このプロジェクトは、ドイツ研究協会（助成HE 4578/3-1）で、部分的にFP7 EU-プロジェクト &quot;GAMBA&quot; NMP3-SL-2010から245993によって賄われていた。

間葉系幹細胞の単離のためのドナーウサギのA.準備（手術室） <ol><li>細胞はオス4ヶ月歳の時にニュージーランドホワイト（NZW）ウサギ、約3キロ体重から分離されています。 </li><li>プロポフォールで麻酔（10 mg / kg体重体重IV）を誘導し、ペントバルビタールナトリウム（100 mg / kg体重体重IV）を生け贄に捧げる。 </li><li>電気クリッパー、真空毛皮で後肢、背中と腹から毛皮を剃る。 </li><li> 70％エタノールで完全に剃毛した領域を消毒する。 </li><li>鈍鉗子、鋭いハサミ（またはメス）と組織や靭帯のための骨のカッターを使用してください。 </li><li>足とふくらはぎの頭蓋の表面に沿って切開を行います。 </li><li>どちら鋭いまたは鈍的切開によって皮膚や皮下組織を反映しています。 </li><li>脛骨と大腿骨とは別の筋肉や靭帯。きれいな解剖を行うことが可能のような骨の近くにカットしてください。この時点で脛骨から大腿骨を分離しないでください。 </li><li> THRをカットウワーッ寛骨臼から大腿骨頭を分離する股関節。 </li><li>脛骨 - 大腿骨複合体を昇格。 </li><li>骨から残りの軟部組織を掻きまたは滅菌布の組織と骨をこするように手術用メスの刃を使用します。この時点で、大腿骨および脛骨はまだ接続されている。 </li><li>切断することにより膝蓋骨を外し、その後最終的に独立した骨に膝関節の靭帯を切った。 </li><li>スプレーは、空気が乾燥させ、70％エタノールで骨を分離し、それらの潤いを保つために、細胞培養培地（DMEM + 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遠心チューブに各ボーンを配置。 </li><li>今無菌細胞培養層流フードの下に切り替える。 </li></ol>骨と拡張からウサギMSCのフラッシュB.（細胞培養フッド） <ol><li>チューブからの骨を収集し、滅菌ピンセットを使用して150ミリメートル皿にそれらを配置します。 </li><li>両方の骨を取り除き、新しい150ミリメートル皿に無菌のこぎり、移動部分で終わる。 </li><li>メディとを10mlの注射器を埋めるUM（DMEM）、18ゲージの針を取り付け、骨髄の開口部に挿入します。 </li><li>その後、皿に骨髄をフラッシュする媒体と骨髄腔をすすいでください。可能であればその後、もう一方の端からすすいでください。必要に応じて、両端から多くを見送った。骨が壊れるべき場合は、単に骨の内部をすすいでください。 </li><li>シリンジに細胞培地懸濁液を吸引除去し、サスペンションは骨髄腔と骨髄血栓が表示されない、さらに骨を通して自由に浮遊するまで繰り返し骨髄をすすいでください。 </li><li>一度骨髄はすべて骨から収集された、18ゲージの針を通過することによって骨髄塊を乱す：針付属とシリンジを記入し、培地中に追い出す。 </li><li>その後、50ミリリットルチューブに細胞フィルターを通してサスペンションをフィルタリング。セル損失を防止するために10mlの培地で培養皿の2倍を洗浄し、同様にこむ。 </li><li>室温で5分間、500×gでサスペンションを遠心分離。 </li><li>清と再懸セルペルを削除らを10mlの培地（DMEM + 1％ペニシリン/ストレプトマイシン）であった。 </li><li> Biocoll分離ソリューションを使用して、末梢血単核細胞（PBMC）及び間葉系幹細胞（MSC）から独立した血液細胞。 </li><li>を15mlチューブにBiocoll分離溶液5mlを記入し、慎重に室温で20分（ブレーキなし）のために800 xgで上部と遠心分離機に細胞懸濁液を5ミリリットルを追加します。 </li><li> PBMCと間葉系幹細胞は、界面でのままBiocoll、底まで赤血球堆積物よりも高密度であること：可能な結果は、 図1を参照してください。 </li><li>慎重を15mlチューブにインターフェイスを削除し、5 mlのPBSで洗浄する。 </li><li>ステップ22で説明したように、遠心5ミリリットルのPBSに再懸と2〜3倍を繰り返す。 </li><li>その後、室温で10分（ブレーキ付）、350 xgで再び遠心。 </li><li>を10mlの培地に再懸濁し、血球のセルを数える。 </li><li> 150ミリメートルの皿で約5×10 6の初期の播種密度でプレート細胞。 </li><li> 2-3日後、再非接着細胞を移動します。