes from the autologous donor/recipient animal, 2) ex vivo gene delivery via hepatocyte transduction with a lentiviral vector, and 3) autologous transplant of corrected hepatocytes via portal vein injection. Success of the method generally relies upon efficient and sterile removal of the liver resection, careful handling of the excised specimen for isolation of viable hepatocytes sufficient for re-engrafting, high-percentage transduction of the isolated cells, and aseptic surgical procedures throughout to prevent infection. Technical failure at any of these steps will result in low yield of viable transduced hepatocytes for autologous transplant or infection of the donor/recipient animal. The pig model of human type 1 hereditary tyrosinemia (HT-1) chosen for this approach is uniquely amenable to such a method, as even a small percentage of engraftment of corrected cells will lead to repopulation of the liver with healthy cells based on a powerful selective advantage over native-diseased hepatocytes. Although this growth selection will not be true for all indications, this approach is a foundation for expansion into other indications and allows for manipulation of this environment to address additional diseases, both within the liver and beyond, while controlling for exposure to viral vector and opportunity for off-target toxicity and tumorigenicity.

Lentiviral Vector-mediated Gene Therapy of Hepatocytes Ex Vivo for Autologous Transplantation in Swine

This protocol is intended to describe porcine hepatocyte isolation and ex vivo gene delivery to cure models of metabolic diseases via autologous cell transplantation. Although this particular model enjoys unique advantages that favor successful therapy, the application is a relevant foundation to address additional diseases and indications.

豚に自家移植用肝細胞Ex Vivoのレンチ ウイルス媒介性遺伝子治療

Gene therapy is an ideal choice to cure many inborn errors of metabolism of the liver. Ex-vivo, lentiviral vectors have been used successfully in the ...

Transthoracic Echocardiographic Examination in the Rabbit Model

Large animal models such as the rabbit are valuable for translational preclinical research. Rabbits have a similar cardiac electrophysiology compared to that of humans and that of other large animal models such as dogs and pigs. However, the rabbit model has the additional advantage of lower maintenance costs compared to other large animal models. The longitudinal evaluation of cardiac function using echocardiography, when appropriately implemented, is a useful methodology for preclinical assessment of novel therapies for heart failure with reduced ejection fraction (e.g. cardiac regeneration). The correct use of this non-invasive tool requires the implementation of a standardized examination protocol following international guidelines. Here we describe, step by step, a detailed protocol supervised by veterinary cardiologists for performing echocardiography in the rabbit model, and demonstrate how to correctly obtain the different echocardiographic views and imaging planes, as well as the different imaging modes available in a clinical echocardiography system routinely used in human and veterinary patients.

Here we describe, step by step, a detailed protocol for performing echocardiography in the rabbit model. We show how to correctly obtain the different echocardiographic views and imaging planes, as well as the different imaging modes available in a clinical echocardiography system routinely used in human and veterinary patients.

ウサギモデルにおける経胸部心エコー検査

Large animal models such as the rabbit are valuable for translational preclinical research. Rabbits have a similar cardiac electrophysiology compared to ...

Retinal Pigment Epithelium Transplantation in a Non-human Primate Model for Degenerative Retinal Diseases

Retinal pigment epithelial (RPE) transplantation holds great promise for the treatment of inherited and acquired retinal degenerative diseases. These conditions include retinitis pigmentosa (RP) and advanced forms of age-related macular degeneration (AMD), such as geographic atrophy (GA). Together, these disorders represent a significant proportion of currently untreatable blindness globally. These unmet medical needs have generated heightened academic interest in developing methods of RPE replacement. Among the animal models commonly utilized for preclinical testing of therapeutics, the non-human primate (NHP) is the only animal model that has a macula. As it shares this anatomical similarity with the human eye, the NHP eye is an important and appropriate preclinical animal model for the development of advanced therapy medicinal products (ATMPs) such as RPE cell therapy.
This manuscript describes a method for the submacular transplantation of an RPE monolayer, cultured on a polyethylene terephthalate (PET) cell carrier, underneath the macula onto a surgically created RPE wound in immunosuppressed NHPs. The fovea-the central avascular portion of the macula-is the site of the greatest mechanical weakness during the transplantation. Foveal trauma will occur if the initial subretinal fluid injection generates an excessive force on the retina. Hence, slow injection under perfluorocarbon liquid (PFCL) vitreous tamponade is recommended with a dual-bore subretinal injection cannula at low intraocular pressure (IOP) settings to create a retinal bleb. 
Pretreatment with an intravitreal plasminogen injection to release parafoveal RPE-photoreceptor adhesions is also advised. These combined strategies can reduce the likelihood of foveal tears when compared to conventional techniques. The NHP is a key animal model in the preclinical phase of RPE cell therapy development. This protocol addresses the technical challenges associated with the delivery of RPE cellular therapy in the NHP eye.

The non-human primate (NHP) is an ideal model for studying human retinal cellular therapeutics due to the anatomical and genetic similarities. This manuscript describes a method for submacular transplantation of retinal pigment epithelial cells in the NHP eye and strategies to prevent intraoperative complications associated with macular manipulation.

ヒト以外の霊長類モデルにおける変性網膜疾患の網膜色素上皮移植

Retinal pigment epithelial (RPE) transplantation holds great promise for the treatment of inherited and acquired retinal degenerative diseases. These ...

