NIDDK T32-DK60442（フリーマン）; NIDDK 1K99-DK083616（Mauney）これらの研究は、ボストン小児病院泌尿器科基金の歳入基金と健康補助金NIBIB P41-EB002520（カプラン）の国立研究所によって、部分的に資金を供給した。我々は、膀胱の管の配置と膀胱内圧測定する技術を確立するための支援のためにバーモント大学からピーターZvaraを認める。

<ol>
	<li>Atala, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+engineering+for+bladder+substitution.">Tissue engineering for bladder substitution.</a> World. J. Urol. 18, 364-370 (2000).</li><li>Roehrborn, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Male+lower+urinary+tract+symptoms+(LUTS)+and+benign+prostatic+hyperplasia+(BPH).">Male lower urinary tract symptoms (LUTS) and benign prostatic hyperplasia (BPH).</a> Med. Clin. North Am. 95, 87-100 (2011).</li><li>Niknejad, K. G., Atala, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bladder+augmentation+techniques+in+women.">Bladder augmentation techniques in women.</a> Int. Urogynecol. J. Pelvic Floor Dysfunct. 11, 156-169 (2000).</li><li>Hensle, T. W., Gilbert, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+metabolic+consequences+and+long-term+complications+of+enterocystoplasty+in+children.">A review of metabolic consequences and long-term complications of enterocystoplasty in children.</a> Curr. Urol. Rep. 8, 157-162 (2007).</li><li>Somani, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bowel+dysfunction+after+transposition+of+intestinal+segments+into+the+urinary+tract:+8-year+prospective+cohort+study.">Bowel dysfunction after transposition of intestinal segments into the urinary tract: 8-year prospective cohort study.</a> J. Urol. 177, 1793-1798 (2007).</li><li>Atala, A., Bauer, S. B., Soker, S., Yoo, J. J., Retik, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-engineered+autologous+bladders+for+patients+needing+cystoplasty.">Tissue-engineered autologous bladders for patients needing cystoplasty.</a> Lancet. 367, 1241-1246 (2006).</li><li>Sharma, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+nonhuman+primate+model+for+urinary+bladder+regeneration+using+autologous+sources+of+bone+marrow-derived+mesenchymal+stem+cells.">A nonhuman primate model for urinary bladder regeneration using autologous sources of bone marrow-derived mesenchymal stem cells.</a> Stem Cells. 29, 241-250 (2011).</li><li>Chung, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bladder+reconstitution+with+bone+marrow+derived+stem+cells+seeded+on+small+intestinal+submucosa+improves+morphological+and+molecular+composition.">Bladder reconstitution with bone marrow derived stem cells seeded on small intestinal submucosa improves morphological and molecular composition.</a> J. Urol. 174, 353-359 (2005).