あなたは、細胞の破片を除去するためにPBS最初ですすぎが必要になる場合があります。その後、新鮮な完全培地（DMEM + 10％ウシ胎児血清（FCS）+ 1％ペニシリン/ストレプトマイシン）を追加する。 </li><li> 3〜4日ごとに（ 図2）の細胞を養う。 </li><li> 5-10日後、経過初めて細胞。 </li></ol> in vitroでフィブリン塊のC.の準備<ol><li>移植の日には、0.25％トリプシン-EDTAに3分露出でフラスコ/皿から接着細胞を解放。完全培地を追加することで、トリプシン処理を停止します。 </li><li> 50ミリリットルファルコンチューブで細胞を配布し、PBSで2回、それらを洗ってください。 </li><li>トリパンブルー染色により細胞の生存と数字を決定します。 </li><li> 50,000細胞/マイクロ遠心チューブを追加し、室温で5分間、500×gで遠心分離によりペレットを収集します。必要に応じて、少なくとももう1つの塊のためにマスターミックスを準備します。 </li><li> 17μlのPBSで細胞ペレットを再懸濁し、TISSUのフィブリノゲン成分を25μlのを混ぜてこの17μlのMSCサスペンションとCOL-キット。 </li><li> in vivoでのドリル穴（ 図3）に基づき、事前ドリル穴（3x3.6ミリ）で滅菌プレートを取る。 </li><li>まず、一番上に42μlのフィブリノゲン - 細胞懸濁し、再び4μl​​のトロンビン溶液の即時加え、続いて一つの穴の中に4μlのトロンビン溶液（500 IU / ml）を接種。ピペットチップ内凝固を避けるために、サスペンションを混在させないでください。まず、分取フィブリノーゲン細胞懸濁液50μlの体積は、表面張力により溶融せずにプレドリル穴の縁に突出します。しかし、完全に凝固した後（60分後）血栓は縮小していると事前ドリル穴に収まります。 </li><li> PBSでマイクロ遠心チューブに鈍鉗子と場所を使用して慎重に血栓を削除します。 2血栓/動物を準備します。 </li><li>手術室に血栓を取る。 </li></ol>フィブリン塊における同種間葉系幹細胞のD.の移植<ol><li>プロポフォールの静脈注射（10 mg / kg体重）でウサギ（NZW、男性、3.5〜4.0キロの体重、5-6ヶ月）に麻酔を誘導する。 </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。前に指定されたすべての手順は、手術室の無菌環境の汚染を避けるために手術の準備室で行われる。 </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体重/分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>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、膝蓋骨に内側の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。どんな小さな表在血管を切断しないようにしてください。 </li><li> （ 図4）横方向に膝蓋骨を置換。 </li><li> inspe後に任意の付随軟骨病変や関節の異常のために膝関節のctionは、ストップデバイスと無菌空気オペレーティングパワードリルと滑車溝に2つの骨軟骨欠損（ディープ3ミリメートル、8字型の図）（直径3.6ミリメートル）を作成（ 図5）。 </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>移植の前に、欠陥にフィブリン糊の20μLを記入し、欠陥の底に均等に分配する。 </li><li>その後血栓フィギュア8字型欠陥に圧入植え込む。 </li><li>凝固後、滑車溝内膝蓋骨を再配置し、数回曲げると膝を伸ばす。 </li><li>もう一度横膝蓋骨を置換し、フィブリノーゲン細胞塊の場所に残っているかどうかを確認します。 </li><li>再び膝蓋骨を交換し、（両方吸収性縫合糸を持つ単一のボタン縫合（4-0 VICRYL）および連続皮膚縫合（4-0モノクリル）と層における創傷閉鎖で動作を終える材料）。 </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><li>術後ケアのため、傷は7日間毎日チェックされます。ウサギは術後鎮痛カルプロフェン4 mg / kgを皮下毎24時間（4日間）とブプレノルフィン0.03 mg / kgを皮下毎12時間（2.5日間）のために受け取る。膝の安定化（ 例えばドレッシング）は必要ありません。 </li></ol>

