The project was supported by CTSA (UL1TR000058) from the National Center for Advancing Translational Sciences) and the CCTR Endowment Fund of Virginia Commonwealth University. The contents are solely the responsibility of the authors and do not necessarily represent official views of the National Center for Advancing Translational Sciences or the National Institutes of Health.

すべての血液描画手順はライセンスと認定専門家によって行われるべきです。研究のためのヒト被験者の使用は、治験審査委員会またはその他の適切な権限の承認を必要とします。インフォームドコンセントに関する参加者の識別情報を保護する特別な予防措置が続いする必要があります。このプロトコルに記載されているすべての実験は、ヒトの血液および/または血液製剤及び適切な個人用保護具の取り扱いは常に着用する必要が伴います。廃棄物はバイオハザードと見なされ、規則に従って廃棄する必要があります。 1.静脈穿刺<ol><li>患者/参加者を特定し、既存のレコードで確定します。詳細に研究を説明し、インフォームドコンセントを得ます。 </li><li>平らな安定した面にマーキング管を個々の患者のために設定トレイを持っています。 </li><li>手順を説明し、支持されたアームで快適に患者席を作ります。患者彼またはSHに知らせますeは小さなピンチを感じるだろうし、手順全体でまだ残っている必要があります。 </li><li>アダプタに針を取り付けます。 </li><li>手を洗うと手袋を着用してください。 </li><li>中心から外側同心円状に70％のイソプロピルアルコールで洗浄することにより、静脈穿刺のための肘前窩を準備します。 30秒間空気乾燥にサイトを許可します。 </li><li>触診によって適切な静脈を識別します。 </li><li> （まだラジアルパルスを感じながら）それはあまりにもタイトではないことを確認して穿刺部位以上に止血帯3-4を適用します。 </li><li> 1滑らかな動きで皮膚に針15〜30度のベベルを挿入することにより、静脈穿刺を実行します。針を通して採血管を押して、全血の9ミリリットルを収集します。 </li><li>止血帯を削除します。針からチューブを外します。 </li><li>針を撤回し、針を除去する前に穿刺部位にガーゼ正方形を適用します。適切なバイオハザード容器に針を廃棄してください。 </li><li>広報を維持するために患者を依頼穿刺部位でessure。 </li><li>チューブにラベルを付けます</li><li>出血が停止していることを確認するために穿刺部位を確認してください。 </li><li>患者は大丈夫（痛み、腫れや光ふらつき）を感じていないかどうか尋ねる、ガーゼ正方形の上に接着剤包帯やテープで固定します</li><li>退院前に患者/参加ありがとうございました。 </li></ol> 2. L-PRF準備<ol><li>すぐに赤張りの乾燥したガラス管の中に静脈採血後、遠心に配置します。 </li><li>適切なカウンターバランスを配置した後、室温で12分間、400×gで遠心分離します。 </li><li>サイクルの終わりにチューブを外します。乏血小板血漿（PPP）、多血小板フィブリン（L-PRF）とRBCベース（ 図1）：三層に注目してください。 </li><li>ピペットを使用して、PPPを吸引します。ピンセットを使用すると、静かに、L-PRFを引き出し、滅菌、パンチングメタルメッシュに配置します。 </li><li>外科用メスを使用して、慎重にバフを残すRBC層の大部分をこすりYコートそのまま。 </li><li>穏やかに30秒間（無菌の金属板を用いて、およその重量225 g）のL-PRF塊を圧縮します。乏血小板血漿が絞り出されます。 </li><li>プレートを外し、ゆっくりとL-PRF膜を持ち上げます。 L-PRF膜は、実験12で使用するための準備ができています。 </li></ol> 3.一軸引張試験<ol><li>取り扱いを容易にするため、濾紙の上に置いて、L-PRF膜（N = 6）を、カスタムメイドの金属を使用して「ドッグボーン」にパンチ（7.5ミリメートルのゲージ長との狭いポイントで広い2.75ミリメートル）死にます。 </li><li> 3つのスポットで、各サンプルの厚さを測定し、平均値を取ります。 </li><li>一軸試験システムのジョーグリップの中央にL-PRF膜を慎重に係合します。 </li><li>注意深くL-PRF膜を露出させるためにファイラ紙支持体を引き裂きます。 </li><li>プログラム楽器可動ヘッドが一定の割合（10.0ミリメートル/分）で動作しているので、いつL-実験を開始PRFがまだ湿っています。 </li><li>弾性率、破断エネルギー、および一軸テストシステムのソフトウエアから破断時のひずみを記録します。これらの値は自動的に計算されているとは、ユーザー定義の入力は必要ありません。 図3を参照してください。 </li></ol> 4.縫合糸保持力<ol><li>取り扱いを容易にするため、濾紙の上に置いて、L-PRF膜（n = 3）で、手術用メスを用いて、（X 25ミリメートル10ミリメートル）を測定長方形のサンプルに切断。 </li><li>各サンプル（3の平均）の厚さを測定します。 </li><li>ステンレス製歯列矯正リガチャーワイヤー（直径220μm）を用いて、サンプルの中央にピンホールを作成します。 </li><li>ループを形成し、引張試験機にそれを修正するためにピンホールを介して結紮ワイヤを渡します。下顎のグリップ13にL-PRF膜の端を置きます。 </li><li>プログラム可動ヘッドが一定の速度（10 mm /分）と開始時に動作しているように、器具実験。 </li><li>弾性率、破断エネルギー、および一軸テストシステムのソフトウエアから破断時のひずみを記録します。これらの値は自動的に計算されているとは、ユーザー定義の入力は必要ありません。 図3を参照してください。 </li></ol> 5.形態学的検査<ol><li> 10ミリメートルの皮膚生検パンチを使用して、SEM観察用のL-PRFのサンプルを調製し、24ウェルプレートに配置します。 </li><li> PBSでサンプルを洗浄し、20分間（PBS中）、2.5％のグルタルアルデヒドで固定します。 </li><li> 5分ごとに（50％、70％、80％、90％及び100％）エタノールの濃度を増加させ、順次に浸漬することによって試験片を脱水します。 </li><li> 5分間、100％のHMDS（ヘキサメチルジシラザン）0.5mlで処理します。過剰のHMDS 14を除去するために 、O / Nを通気します。 </li><li>両面テープを使用して、スタブのマウントサンプルは、70秒間白金コートスパッタやアクセルで動作する走査型電子顕微鏡で調べますerationの電圧20kV（または適切な設定）。 </li></ol> 6. L-PRFのゲニピン架橋、トリプシン感受性およびニンヒドリンアッセイ<ol><li>ゲニピン架橋されたL-PRFを調製するために、PBSで膜を洗浄し、48時間（70％エタノール中）1％のゲニピン溶液4mlに浸します。過剰ゲニピン15、16を除去するために、実験前にPBSですすいでください。 </li><li>トリプシンによる分解に対する耐性により、L-PRFのゲニピン架橋の安定性を評価します。置き、L-PRF膜（n = 3）を0.01％トリプシン500μlの架橋のL-PRFゲニピンとトリプシンの日々の変化に伴って37℃で3日間インキュベートしました。 <ol><li>露出を酵素に先立って1日目にサンプルを秤量し、3日目に開始と終了の重量差は、酵素分解17を表しています 。 </li></ol></li><li>ニンヒドリンアッセイにより、L-PRF（G-PRF）で処理ゲニピンで架橋の量を定量化します。まず標準CURVを準備電子グリシンを用いて、（1 MM-0.031 mM）の曲線は、遊離アミノ酸濃度（FAA）​​と吸光度との関係を確立します。 <ol><li> 2％の1 mlの熱PRF試料（w / v）の100℃で15分間ニンヒドリン。 </li><li>ソリューションは、室温まで冷却し、50％エタノール1.5ミリリットルを追加することができます。 </li><li>適切な分光光度計を用いて570 nmの吸光度を分析します。 </li><li> 15以下の式を使用して架橋の割合を決定します</li></ol></li></ol><img alt="式（1）" src="/files/ftp_upload/53221/53221eq1.jpg" /> 7. MTS細胞増殖アッセイ<ol><li> 80％のコンフルエントな単層が得られるまで、T-75フラスコ中で最小必須培地 - アルファの変更（αMEM）にMC3T3（マウス頭蓋冠前骨芽細胞）を成長させます。 </li><li>新鮮な、無菌のL-PRF膜を準備し（細胞培養フードの内側にL-PRFチューブを開く）と、新たな細胞培養皿上に膜を転送します。 </li><li>吸引フラスコからメディアと、PBSで単層をリンス5ミリリットル0.05％トリプシンを追加し、5分間37 O Cのインキュベーター内でフラスコを置き、5用の400×gで遠心チューブと遠心分離機にフラスコの内容物をピペット分。 </li><li>優しく、上清を除去し、細胞ペレットを破るためにチューブをタップして、新鮮なαMEMの4mlで再懸濁、血球計算板に細胞懸濁液（50μL）およびトリパンブルー（50μL）の混合物を分配し、数を数えます細胞</li><li>膜内に細胞を保持するために、L-PRF膜の上に置か10ミリメートルのガラスクローニングリング内の4×10 5個の細胞種（環は、24時間後に削除することができます）。 </li><li> 4日目に、10分間のPBS三度を有する構築物をすすぎます。 </li><li>各ウェルに無血清培地の1ミリリットルと200μlのMTS試薬を加え、37℃で2時間インキュベートします。 </li><li> 490 nmで200μlのアリコートからの吸光度を測定します。 </li></ol>