</li><li>Ashley, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regional+variations+in+small+intestinal+submucosa+evoke+differences+in+inflammation+with+subsequent+impact+on+tissue+regeneration+in+the+rat+bladder+augmentation+model.">Regional variations in small intestinal submucosa evoke differences in inflammation with subsequent impact on tissue regeneration in the rat bladder augmentation model.</a> BJU Int. 105, 1462-1468 (2010).</li><li>Zhang, Y., Frimberger, D., Cheng, E. Y., Lin, H. K., Kropp, B. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Challenges+in+a+larger+bladder+replacement+with+cell-seeded+and+unseeded+small+intestinal+submucosa+grafts+in+a+subtotal+cystectomy+model.">Challenges in a larger bladder replacement with cell-seeded and unseeded small intestinal submucosa grafts in a subtotal cystectomy model.</a> BJU Int. 98, 1100-1105 (2006).</li><li>Gomez, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+manipulation+of+silk+scaffold+fabrication+parameters+on+matrix+performance+in+a+murine+model+of+bladder+augmentation.">The effect of manipulation of silk scaffold fabrication parameters on matrix performance in a murine model of bladder augmentation.</a> Biomaterials. 32, 7562-7570 (2011).</li><li>Mauney, J. R. <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+gel+spun+silk-based+biomaterials+in+a+murine+model+of+bladder+augmentation.">Evaluation of gel spun silk-based biomaterials in a murine model of bladder augmentation.</a> Biomaterials. 32, 808-818 (2011).</li><li>Persson, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spinal+and+peripheral+mechanisms+contributing+to+hyperactive+voiding+in+spontaneously+hypertensive+rats.">Spinal and peripheral mechanisms contributing to hyperactive voiding in spontaneously hypertensive rats.</a> Am. J. Physiol. 275, 1366-1373 (1998).</li><li>Yaksh, T. L., Durant, P. A., Brent, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micturition+in+rats:+a+chronic+model+for+study+of+bladder+function+and+effect+of+anesthetics.">Micturition in rats: a chronic model for study of bladder function and effect of anesthetics.</a> Am. J. Physiol. 251, 1177-1185 (1986).</li><li>Pandita, R. K., Fujiwara, M., Alm, P., Andersson, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cystometric+evaluation+of+bladder+function+in+non-anesthetized+mice+with+and+without+bladder+outlet+obstruction.">Cystometric evaluation of bladder function in non-anesthetized mice with and without bladder outlet obstruction.</a> J. Urol. 164, 1385-1389 (2000).</li><li>Soler, R., Fullhase, C., Lu, B., Bishop, C. E., Andersson, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bladder+dysfunction+in+a+new+mutant+mouse+model+with+increased+superoxide--lack+of+nitric+oxide.">Bladder dysfunction in a new mutant mouse model with increased superoxide--lack of nitric oxide.