<ol>
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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells+in+arthritic+diseases.">Mesenchymal stem cells in arthritic diseases.</a> Arthritis Res. Ther. 10, 223 (2008).</li><li>Le Blanc, K., Tammik, C., Rosendahl, K., Zetterberg, E., Ringden, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HLA+expression+and+immunologic+properties+of+differentiated+and+undifferentiated+mesenchymal+stem+cells.">HLA expression and immunologic properties of differentiated and undifferentiated mesenchymal stem cells.</a> Exp. Hematol. 31, 890-896 (2003).</li></ol>

著者らは、開示することは何もありません。

近年では、複雑な関節骨軟骨欠損治療の可能性 - そのような骨軟骨炎の離断、骨壊死や外傷に起因するものなどを - 組織工学的アプローチでは、ますます魅力的になりました。前述した病的なエンティティでは、組織の損傷は軟骨下骨に延びており、別の本質的な癒しの容量1によって特徴付けられる2組織が ​​含まれます。骨軟骨関節損傷11,23の病原性プロセスの軟骨下骨?...

<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>&nbsp;</td>
		</tr>
		<tr>
			<td>FCS</td>
			<td>PAN Biotech GmbH</td>
			<td>0401</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Propofol</td>
			<td>Fresenius Kabi</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Biochrom AG</td>
			<td>A 2210</td>
			<td>1,000 units/10 &mu;g/&mu;l in 0.9% NaCl</td>
		</tr>
		<tr>
			<td>PBS Dulbecco (1X)</td>
			<td>Biochrom AG</td>
			<td>L1815</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ethanol (70%)</td>
			<td>Merck KGaA</td>
			<td>410230</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Trypan Blue Solution (0.4%)</td>
			<td>Sigma-Aldrich</td>
			<td>T8154</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Biocoll Separation Sol.</td>
			<td>Biochrom AG</td>
			<td>L6115</td>
			<td>Isotonic solution Density: 1,077 g/ml</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA 0.05%</td>
			<td>Invitrogen GmbH</td>
			<td>25300-054</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fentanyl</td>
			<td>DeltaSelectGmBH</td>
			<td>1819340</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NaCl solution (0.9%)</td>
			<td>BBraun</td>
			<td>8333A193</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Syringes (Injekt)</td>
			<td>BBraun</td>
			<td>4606108V</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Needles (Sterican)</td>
			<td>BBraun</td>
			<td>4657519</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Forceps (blunt/sharp)</td>
			<td>Aesculap</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Aesculap</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Scalpels</td>
			<td>Feather Safety Razor Co</td>
			<td>02.001.30.022</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pipettes research</td>
			<td>Eppendorf</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Bone Cutter</td>
			<td>Aesculap</td>
			<td>&nbsp;</td>
			<td>&nbsp;</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>CO2 Incubator</td>
			<td>Forma Scientific Inc.</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cell culture laminar flow hood Hera Safe</td>
			<td>Heraeus Instruments</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sterile saw</td>
			<td>Aesculap</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Centrifuge Megafuge 2.0 R</td>
			<td>Heraeus Instruments</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>Brand GmbH+Co KG</td>
			<td>717810</td>
			<td>Neubauer</td>
		</tr>
		<tr>
			<td>Air operated power drill</td>
			<td>Aesculap</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>TISSUCOL-Kit 1.0 ml Immuno</td>
			<td>Baxter</td>
			<td>2546648</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fibers (4-0 Monocryl, 4-0 Vicryl)</td>
			<td>Ethicon</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Spray dressing (OpSite)</td>
			<td>Smith&amp;Nephew</td>
			<td>66004978</td>
			<td>Permeable for water vapor</td>
		</tr>
	</tbody>
</table>

treatment of osteochondral defects in the rabbit's knee joint by implantation of allogeneic mesenchymal stem cells in fibrin clots