<ol>
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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=Flow+cytometric+and+morphological+characterization+of+platelet-rich+plasma+gel.">Flow cytometric and morphological characterization of platelet-rich plasma gel.</a> Clin Oral Implants Re. 17 (6), 687-693 (2006).</li><li>Li, Z., Guan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogels+for+cardiac+tissue+engineering.">Hydrogels for cardiac tissue engineering.</a> Polymer. 3 (2), 740-761 (2011).</li><li>Zhu, J., Cai, B., Ma, Q., Chen, F., Wu, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+bricks-enriched+platelet-rich+plasma+gel+for+injectable+cartilage+engineering+&#8211;+an+in+vivo+experiment+in+nude+mice.">Cell bricks-enriched platelet-rich plasma gel for injectable cartilage engineering &#8211; an in vivo experiment in nude mice.</a> J Tissue Eng Regen Med. 7 (10), 819-830 (2013).</li><li>Dohan, D. 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=Platelet-rich+fibrin+(PRF):+a+second-generation+platelet+concentrate.+Part+I:+technological+concepts+and+evolution.">Platelet-rich fibrin (PRF): a second-generation platelet concentrate. Part I: technological concepts and evolution.</a> Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 101 (3), e37-e44 (2006).</li><li>Dohan, D. 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=Platelet-rich+fibrin+(PRF):+a+second-generation+platelet+concentrate.+Part+II:+platelet-related+biologic+features.">Platelet-rich fibrin (PRF): a second-generation platelet concentrate. Part II: platelet-related biologic features.</a> Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 101 (3), e45-e50 (2006).</li><li>Choukroun, J., 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=Platelet-rich+fibrin+(PRF):+a+second-generation+platelet+concentrate.+Part+V:+histologic+evaluations+of+PRF+effects+on+bone+allograft+maturation+in+sinus+lift.">Platelet-rich fibrin (PRF): a second-generation platelet concentrate. Part V: histologic evaluations of PRF effects on bone allograft maturation in sinus lift.</a> Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 101 (3), 299-303 (2006).</li><li>Dohan Ehrenfest, D. M., Bielecki, T., 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=Do+the+Fibrin+Architecture+and+Leukocyte+Content+Influence+the+Growth+Factor+Release+of+Platelet+Concentrates?+An+Evidence-based+Answer+Comparing+a+Pure+Platelet-Rich+Plasma+(P-PRP)+Gel+and+a+Leukocyte-+and+Platelet-Rich+Fibrin+(L-PRF).">Do the Fibrin Architecture and Leukocyte Content Influence the Growth Factor Release of Platelet Concentrates? An Evidence-based Answer Comparing a Pure Platelet-Rich Plasma (P-PRP) Gel and a Leukocyte- and Platelet-Rich Fibrin (L-PRF).</a> Curr Pharma Biotechno. 13 (7), 1145-1152 (2012).</li><li>Dohan Ehrenfest, D. 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A., 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=Cross-linking+methods+of+electrospun+fibrinogen+scaffolds+for+tissue+engineering+applications.">Cross-linking methods of electrospun fibrinogen scaffolds for tissue engineering applications.</a> Biomed Mater. 3 (4), (2008).</li><li>Gorczyca, G., 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=Preparation+and+characterization+of+genipin+cross-linked+porous+chitosan-collagen-gelatin+scaffolds+using+chitosan-CO2+solution.">Preparation and characterization of genipin cross-linked porous chitosan-collagen-gelatin scaffolds using chitosan-CO2 solution.</a> Carbohydr Poly. 102, 901-911 (2014).</li><li>Rozman, P., Semenic, D., Wislet-Gendebien, S. a. b. i. n. e. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+15.+The+Role+of+Platelet+Gel+in+Regenerative+Medicine.">Chapter 15. 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自己血小板濃縮物が原因の増殖因子の存在量の18再生医療の分野において有望です。しかしながら、これらの製剤は、多くの場合、外科的操作が非常に困難に定義された構造を欠いていました。多くの場合、懸濁液およびゲルは、予測不可能な結果が得られ、送達部位に効果的に保持されません。 L-PRFは、血小板の進化に大きな進歩が、それは本質的に捕捉された血小板としっかりフ...