</a> J. Urol. 183, 780-785 (2010).</li><li>Matsuura, S., Downie, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+anesthetics+on+reflex+micturition+in+the+chronic+cannula-implanted+rat.">Effect of anesthetics on reflex micturition in the chronic cannula-implanted rat.</a> Neurourol. Urodyn. 19, 87-99 (2000).</li><li>Andersson, K. E., Soler, R., Fullhase, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rodent+models+for+urodynamic+investigation.">Rodent models for urodynamic investigation.</a> Neurourol. Urodyn. 30, 636-646 (2011).</li><li>Soler, R., Fullhase, C., Santos, C., Andersson, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+bladder+dysfunction+in+a+rat+model+of+dopaminergic+brain+lesion.">Development of bladder dysfunction in a rat model of dopaminergic brain lesion.</a> Neurourol Urodyn. 30, 188-193 (2011).</li><li>Streng, T., Santti, R., Andersson, K. E., Talo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+the+rhabdosphincter+in+female+rat+voiding.">The role of the rhabdosphincter in female rat voiding.</a> BJU Int. 94, 138-142 (2004).</li><li>Malmgren, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cystometrical+evaluation+of+bladder+instability+in+rats+with+infravesical+outflow+obstruction.">Cystometrical evaluation of bladder instability in rats with infravesical outflow obstruction.</a> J. Urol. 137, 1291-1294 (1987).</li><li>Smith, P. P., Kuchel, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+uroflow+cystometry+in+the+urethane-anesthetized+mouse.">Continuous uroflow cystometry in the urethane-anesthetized mouse.</a> Neurourol. Urodyn. 29, 1344-1349 (2010).</li><li>Cannon, T. W., Damaser, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+anesthesia+on+cystometry+and+leak+point+pressure+of+the+female+rat.">Effects of anesthesia on cystometry and leak point pressure of the female rat.</a> Life Sci. 69, 1193-1202 (2001).</li><li>Smith, P. P., Hurtado, E., Smith, C. P., Boone, T. B., Somogyi, G. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+cystometric+methods+in+female+rats.">Comparison of cystometric methods in female rats.</a> Neurourol. Urodyn. 27, 324-329 (2008).</li></ol>

利害の衝突が宣言されません。

小さな動物モデルでの注入、膀胱増強後に生体の構成の内圧の評価は、臨床場面で使用するためのマトリックス設計の最適構造と機械的特性を識別する上で重要な検証ステップを表しています。本研究では、機能評価のために設計された臓器の尿流動態特性を決定するために膀胱のマウスおよびラットの増強だけでなく、内圧のテクニックを実行するための手術方法について説明します。我々は重大な問題なしに30 +のげっ歯類から成るそれぞれの実験で、マウスとラットの両方を含む複数の実験では、これらの技術を利用しています。私たちの研究室では、基本的な科学者や医師、外科医の多様なコングロマリットであり、大学院外科研修の少なくとも5-6年の外科医は、これらの実験の手続き的側面を行った。 かかわらず、生体材料使用され、主要なディのタイプのラット対マウスの膀胱を増大させる間にfferenceは、膀胱の大きさです。小さな膀胱の大きさに起因する、生体の解剖と取り込みはマウスでより技術的に困難である。