The treatment of osteochondral articular defects has been challenging physicians for many years. The better understanding of interactions of articular cartilage and subchondral bone in recent years led to increased attention to restoration of the entire osteochondral unit. In comparison to chondral lesions the regeneration of osteochondral defects is much more complex and a far greater surgical and therapeutic challenge. The damaged tissue does not only include the superficial cartilage layer but also the subchondral bone. For deep, osteochondral damage, as it occurs for example with osteochondrosis dissecans, the full thickness of the defect needs to be replaced to restore the joint surface 1. Eligible therapeutic procedures have to consider these two different tissues with their different intrinsic healing potential 2. In the last decades, several surgical treatment options have emerged and have already been clinically established 3-6.
Autologous or allogeneic osteochondral transplants consist of articular cartilage and subchondral bone and allow the replacement of the entire osteochondral unit. The defects are filled with cylindrical osteochondral grafts that aim to provide a congruent hyaline cartilage covered surface 3,7,8. Disadvantages are the limited amount of available grafts, donor site morbidity (for autologous transplants) and the incongruence of the surface; thereby the application of this method is especially limited for large defects.
New approaches in the field of tissue engineering opened up promising possibilities for regenerative osteochondral therapy. The implantation of autologous chondrocytes marked the first cell based biological approach for the treatment of full-thickness cartilage lesions and is now worldwide established with good clinical results even 10 to 20 years after implantation 9,10. However, to date, this technique is not suitable for the treatment of all types of lesions such as deep defects involving the subchondral bone 11.
The sandwich-technique combines bone grafting with current approaches in Tissue Engineering 5,6. This combination seems to be able to overcome the limitations seen in osteochondral grafts alone. After autologous bone grafting to the subchondral defect area, a membrane seeded with autologous chondrocytes is sutured above and facilitates to match the topology of the graft with the injured site. Of course, the previous bone reconstruction needs additional surgical time and often even an additional surgery. Moreover, to date, long-term data is missing 12.
Tissue Engineering without additional bone grafting aims to restore the complex structure and properties of native articular cartilage by chondrogenic and osteogenic potential of the transplanted cells. However, again, it is usually only the cartilage tissue that is more or less regenerated. Additional osteochondral damage needs a specific further treatment. In order to achieve a regeneration of the multilayered structure of osteochondral defects, three-dimensional tissue engineered products seeded with autologous/allogeneic cells might provide a good regeneration capacity 11.
Beside autologous chondrocytes, mesenchymal stem cells (MSC) seem to be an attractive alternative for the development of a full-thickness cartilage tissue. In numerous preclinical in vitro and in vivo studies, mesenchymal stem cells have displayed excellent tissue regeneration potential 13,14. The important advantage of mesenchymal stem cells especially for the treatment of osteochondral defects is that they have the capacity to differentiate in osteocytes as well as chondrocytes. Therefore, they potentially allow a multilayered regeneration of the defect.
In recent years, several scaffolds with osteochondral regenerative potential have therefore been developed and evaluated with promising preliminary results 1,15-18. Furthermore, fibrin glue as a cell carrier became one of the preferred techniques in experimental cartilage repair and has already successfully been used in several animal studies 19-21 and even first human trials 22.
The following protocol will demonstrate an experimental technique for isolating mesenchymal stem cells from a rabbit's bone marrow, for subsequent proliferation in cell culture and for preparing a standardized in vitro-model for fibrin-cell-clots. Finally, a technique for the implantation of pre-established fibrin-cell-clots into artificial osteochondral defects of the rabbit's knee joint will be described.

記載の外科技術は、人工骨軟骨欠損部に同種間葉系幹細胞の単離及び移植の成功を可能にする。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。 欠陥が周囲の軟骨に比べて類似の生体力学的特性と類似した耐久性と修復組織で充填した。フィブリン血餅細胞が骨軟骨欠損（ 図3）と同じサイズを有していた事前ドリル穴を有する滅菌プレー?...

Watch this Scientific Journal Video about Treatment of Osteochondral Defects in the Rabbit's Knee Joint by Implantation of Allogeneic Mesenchymal Stem Cells in Fibrin Clots at JoVE.com

Treatment of Osteochondral Defects in the Rabbit&#039;s Knee Joint by Implantation of Allogeneic Mesenchymal Stem Cells in Fibrin Clots

ウサギの膝関節における骨軟骨欠損の治療のための実験技術が記載されている。骨軟骨欠損に同種間葉系幹細胞の移植は、組織工学の分野で有望な開発を提供しています。フィブリン細胞塊の準備<em&gt; in vitroで</em&gt;移植のための標準化された方法を提供しています。

フィブリン塊における同種間葉系幹細胞の注入によってウサギの膝関節における骨軟骨欠損の治療

The treatment of osteochondral articular defects has been challenging physicians for many years. The better understanding of interactions of articular ...