The use of blood and blood-derived products to seal wounds and improve healing in different clinical situations started with fibrin glues, which are mainly fibrinogen concentrates. Addition of platelets to fibrin glue not only improved their strength but also promoted neoangiogenesis and regeneration. These benefits are attributed to the release of a variety of peptide growth factors from the alpha-granules of platelets upon activation1. Platelet concentrates (PC) were seen as a practical way to deliver growth factors2 and its use was driven by commercial interests rather than research characterization3. In fact, PCs are difficult to characterize unlike homogenous and defined pharmacological preparations, they are a potpourri of signaling molecules and blood cells (platelet and leukocytes) entrapped within a fibrin matrix. Different commercial and proprietary preparations yield a variety of PC that are different in cellular composition, growth factor recovery and kinetics of release4. 
It is important to realize that in most oral surgeries, platelet-rich plasma (PRP) preparations are used as a gel in open surgical wounds and not as platelet suspensions. In these situations, the gelation is induced by the addition of thrombin, calcium chloride, batroxobin or other agents and directly placed in the site of injury5. Due to rapid activation, fibrinogen polymerization is often incomplete and results in friable fibrin gels with very little mechanical strength. In addition, injectable PRP gels undergo rapid fibrinolysis6,7.
In contrast, the processes of blood coagulation (fibrinogen polymerization), platelet enrichment and activation occur simultaneously in the preparation of L-PRF8. The coagulation cascade is triggered when whole blood contacts the walls of a dry glass tube and continues throughout the centrifugation process. This results in the formation of a mechanically-strong blood clot (L-PRF) that can be surgically handled and used. 
Even though L-PRF has been investigated in terms of optimal methods of preparation, growth factor release and cell distribution9-11, detailed mechanical characterization of these membranes are lacking. This is significant gap in knowledge, given the popularity of these membranes in clinical practice as well as its potential to be used as a biomaterial. Current study focusses on the protocol for deriving L-PRF as well as methods that can be employed to study its mechanical properties. This data is intended to serve as baseline for ongoing studies investigating the viscoelastic properties of this interesting natural biomaterial.