可視化を支援するために、手術用顕微鏡を使用することができます。ラットの膀胱の大きさが大きいので、複数の手順が膀胱（例えば増強および膀胱カテーテルの配置）を実行する必要がある状況に、より適している。さらに、上記のプロトコルは、ラット13、PE-50チューブの使用方法について説明します、しかし、最大PE-100にも大きなサイズのカテーテルは、特に長期試験14に、使用されています。マウスでは、そのようなPE-10チューブなどの小さい口径が15,16を利用することができますが、それはより小さく、より柔軟なチューブを正確にトランスデューサに圧力変化を転送することもできませんことに留意する必要があります。また、（*上記の手順8）を背にカテーテルを固定する別の方法は、マイルで行われます。その小さな体の大きさと鈍先端の針とIVキャップに起因するCEは、あまりにも面倒です。これの欠点は、膀胱内圧測定の前に皮下ポーチにカテーテルの端を抽出するための麻酔の必要性です。 研究では、カテーテルの留置後最初の最初の日（0-4日）で、膀胱内圧測定は、低ボイドボリュームで高い膀胱圧と過活動を明らかにしたことが示されている。これらの知見は、七日目14,17に第六の周りに安定させるために登場し、それゆえ、おそらく内圧評価のための理想的なタイミングです。しかし、文学のほとんどのレポートでは、カテーテル18の第3日以内に膀胱内圧測定を実行して、時間に比例して、上記のパラメータに大きなばらつきは、このアカウント。 3日より長い期間の恥骨上カテーテルを残すことはそれのような結石のリスク、dislodgement、感染症、血尿及び残骸とカテーテルの閉塞などの合併症を運ぶ。 &lt;膀胱内圧測定時のpのクラス= &quot;jove_content&quot;&gt;別の注入速度は、マウスとラット15,16 13,19,20用10-11mL/hrため1-3mL/hrから記載されている。超生理学的注入率は見かけ上の高い圧力は14引き起こす可能性があります。我々は、マウス、我々のセットアップでは、ラットの100μL/分（6 / mLの時間）は12.5μL/分（0.75 mLの/時間）の注入速度を使用していますが、低いレートも利用することができます。暖かい（37℃）生理食塩水が冷溶液を浸透させると誘発膀胱過活​​動を避けるために、より最適ですが、生理食塩水の温度は、少なくとも室温でなければなりません。目を覚まし膀胱内圧測定では、動物が我々の経験で〜10〜20分の期間を必要とケージに調整になると、排尿パターンの安定化を可能にするために重要である。この後、定期的な排尿サイクルが45から120分間、または最小3月4日の排尿サイクルで記録することができます。動物が自由にmovinをあるので、動物は、リアルタイムで観察すべきであるカテーテルのねじれやキンクなどのgと合併症は内圧の解析を変更することができます。膀胱内圧測定時の環境騒音を制限することで、動物の動きとその後のアーティファクトを減少させることが望まれている。無意識の膀胱内圧測定では、目を覚まし膀胱内圧測定などの付随する問題を抱えていないが、複数の麻酔薬は、自発的な膀胱の収縮を阻害することが示されている。この阻害は、麻酔効果がおさまるが、自発的な収縮が14を再開するとき、すなわち麻酔薬の作用の予想される期間に直接対応しています。また、膀胱がオーバーフローしたときに測定した圧力は、膀胱壁のパッシブコンプライアンスの特性への影響を示す、生きていると事後の両方、麻酔ラットにおいて統計的に大きかった。この効果は、吸入ハロタンおよび髄腔内nesacaine 14に加えて、21ペントバルビタールケタミン、およびクロラロースIM / IP、で見られます。様々な麻酔薬、確認のより広範な研究mは、この適度な麻酔レベルは17歳未満の吸入（イソフルランおよびメトキシ）、およびバルビツレート（ペントバルビタールとthiobutabarbital）麻酔薬の両方の排尿反射の抑制を求める。この効果は、フェンタニルとドロペリドールケタミンジアゼパムなどの薬と麻酔のも光や鎮静レベルで観察し、麻酔効果が沈静化として、以前の研究では、したがって、阻害17日でした。それはまた、適切な麻酔17,22を可能にしながら反射排尿が保持されることが実証されているので、この手順では、ウレタンの腹腔内注射を使用することができます。また、全く効果は排尿圧23〜に関して観察されなかった。尿道カテーテルは、より高い膀胱圧の曲線と相対的な膀胱出口部閉塞24と一貫性のある低流量を有することが示されているので、膀胱内圧測定のために恥骨上カテーテルの配置は、ここで説明されています。また、尿道カテーテルは、麻酔動物でのみ使用可能であり、さらにその後、カテーテルは、特に雄のげっ歯類やマウスでは、困難な場合があります。 結論としては、膀胱の増強および/または内圧分析に使用するモデルの選択は、特定の研究の目標に依存しています。技術的な観点からモデルラットは、明らかに前述の理由から、優位性を保持しています。ただし、マウスモデルは遺伝子操作のために彼らの感受性による尿路系疾患、内の特定の遺伝子でエンコードされた最終製品の役割を評価する研究で使用することができます。これは、ラットで一般的に現実的ではありません。 目を覚まし膀胱内圧測定は、最も正確にこれらの動物は、その排尿サイクルを受けるている正常な生理学的状態を模倣するなど、膀胱機能のより信頼性の生理的な決定を与える可能性があります。また、直接の効果の交絡変数膀胱機能のnestheticsは回避されます。