treatment-osteochondral-defects-rabbit-s-knee-joint-implantation

Research

JoVE Journal

Medicine

Treatment of Osteochondral Defects in the Rabbit's Knee Joint by Implantation of Allogeneic Mesenchymal Stem Cells in Fibrin Clots

17.3K ビュー.  Klinikum rechts der Isar der Technischen Universität München. ウサギの膝関節における骨軟骨欠損の治療のための実験技術が記載されている。骨軟骨欠損に同種間葉系幹細胞の移植は、組織工学の分野で有望な開発を提供しています。フィブリン細胞塊の準備 in vitroで移植のための標準化された方法を提供しています。

ビデオ: Treatment of Osteochondral Defects in the Rabbit's Knee Joint by Implantation of Allogeneic Mesenchymal Stem Cells in Fibrin Clots

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

単離、特徴付けと二つの異なる方法を使用して永久歯に由来するヒト歯髄幹細胞の分化の比較

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

医学

Implantation of Fibrin Gel on Mouse Lung to Study Lung-specific Angiogenesis

Recent significant advances in stem cell research and bioengineering techniques have made great progress in utilizing biomaterials to regenerate and repair damage in simple tissues in the orthopedic and periodontal fields. However, attempts to regenerate the structures and functions of more complex three-dimensional (3D) organs such as lungs have not been very successful because the biological processes of organ regeneration have not been well explored. It is becoming clear that angiogenesis, the formation of new blood vessels, plays key roles in organ regeneration. Newly formed vasculatures not only deliver oxygen, nutrients and various cell components that are required for organ regeneration but also provide instructive signals to the regenerating local tissues. Therefore, to successfully regenerate lungs in an adult, it is necessary to recapitulate the lung-specific microenvironments in which angiogenesis drives regeneration of local lung tissues. Although conventional in vivo angiogenesis assays, such as subcutaneous implantation of extracellular matrix (ECM)-rich hydrogels (e.g., fibrin or collagen gels or Matrigel - ECM protein mixture secreted by Engelbreth-Holm-Swarm mouse sarcoma cells), are extensively utilized to explore the general mechanisms of angiogenesis, lung-specific angiogenesis has not been well characterized because methods for orthotopic implantation of biomaterials in the lung have not been well established. The goal of this protocol is to introduce a unique method to implant fibrin gel on the lung surface of living adult mouse, allowing for the successful recapitulation of host lung-derived angiogenesis inside the gel. This approach enables researchers to explore the mechanisms by which the lung-specific microenvironment controls angiogenesis and alveolar regeneration in both normal and pathological conditions. Since implanted biomaterials release and supply physical and chemical signals to adjacent lung tissues, implantation of these biomaterials on diseased lung can potentially normalize the adjacent diseased tissues, enabling researchers to develop new therapeutic approaches for various types of lung diseases.

Recapitulation of the organ-specific microenvironment, which stimulates local angiogenesis, is indispensable for successful regeneration of damaged tissues. This report demonstrates a novel method to implant fibrin gels on the lung surface of living mouse in order to explore how the lung-specific microenvironment modulates angiogenesis and alveolar regeneration in adult mouse.

肺特異的血管形成を研究するためにマウスの肺にフィブリンゲルの移植

Recent significant advances in stem cell research and bioengineering techniques have made great progress in utilizing biomaterials to regenerate and ...

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest improved outcome measures in both experimental and clinical studies. Although the BMMSC are implanted into the tendon lesion in large numbers (usually 10 - 20 million cells), only a relatively small number survive (&#60;10%) although these can persist for up to 5 months after implantation. This appears to be a common observation in other species where BMMSC have been implanted into other tissues and it is important to understand when this loss occurs, how many survive the initial implantation process and whether the cells are cleared into other organs. Tracking the fate of the cells can be achieved by radiolabeling the BMMSC prior to implantation which allows non-invasive in vivo imaging of cell location and quantification of cell numbers.
This protocol describes a cell labeling procedure that uses Technetium-99m (Tc-99m), and tracking of these cells following implantation into injured flexor tendons in horses. Tc-99m is a short-lived (t1/2 of 6.01 hr) isotope that emits gamma rays and can be internalized by cells in the presence of the lipophilic compound hexamethylpropyleneamine oxime (HMPAO). These properties make it ideal for use in nuclear medicine clinics for the diagnosis of many different diseases. The fate of the labeled cells can be followed in the short term (up to 36 hr) by gamma scintigraphy to quantify both the number of cells retained in the lesion and distribution of the cells into lungs, thyroid and other organs. This technique is adapted from the labeling of blood leukocytes and could be utilized to image implanted BMMSC in other organs.