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			<td>Needle 19G</td>
			<td>BD</td>
			<td>305186</td>
			<td></td>
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		<tr>
			<td>Needle Disposal Container</td>
			<td>Fisherbrand</td>
			<td>14-827-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Red-Topped Glass Collection Tube</td>
			<td>BD</td>
			<td>8020129</td>
			<td></td>
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		<tr>
			<td>Gauze Pads</td>
			<td>Tyco</td>
			<td>5750</td>
			<td></td>
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			<td>Bandage</td>
			<td>Johnson &#38; Johnson</td>
			<td>5005989</td>
			<td></td>
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		<tr>
			<td>Surshield</td>
			<td>Terumo</td>
			<td>SV*S19BL</td>
			<td>Safety winged infusion set</td>
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			<td>Blood Collection Assembly</td>
			<td>BD</td>
			<td>303380</td>
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			<td>Tourniquets</td>
			<td>BD</td>
			<td>367203</td>
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			<td>Vacutainer</td>
			<td>L42179</td>
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			<td>Intra-Spin System&#160;</td>
			<td>Intra-Lock International</td>
			<td>ISS110</td>
			<td>Centrifuge and Xpression L-PRF FabricationKit&#160;</td>
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			<td>Pipettes (Serological &#38; Micro)</td>
			<td>Corning</td>
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			<td>Scalpel</td>
			<td>Exelint</td>
			<td>29552</td>
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			<td>MTS Bionix 200</td>
			<td>MTS Systems Corporation</td>
			<td></td>
			<td>Material testing systems</td>
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			<td>MTS Test Works 4</td>
			<td>MTS Systems Corporation</td>
			<td></td>
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			<td>Whatman Filter Paper</td>
			<td>Whatman</td>
			<td>1004 070</td>
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			<td>SS Orthodontic ligature wire</td>
			<td>Patterson Dental</td>
			<td>628-4228</td>
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			<td>200 Proof Ethanol</td>
			<td>Koptec</td>
			<td>V1001</td>
			<td></td>
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			<td>Hexamethyldisilazane (HMDS)</td>
			<td>Aldrich</td>
			<td>440191</td>
			<td></td>
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		<tr>
			<td>Aluminium Mounting Stubs</td>
			<td>Ted Pella</td>
			<td>16324</td>
			<td></td>
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		<tr>
			<td>Double Sided Carbon Tape</td>
			<td>PELCO Tabs</td>
			<td>16084-1</td>
			<td></td>
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			<td>Scanning Electron Microscope</td>
			<td>JEOL</td>
			<td>LV 5610</td>
			<td></td>
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			<td>Trypsin</td>
			<td>HyClone</td>
			<td>SH30042.01</td>
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			<td>Cell Culture Incubator</td>
			<td>Thermo Fisher Scientific Inc</td>
			<td>51026282</td>
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			<td>Gibco</td>
			<td>15240-062</td>
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			<td>Genipin</td>
			<td>Wako</td>
			<td>078-03021</td>
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			<td>Cell Culture Media</td>
			<td>Gibco</td>
			<td>12000-022</td>
			<td>Minimum Essential Medium-Alpha</td>
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			<td>MTS Reagent</td>
			<td>Promega</td>
			<td>G1118</td>
			<td></td>
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			<td>PMS Reagent</td>
			<td>Sigma</td>
			<td>P9625</td>
			<td></td>
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			<td>Spectrophotometer</td>
			<td>BioTek</td>
			<td></td>
			<td>Epoch Spectrophotometer</td>
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			<td>10mm Glass Cloning Rings</td>
			<td>Corning</td>
			<td>3166-10</td>
			<td></td>
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		<tr>
			<td>T-75 Flask</td>
			<td>Corning</td>
			<td>430641</td>
			<td></td>
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			<td>DPBS</td>
			<td>Corning</td>
			<td>55-031-PB</td>
			<td></td>
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			<td>Ninhydrin 98%</td>
			<td>Aldrich</td>
			<td>454044</td>
			<td></td>
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		<tr>
			<td>24 Well Plate</td>
			<td>Corning</td>
			<td>3987</td>
			<td></td>
		</tr>
		<tr>
			<td>Biopsy Punch</td>
			<td>Acu Punch</td>
			<td>P1025</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital Micrometer</td>
			<td>Pittsburgh</td>
			<td>68305</td>
			<td></td>
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		<tr>
			<td>Glutaraldehyde</td>
			<td>Sigma</td>
			<td>G6257</td>
			<td></td>
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		<tr>
			<td>12 Well Plate</td>
			<td>Corning</td>
			<td>3336</td>
			<td></td>
		</tr>
		<tr>
			<td>96 Well Plate</td>
			<td>Corning</td>
			<td>3596</td>
			<td></td>
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characterization of leukocyte-platelet rich fibrin, a novel biomaterial