A Simplified Model for Heterotopic Heart Valve Transplantation in Rodents

There is an urgent clinical need for heart valve replacements that can grow in children. Heart valve transplantation is proposed as a new type of transplant with the potential to deliver durable heart valves capable of somatic growth with no requirement for anticoagulation. However, the immunobiology of heart valve transplants remains unexplored, highlighting the need for animal models to study this new type of transplant. Previous rat models for heterotopic aortic valve transplantation into the abdominal aorta have been described, though they are technically challenging and costly. For addressing this challenge, a renal subcapsular transplant model was developed in rodents as a practical and more straightforward method for studying heart valve transplant immunobiology. In this model, a single aortic valve leaflet is harvested and inserted into the renal subcapsular space. The kidney is easily accessible, and the transplanted tissue is securely contained in a subcapsular space that is well vascularized and can accommodate a variety of tissue sizes. Furthermore, because a single rat can provide three donor aortic leaflets and a single kidney can provide multiple sites for transplanted tissue, fewer rats are required for a given study. Here, the transplantation technique is described, providing a significant step forward in studying the transplant immunology of heart valve transplantation.

This protocol describes a simple and efficient method for the transplantation of aortic valve leaflets under the renal capsule to allow for the study of alloreactivity of heart valves.

げっ歯類におけるヘテロトピック心臓弁移植のための簡略モデル

There is an urgent clinical need for heart valve replacements that can grow in children. Heart valve transplantation is proposed as a new type of ...

Left Lung Orthotopic Transplantation in a Juvenile Porcine Model for ESLP

Lung transplantation is the gold-standard treatment for end-stage lung disease, with over 4,600 lung transplantations performed worldwide annually. However, lung transplantation is limited by a shortage of available donor organs. As such, there is high waitlist mortality. Ex situ lung perfusion (ESLP) has increased donor lung utilization rates in some centers by 15%-20%. ESLP has been applied as a method to assess and recondition marginal donor lungs and has demonstrated acceptable short- and long-term outcomes following transplantation of extended criteria donor (ECD) lungs. Large animal (in vivo) transplantation models are required to validate ongoing in vitro research findings. Anatomic and physiologic differences between humans and pigs pose significant technical and anesthetic challenges. An easily reproducible transplant model would permit the&#160;in vivo&#160;validation of current ESLP strategies and the preclinical evaluation of various interventions designed to improve donor lung function. This protocol describes a porcine model of orthotopic left lung allotransplantation. This includes anesthetic and surgical techniques, a customized surgical checklist, troubleshooting, modifications, and the benefits and limitations of the approach.

This protocol describes a juvenile porcine model of orthotopic left lung allotransplantation designed for use with ESLP research. Focus is made on anesthetic and surgical techniques, as well as critical steps and troubleshooting.

ESLPのブタ幼体モデルにおける左肺同所性移植

Lung transplantation is the gold-standard treatment for end-stage lung disease, with over 4,600 lung transplantations performed worldwide annually. ...

Transcatheter Pulmonary Valve Replacement from Autologous Pericardium with a Self-Expandable Nitinol Stent in an Adult Sheep Model

Transcatheter pulmonary valve replacement has been established as a viable alternative approach for patients suffering from right ventricular outflow tract or bioprosthetic valve dysfunction, with excellent early and late clinical outcomes. However, clinical challenges such as stented heart valve deterioration, coronary occlusion, endocarditis, and other complications must be addressed for lifetime application, particularly in pediatric patients. To facilitate the development of a lifelong solution for patients, transcatheter autologous pulmonary valve replacement was performed in an adult sheep model. The autologous pericardium was harvested from the sheep via left anterolateral minithoracotomy under general anesthesia with ventilation. The pericardium was placed on a 3D shaping heart valve model for non-toxic cross-linking for 2 days and 21 h. Intracardiac echocardiography (ICE) and angiography were performed to assess the position, morphology, function, and dimensions of the native pulmonary valve (NPV). After trimming, the crosslinked pericardium was sewn onto a self-expandable Nitinol stent and crimped into a self-designed delivery system. The autologous pulmonary valve (APV) was implanted at the NPV position via left jugular vein catheterization. ICE and angiography were repeated to evaluate the position, morphology, function, and dimensions of the APV. An APV was successfully implanted in&#160;sheep J. In this paper, sheep J was selected to obtain representative results. A 30 mm APV with a Nitinol stent was accurately implanted at the NPV position without any significant hemodynamic change. There was no paravalvular leak, no new pulmonary valve insufficiency, or stented pulmonary valve migration. This study demonstrated the feasibility and safety, in a long-time follow-up, of developing an APV for implantation at the NPV position with a self-expandable Nitinol stent via jugular vein catheterization in an adult sheep model.

This study demonstrates the feasibility and safety of developing an autologous pulmonary valve for implantation at the native pulmonary valve position by using a self-expandable Nitinol stent in an adult sheep model. This is a step toward developing transcatheter pulmonary valve replacement for patients with right ventricular outflow tract dysfunction.

成体ヒツジモデルにおける自己拡張性ニチノールステントを有する自家心膜からの経カテーテル肺弁置換

Transcatheter pulmonary valve replacement has been established as a viable alternative approach for patients suffering from right ventricular outflow ...