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

This protocol describes the radiolabeling of equine mesenchymal stem cells and their implantation into tendon injuries in the horse in order to determine cell survival and tissue distribution using gamma scintigraphy.

馬腱でテクネチウム-99m標識骨髄間葉系幹細胞のin vivoイメージングと追跡中

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

新しい治療戦略を開発するための膵臓癌幹細胞特性を研究

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Preparation, Procedures and Evaluation of Platelet-Rich Plasma Injection in the Treatment of Knee Osteoarthritis

Knee osteoarthritis (KOA) is one of the most frequently encountered diseases in the orthopedic department. Existing non-surgical treatments have a limited effect on the repair of cartilage and on bone regeneration. Platelet-rich plasma (PRP) is an autologous bioactive substance that can repair cartilage injury and accelerate bone regeneration effectively. However, reporting of PRP preparation protocols in clinical studies is highly inconsistent, with the majority of studies providing insufficient information to allow the protocol to be reproduced. We describe a repeatable method of preparing PRP visually, the treatment of KOA using PRP intra-articular injection, and methods of evaluating the outcome. PRP was prepared using manual double centrifugation. The PRP layer was extracted from peripheral blood and used for knee joint cavity injection. Evaluations included assessments of blood platelet concentrations and clinical outcomes. Preparation of PRP by manual centrifugation requires less apparatus and is less costly than plasma filtration or centrifugation using equipment. The centrifugation time of our double centrifugation method was 6 and 5 minutes for the respective centrifugations at forces of 800 and 1400 x g, respectively, to allow for the consistent preparation of standardized PRP. However, a manual method is susceptible to operator error, and PRP batch preparation is not available. Intra-articular injection of PRP proved to be an effective treatment for knee osteoarthritis. The entire treatment procedure took less than 30 minutes, the blood platelet concentration of PRP could be standardized, and treatment was proven to be effective when evaluated by follow-up.

Knee osteoarthritis is frequently seen in the orthopedic department. We introduce in detail the entire knee osteoarthritis treatment process with platelet-rich plasma injection, including preparation, procedures, and evaluation.

準備・手順・膝変形性関節症の治療における多血小板血漿注入の評価

Knee osteoarthritis (KOA) is one of the most frequently encountered diseases in the orthopedic department. Existing non-surgical treatments have a limited ...

Using a Knee Arthrometer to Evaluate Tissue-specific Contributions to Knee Flexion Contracture in the Rat

Normal knee range of motion (ROM) is critical to well-being and allows one to perform basic activities such as walking, climbing stairs and sitting. Lost ROM is called a joint contracture and results in increased morbidity. Due to the difficulty of reversing established knee contractures, early detection is important, and hence, knowing risk factors for their development is essential. The rat represents a good model with which the effect of an intervention can be studied due to the similarity of rat knee anatomy to that of humans, the rat's ability to tolerate long durations of knee immobilization in flexion, and because mechanical data can be correlated with histologic and biochemical analysis of knee tissue.
Using an automated arthrometer, we demonstrate a validated, precise, reproducible, user-independent method of measuring the extension ROM of the rat knee joint at specific torques. This arthrometer can be used to determine the effects of interventions on knee joint ROM in the rat.

The goal of the protocol is to measure the extension range of motion of the rat knee. The effects of various diseases that increase the stiffness of the knee joint and the effectiveness of treatments can be quantified.

膝 Arthrometer を使用してラットの膝関節屈曲拘縮への組織固有の貢献を評価するには

Normal knee range of motion (ROM) is critical to well-being and allows one to perform basic activities such as walking, climbing stairs and sitting. Lost ...