Autologous platelet concentrates represent promising innovative tools in the field of regenerative medicine and have been extensively used in oral surgery. Unlike platelet rich plasma (PRP) that is a gel or a suspension, Leukocyte-Platelet Rich Fibrin (L-PRF) is a solid 3D fibrin membrane generated chair-side from whole blood containing no anti-coagulant. The membrane has a dense three dimensional fibrin matrix with enriched platelets and abundant growth factors. L-PRF is a popular adjunct in surgeries because of its superior handling characteristics as well as its suturability to the wound bed. The goal of the study is to demonstrate generation as well as provide detailed characterization of relevant properties of L-PRF that underlie its clinical success.

（上部、中央および底部）層を、 図2に示されている別のセクションのL- PRFクロットの走査型電子顕微鏡画像。図から分かるように、頂部は無細胞で主にフィブリンネットワークの構成されています。中間層は、それらの活性化と脱顆粒の証拠と血小板と濃縮されています。下層は、白血球およびフィブリンマトリックス内に捕捉された赤血球の混合物を有しています。 <p cla...

Watch this Scientific Journal Video about Characterization of Leukocyte-platelet Rich Fibrin, A Novel Biomaterial at JoVE.com

Characterization of Leukocyte-platelet Rich Fibrin, A Novel Biomaterial

Leucocyte-Platelet Rich Fibrin (L-PRF) represents an FDA cleared preparation of autologous platelet concentrates that possesses unique fibrin architecture, enriched platelets and abundant growth factors. Here, we present a protocol for chair-side generation of L-PRF as well as evaluate its mechanical properties including uniaxial testing and suture retention strength testing.

白血球、血小板リッチフィブリン、新規生体材料の特性評価

Autologous platelet concentrates represent promising innovative tools in the field of regenerative medicine and have been extensively used in oral ...

characterization-of-leukocyte-platelet-rich-fibrin-a-novel-biomaterial

Research

JoVE Journal

Bioengineering

12.9K ビュー.  School of Dentistry, Virginia Commonwealth University. Leucocyte-Platelet Rich Fibrin (L-PRF) represents an FDA cleared preparation of autologous platelet concentrates that possesses unique fibrin architecture, enriched platelets and abundant growth factors. Here, we present a protocol for chair-side generation of L-PRF as well as evaluate its mechanical properties including uniaxial testing and suture retention strength testing. 

ビデオ: Characterization of Leukocyte-platelet Rich Fibrin, A Novel Biomaterial

Encapsulation of Cardiomyocytes in a Fibrin Hydrogel for Cardiac Tissue Engineering

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying cellular responses under various culture conditions in vitro. The three dimensional environment more closely mimics what the cells observe in vivo due to the application of mechanical and chemical stimuli in all dimensions 1. Three-dimensional hydrogels can either be made from synthetic polymers such as PEG-DA 2 and PLGA 3 or a number of naturally occurring proteins such as collagen 4, hyaluronic acid 5 or fibrin 6,7. Hydrogels created from fibrin, a naturally occurring blood clotting protein, can polymerize to form a mesh that is part of the body's natural wound healing processes 8. Fibrin is cell-degradable and potentially autologous 9, making it an ideal temporary scaffold for tissue engineering.
Here we describe in detail the isolation of neonatal cardiomyocytes from three day old rat pups and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. Neonatal myocytes are a common cell source used for in vitro studies in cardiac tissue formation and engineering 4. Fibrin gel is created by mixing fibrinogen with the enzyme thrombin. Thrombin cleaves fibrinopeptides FpA and FpB from fibrinogen, revealing binding sites that interact with other monomers 10. These interactions cause the monomers to self-assemble into fibers that form the hydrogel mesh. Because the timing of this enzymatic reaction can be adjusted by altering the ratio of thrombin to fibrinogen, or the ratio of calcium to thrombin, one can injection mold constructs with a number of different geometries 11,12. Further we can generate alignment of the resulting tissue by how we constrain the gel during culture 13.
After culturing the engineered cardiac tissue constructs for two weeks under static conditions, the cardiac cells have begun to remodel the construct and can generate a contraction force under electrical pacing conditions 6. As part of this protocol, we also describe methods for analyzing the tissue engineered myocardium after the culture period including functional analysis of the active force generated by the cardiac muscle construct upon electrical stimulation, as well as methods for determining final cell viability (Live-Dead assay) and immunohistological staining to examine the expression and morphology of typical proteins important for contraction (Myosin Heavy Chain or MHC) and cellular coupling (Connexin 43 or Cx43) between myocytes.

We describe the isolation of neonatal cardiomyocytes and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. We describe methods for analyzing the tissue engineered myocardium after the culture period including active force generated upon electrical stimulation and cell viability and immunohistological staining.

心臓組織工学のためのフィブリンゲルにおける心筋細胞のカプセル化

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying ...

生物工学

Silk Film Culture System for in vitro Analysis and Biomaterial Design

Silk films are promising protein-based biomaterials that can be fabricated with high fidelity and economically within a research laboratory environment 1,2 . These materials are desirable because they possess highly controllable dimensional and material characteristics, are biocompatible and promote cell adhesion, can be modified through topographic patterning or by chemically altering the surface, and can be used as a depot for biologically active molecules for drug delivery related applications 3-8 . In addition, silk films are relatively straightforward to custom design, can be designed to dissolve within minutes or degrade over years in vitro or in vivo, and are produce with the added benefit of being transparent in nature and therefore highly suitable for imaging applications 9-13. The culture system methodology presented here represents a scalable approach for rapid assessments of cell-silk film surface interactions. Of particular interest is the use of surface patterned silk films to study differences in cell proliferation and responses of cells for alignment 12,14 . The seeded cultures were cultured on both micro-patterned and flat silk film substrates, and then assessed through time-lapse phase-contrast imaging, scanning electron microscopy, and biochemical assessment of metabolic activity and nucleic acid content. In summary, the silk film in vitro culture system offers a customizable experimental setup suitable to the study of cell-surface interactions on a biomaterial substrate, which can then be optimized and then translated to in vivo models. Observations using the culture system presented here are currently being used to aid in applications ranging from basic cell interactions to medical device design, and thus are relevant to a broad range of biomedical fields.

Silk Film Culture System for in vitro Analysis and Biomaterial Design

Silk films are a novel class of biomaterials readily customizable for an array of biomedical applications. The presented silk film culture system is highly adaptable to a variety of in vitro analyses. This system represents a biomaterial design platform offering in vitro optimization before direct translation to in vivo models.

用絹フィルム培養システム in vitroで分析とマテリアルデザイン

Silk films are promising protein-based biomaterials that can be fabricated with high fidelity and economically within a research laboratory environment ...

Observing and Quantifying Fibroblast-mediated Fibrin Gel Compaction

Cells embedded in collagen and fibrin gels attach and exert traction forces on the fibers of the gel. These forces can lead to local and global reorganization and realignment of the gel microstructure. This process proceeds in a complex manner that is dependent in part on the interplay between the location of the cells, the geometry of the gel, and the mechanical constraints on the gel. To better understand how these variables produce global fiber alignment patterns, we use time-lapse differential interference contrast (DIC) microscopy coupled with an environmentally controlled bioreactor to observe the compaction process between geometrically spaced explants (clusters of fibroblasts). The images are then analyzed with a custom image processing algorithm to obtain maps of the strain. The information obtained from this technique can be used to probe the mechanobiology of various cell-matrix interactions, which has important implications for understanding processes in wound healing, disease development, and tissue engineering applications.

Time-lapse microscopy and image processing techniques were used to observe and analyze fibroblast-mediated gel compaction and fibrin fiber realignment in an environmentally controlled bioreactor over a 48 hr&#160;period.

線維芽細胞媒介フィブリンゲル圧縮の観察と定量

Cells embedded in collagen and fibrin gels attach and exert traction forces on the fibers of the gel. These forces can lead to local and global ...

Construction and Characterization of a Novel Vocal Fold Bioreactor

In vitro engineering of mechanically active tissues requires the presentation of physiologically relevant mechanical conditions to cultured cells. To emulate the dynamic environment of vocal folds, a novel vocal fold bioreactor capable of producing vibratory stimulations at fundamental phonation frequencies is constructed and characterized. The device is composed of a function generator, a power amplifier, a speaker selector and parallel vibration chambers. Individual vibration chambers are created by sandwiching a custom-made silicone membrane between a pair of acrylic blocks. The silicone membrane not only serves as the bottom of the chamber but also provides a mechanism for securing the cell-laden scaffold. Vibration signals, generated by a speaker mounted underneath the bottom acrylic block, are transmitted to the membrane aerodynamically by the oscillating air. Eight identical vibration modules, fixed on two stationary metal bars, are housed in an anti-humidity chamber for long-term operation in a cell culture incubator. The vibration characteristics of the vocal fold bioreactor are analyzed non-destructively using a Laser Doppler Vibrometer (LDV). The utility of the dynamic culture device is demonstrated by culturing cellular constructs in the presence of 200-Hz sinusoidal vibrations with a mid-membrane displacement of 40 &#181;m. Mesenchymal stem cells cultured in the bioreactor respond to the vibratory signals by altering the synthesis and degradation of vocal fold-relevant, extracellular matrix components. The novel bioreactor system presented herein offers an excellent in vitro platform for studying vibration-induced mechanotransduction and for the engineering of functional vocal fold tissues.