Isolation and Cultivation of Mandibular Bone Marrow Mesenchymal Stem Cells in Rats

Here we present an efficient method for isolating and culturing mandibular bone marrow mesenchymal stem cells (mBMSCs) in vitro to rapidly obtain numerous high-quality cells for experimental requirements. mBMSCs could be widely used in therapeutic applications as tissue engineering cells in case of craniofacial diseases and cranio-maxillofacial regeneration in the future due to the excellent self-renewal ability and multi-lineage differentiation potential. Therefore, it is important to obtain mBMSCs in large numbers.
In this study, bone marrow was flushed from the mandible and primary mBMSCs were isolated through whole bone marrow adherent cultivation. Furthermore, CD29+CD90+CD45&#8722; mBMSCs were purified through fluorescent cell sorting. The second generation of purified mBMSCs were used for further study and displayed potential in differentiating into osteoblasts, adipocytes, and chondrocytes. Utilizing this in vitro model, one can obtain a high number of proliferative mBMSCs, which may facilitate the study of the biological characteristics, the subsequent reaction to the microenvironment, and other applications of mBMSCs.

This article presents a method that combines whole bone marrow adherence and flow cytometry sorting for isolating, cultivating, sorting, and identifying bone marrow mesenchymal stem cells from rat mandibles.

ラットにおける骨髄間葉系幹細胞の単離と培養

Here we present an efficient method for isolating and culturing mandibular bone marrow mesenchymal stem cells (mBMSCs) in vitro to rapidly obtain numerous ...

Orthopedic Robot-Assisted Femoral Neck System in the Treatment of Femoral Neck Fracture

Cannulated screw fixation is the main therapy for femoral neck fractures, especially in young patients. The traditional surgical procedure uses C-arm fluoroscopy to place the screw freehand and requires several guide wire adjustments, which increases the operation time and radiation exposure. Repeated drilling can also cause damage to the blood supply and bone quality of the femoral neck, which can be followed by complications such as screw loosening, nonunion, and femoral head necrosis. In order to make fixation more precise and reduce the incidence of complications, our team applied robot-assisted orthopedic surgery for screw placement using the femoral neck system to modify the traditional procedure. This protocol introduces how to import a patient's X-ray information into the system, how to perform screw path planning in software, and how the robotic arm assists in screw placement. Using this method, the surgeons can place the screw successfully the first time, improve the accuracy of the procedure, and avoid radiation exposure. The whole protocol includes the diagnosis of femoral neck fracture; the collection of intraoperative X-ray images; screw path planning in the software; precise placement of the screw under the assistance of the robotic arm by the surgeon; and verification of the implant placement.

This article introduces a method of robot-assisted orthopedic surgery for screw placement during the treatment of femoral neck fracture using the femoral neck system, which allows for more accurate screw placement, improved surgical efficiency, and fewer complications.

大腿骨頸部骨折の治療における整形外科ロボット支援大腿骨頸部システム

Cannulated screw fixation is the main therapy for femoral neck fractures, especially in young patients. The traditional surgical procedure uses C-arm ...

修正Videman法を用いたウサギのKOAモデルの確立

Application of Acupotomy in a Knee Osteoarthritis Model in Rabbit

Knee osteoarthritis (KOA) is one of the most frequently encountered diseases in the orthopedic department, which seriously reduces the quality of life of people with KOA. Among several pathogenic factors, the biomechanical imbalance of the knee joint is one of the main causes of KOA. Acupotomology believes that restoring the mechanical balance of the knee joint is the key to treating KOA. Clinical studies have shown that acupotomy can effectively reduce pain and improve knee mobility by reducing adhesion, contracture of soft tissues, and stress concentration points in muscles and tendons around the knee joint. 
In this protocol, we used the modified Videman method to establish a KOA model by immobilizing the left hindlimb in a straight position. We have outlined the method of operation and the precautions related to acupotomy in detail and evaluated the efficacy of acupotomy in conjunction with the theory of "Modulating Muscles and Tendons to Treat Bone Disorders" through the detection of the mechanical properties of quadriceps femoris and tendon, as well as cartilage mechanics and morphology. The results show that acupotomy has a protective effect on cartilage by adjusting the mechanical properties of the soft tissues around the knee joint, improving the cartilage stress environment, and delaying cartilage degeneration.

著者スポットライト:変形性膝関節症の治療における鍼切術の作用機序の調査

In this protocol, a knee osteoarthritis model was prepared using the modified Videman method, and the operation procedures and precautions of acupotomy are detailed. The effectiveness of acupotomy has been demonstrated by testing the mechanical properties of quadriceps femoris and tendon and the mechanical and morphological properties of cartilage.

ウサギの変形性膝関節症モデルにおける鍼切開術の適用

Knee osteoarthritis (KOA) is one of the most frequently encountered diseases in the orthopedic department, which seriously reduces the quality of life of ...