A novel vocal fold bioreactor capable of delivering physiologically relevant, vibratory stimulation to cultured cells is constructed and characterized. This dynamic culture device, when combined with a fibrous poly(&#949;-caprolactone) scaffold, creates a vocal fold-mimetic environment that modulates the behaviors of mesenchymal stem cells.

新規声帯バイオリアクターの構築とキャラクタリゼーション

In vitro engineering of mechanically active tissues requires the presentation of physiologically relevant mechanical conditions to cultured cells. To ...

Preparation and Characterization of Novel HDL-mimicking Nanoparticles for Nerve Growth Factor Encapsulation

The objective of this article is to introduce preparation and characterization methods for nerve growth factor (NGF)-loaded, high-density, lipoprotein (HDL)-mimicking nanoparticles (NPs). HDLs are endogenous NPs and have been explored as vehicles for the delivery of therapeutic agents. Various methods have been developed to prepare HDL-mimicking NPs. However, they are generally complicated, time consuming, and difficult for industrial scale-up. In this study, one-step homogenization was used to mix the excipients and form the prototype NPs. NGF is a water-soluble protein of 26 kDa. To facilitate the encapsulation of NGF into the lipid environment of HDL-mimicking NPs, protamine USP was used to form an ion-pair complex with NGF to neutralize the charges on the NGF surface. The NGF/protamine complex was then introduced into the prototype NPs. Apolipoprotein A-I was finally coated on the surface of the NPs. NGF HDL-mimicking NPs showed preferable properties in terms of particle size, size distribution, entrapment efficiency, in vitro release, bioactivity, and biodistribution. With the careful design and exploration of homogenization in HDL-mimicking NPs, the procedure was greatly simplified, and the NPs were made scalable. Moreover, various challenges, such as separating unloaded NGF from the NPs, conducting reliable in vitro release studies, and measuring the bioactivity of the NPs, were overcome.

Simple homogenization was used to prepare novel, high-density, lipoprotein-mimicking nanoparticles to encapsulate nerve growth factor. Challenges, detailed protocols for nanoparticle preparation, in vitro characterization, and in vivo studies are described in this article.

神経成長因子カプセル化のための新規HDL模倣ナノ粒子の調製とキャラクタリゼーション

The objective of this article is to introduce preparation and characterization methods for nerve growth factor (NGF)-loaded, high-density, lipoprotein ...

Surface Engineering of Pancreatic Islets with a Heparinized StarPEG Nanocoating

Cell surface engineering can protect implanted cells from host immune attack. It can also reshape cellular landscape to improve graft function and survival post-transplantation. This protocol aims to achieve surface engineering of pancreatic islets using an ultrathin heparin-incorporated starPEG (Hep-PEG) nanocoating. To generate the Hep-PEG nanocoating for pancreatic islet surface engineering, heparin succinimidyl succinate (Heparin-NHS) was first synthesized by modification of its carboxylate groups using N-(3-dimethylamino propyl)-N'-ethyl carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). The Hep-PEG mixture was then formed by crosslinking of the amino end-functionalized eight-armed starPEG (starPEG-(NH2)8) and Heparin-NHS. For islet surface coating, mouse islets were isolated via collagenase digestion and gradient purification using Histopaque. Isolated islets were then treated with ice cold Hep-PEG solution for 10 min to allow covalent binding between NHS and the amine groups of islet cell membrane. Nanocoating with the Hep-PEG incurs minimal alteration to islet size and volume and heparinization of the islets with Hep-PEG may also reduce instant blood-mediated inflammatory reaction during islet transplantation. This "easy-to-adopt" approach is mild enough for surface engineering of living cells without compromising cell viability. Considering that heparin has shown binding affinity to multiple cytokines, the Hep-PEG nanocoating also provides an open platform that enables incorporation of unlimited functional biological mediators and multi-layered surfaces for living cell surface bioengineering.

This protocol aims to achieve surface engineering of pancreatic islets using a heparin-incorporated starPEG nanocoating via pseudo-bioorthogonal chemistry between the N-hydroxysuccinimide groups of the nanocoating and the amine groups of islet cell membrane.

ヘパリン StarPEG ナノコーティングと膵島の表面加工

Cell surface engineering can protect implanted cells from host immune attack. It can also reshape cellular landscape to improve graft function and ...

Fabrication and Characterization of Optical Tissue Phantoms Containing Macrostructure

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By replicating the imaging environment, costly animal experiments to validate a technique may be circumvented. Predicting and optimizing the performance of in vivo and ex vivo imaging techniques requires testing on samples that are optically similar to tissues of interest. Tissue-mimicking optical phantoms provide a standard for evaluation, characterization, or calibration of an optical system. Homogenous polymer optical tissue phantoms are widely used to mimic the optical properties of a specific tissue type within a narrow spectral range. Layered tissues, such as the epidermis and dermis, can be mimicked by simply stacking these homogenous slab phantoms. However, many in vivo imaging techniques are applied to more spatially complex tissue where three dimensional structures, such as blood vessels, airways, or tissue defects, can affect the performance of the imaging system. 
This protocol describes the fabrication of a tissue-mimicking phantom that incorporates three-dimensional structural complexity using material with optical properties of tissue. Look-up tables provide India ink and titanium dioxide recipes for optical absorption and scattering targets. Methods to characterize and tune the material optical properties are described. The phantom fabrication detailed in this article has an internal branching mock airway void; however, the technique can be broadly applied to other tissue or organ structures.

Optical tissue phantoms are essential tools for calibration and characterization of optical imaging systems and validation of theoretical models. This article details a method for phantom fabrication that includes replication of tissue optical properties and three-dimensional tissue structure.

マクロを含む光の組織ファントムの作製と評価

The rapid development of new optical imaging techniques is dependent on the availability of low-cost, customizable, and easily reproducible standards. By ...

Microdissection of Primary Renal Tissue Segments and Incorporation with Novel Scaffold-free Construct Technology

Kidney transplantation is now a mainstream therapy for end-stage renal disease. However, with approximately 96,000 people on the waiting list and only one-fourth of these patients achieving transplantation, there is a dire need for alternatives for those with failing organs. In order to decrease the harmful consequences of dialysis along with the overall healthcare costs it incurs, active investigation is ongoing in search of alternative solutions to organ transplantation. Implantable tissue-engineered renal cellular constructs are one such feasible approach to replacing lost renal functionality. Here, described for the first time, is the microdissection of murine kidneys for isolation of living corticomedullary renal segments. These segments are capable of rapid incorporation within scaffold-free endothelial-fibroblast constructs which may enable rapid connection with host vasculature once implanted. Adult mouse kidneys were procured from living donors, followed by stereoscope microdissection to obtain renal segments 200 - 300 &#181;m in diameter. Multiple renal constructs were fabricated using primary renal segments harvested from only one kidney. This method demonstrates a procedure which could salvage functional renal tissue from organs that would otherwise be discarded.

Tissue-engineered renal constructs provide a solution for the organ shortage and deleterious effects of dialysis. Here, we describe a protocol to micro dissect murine kidneys for isolation of cortico-medullary segments. These segments are implanted into scaffold-free cellular constructs, forming renal organoids.

腎組織のプライマリ セグメントと新規スキャフォールド フリー構造技術と定款の

Kidney transplantation is now a mainstream therapy for end-stage renal disease. However, with approximately 96,000 people on the waiting list and only ...

A Zebrafish Embryo Model for In Vivo Visualization and Intravital Analysis of Biomaterial-associated Staphylococcus aureus Infection

Biomaterial-associated infection (BAI) is a major cause of the failure of biomaterials/medical devices. Staphylococcus aureus is one of the major pathogens in BAI. Current experimental BAI mammalian animal models such as mouse models are costly and time-consuming, and therefore not suitable for high throughput analysis. Thus, novel animal models as complementary systems for investigating BAI in vivo are desired. In the present study, we aimed to develop a zebrafish embryo model for in vivo visualization and intravital analysis of bacterial infection in the presence of biomaterials based on fluorescence microscopy. In addition, the provoked macrophage response was studied. To this end, we used fluorescent protein-expressing S. aureus and transgenic zebrafish embryos expressing fluorescent proteins in their macrophages and developed a procedure to inject bacteria alone or together with microspheres into the muscle tissue of embryos. To monitor bacterial infection progression in live embryos over time, we devised a simple but reliable method of microscopic scoring of fluorescent bacteria. The results from microscopic scoring showed that all embryos with more than 20 colony-forming units (CFU) of bacteria yielded a positive fluorescent signal of bacteria. To study the potential effects of biomaterials on infection, we determined the CFU numbers of S. aureus with and without 10 &#181;m polystyrene microspheres (PS10) as model biomaterials in the embryos. Moreover, we used the ObjectJ project file &#34;Zebrafish-Immunotest&#34; operating in ImageJ to quantify the fluorescence intensity of S. aureus infection with and without PS10 over time. Results from both methods showed higher numbers of S. aureus in infected embryos with microspheres than in embryos without microspheres, indicating an increased infection susceptibility in the presence of the biomaterial. Thus, the present study shows the potential of the zebrafish embryo model to study BAI with the methods developed here.

A Zebrafish Embryo Model for In Vivo Visualization and Intravital Analysis of Biomaterial-associated Staphylococcus aureus Infection

The present study describes a zebrafish embryo model for in vivo visualization and intravital analysis of biomaterial-associated infection over time based on fluorescence microscopy. This model is a promising system complementing mammalian animal models such as mouse models for studying biomaterial-associated infections in vivo.

ゼブラフィッシュ胚モデルにおける生体内可視化と生体材料の生体解析黄色ブドウ球菌感染症

Biomaterial-associated infection (BAI) is a major cause of the failure of biomaterials/medical devices. Staphylococcus aureus is one of the major ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.