<table border="1"><tbody><tr><td> 材料： </td><td> 説明/用途： </td></tr><tr><td>シェービングばさみ</td><td>手術のラット/マウスの作製</td></tr><tr><td>滅菌ドレープ、betadine、70％エタノール、滅菌ガーゼ</td><td>無菌手術野の準備</td></tr><tr><td></td><td></td><td></td><td colspan="2"> インストゥルメント： </td></tr><tr><td>手術用メスの刃</td><td>皮膚切開</td></tr><tr><td>歯の鉗子</td><td>スキンを操作する</td></tr><tr><td>ファイン鉗子</td><td>非外傷性（無歯）、操作することはセレーションまたは微鋸歯を</td></tr><tr><td>小さな針ドライバ</td><td>シャープ組織解離</td></tr><tr><td> Metzenbaumはさみ</td><td> Bldder切開</td></tr><tr><td>腱はさみ</td><td>撤回縫合糸および皮下トンを開発するunnel（膀胱カテーテル） </td></tr><tr><td>小さな湾曲したクランプ</td><td>皮下トンネル（膀胱カテーテル） </td></tr><tr><td></td><td></td><td></td><td colspan="2"> 縫合： </td></tr><tr><td> 6から0ポリプロピレン縫合糸</td><td>膀胱滞在縫合糸と縫合pursestring </td></tr><tr><td> 7から0ポリグラクチン縫合</td><td>膀胱への足場の吻合</td></tr><tr><td> 4から0ポリグラクチン縫合</td><td>筋肉/皮膚の閉鎖</td></tr><tr><td> 3から0または4から0シルク縫合糸</td><td>皮膚に固定するカテーテル先端</td></tr><tr><td></td><td></td><td></td><td colspan="2"> 注射針や注射器： </td></tr><tr><td> 18ゲージの針</td><td>膀胱カテーテルの膀胱を貫通</td></tr><tr><td> 25と30ゲージの針</td><td>漏れのテスト膀胱</td></tr><tr><td> 1 mLの生理食塩水fはilledシリンジ</td><td></td></tr><tr><td> 22ゲージ鈍先端の針</td><td></td></tr><tr><td></td><td></td><td></td><td colspan="2"> 膀胱カテーテル： </td></tr><tr><td> PE-50チューブ</td><td></td></tr><tr><td>ライター</td><td> PE-50チューブのフレア加工</td></tr><tr><td>小さな湾曲したクランプ</td><td>皮下トンネルを開発</td></tr><tr><td></td><td></td><td></td><td colspan="2"> 膀胱内圧測定： </td></tr><tr><td> MLT844 ADInstruments·データ·キャプチャとLabChartソフトウェア</td><td>圧力データ収集</td></tr><tr><td>ハーバード大学22シリンジポンプ（ハーバード装置、ホリストン、MA） </td><td>輸液ポンプ</td></tr><tr><td></td><td></td><td></td><td colspan="2"> 麻酔薬（無意識の膀胱内圧測定）： </td></tr><tr><td>私はsoflurane </td><td>全身麻酔の導入/メンテナンス</td></tr><tr><td>ウレタン</td><td> Unconconsciousの膀胱内圧測定</td></tr><tr><td> Bupivicaineまたは同等の</td><td>局所麻酔</td></tr><tr><td>メロキシカム</td><td>術後鎮痛</td></tr><tr><td>ブプレノルフィン</td><td>術後鎮痛</td></tr></tbody></table>

evaluation of biomaterials for bladder augmentation using cystometric analyses in various rodent models

尿の腎機能と失禁、低圧力で尿を保存し、正確に収縮を画策してそれを排出する膀胱の適切な機能に決定的に依存しています。二分脊椎/脊髄損傷に続発する後部尿道弁を含む先天性および後天性泌尿器異常、良性前立腺過形成、および神経因性膀胱の数は、障害のあるコンプライアンスと能力の低下を1に主要な病理学的組織の再構築になる可能性があります。尿路の機能的または解剖学的障害は頻繁にこれらの条件に関連付けられており、増加したストレージと排尿圧2から尿失禁と腎臓の損傷につながる可能性があります。臓器の容量を拡大し、膀胱内の圧力を減らすために消化管セグメントの外科的移植は医学的管理は、3が失敗したこれらの疾患の主要な外科的治療法の選択肢を表しています。しかし、このアプローチは妨げである利用可能なドナー組織の制限によるedとは、慢性尿路感染症、代謝摂動、尿路結石の形成、及び4,5 -二次悪性腫瘍を含む重大な合併症に関連付けられています。 膀胱組織工学における現在の研究は大きく、欠陥部位で組織の再生をサポートすることができます生体の構成を識別するに焦点を当てています。このような小腸粘膜下組織とポリグリコール酸などの天然及び合成ポリマーから派生した従来の3-D足場は、尿路上皮および平滑筋再生をサポートするだけでなく、動物モデルの両方で増加した臓器のストレージ容量を容易にし、いくつかの短期的な成功を示しているクリニック6,7。しかし、足場機械的完全性と生体適合性の不足は、しばしばこのようにインプラント失敗のリスクを増加させる有害な線維症8、移植拘9、および石灰化10に示す結果とfoを必要とするrの二次手術。さらに、補強膀胱拡大術の標準的な生体構造を利用した通常の排尿特性の回復が達成されていないが、、この役割を果たすことができる新規なマトリックスの、したがって研究開発が必要とされる。 首尾よく開発し、臨床的膀胱増強のために最適なマテリアルを評価するために、有効性の研究は最初の詳細な手術法と機能的転帰の評価を用いて標準化された動物モデルで実行する必要があります。我々は以前に組織再生と機能の排尿特性を仲介する絹フィブロインベースの足場の可能性を決定するためにマウスの膀胱補強モデルの使用が報告されています。このモデルの11,12内圧の解析は、構造的および機械的インプラントの特性のばらつきが示されている組織工学膀胱11,12の結果尿流動態機能に影響を与えることができます。正の相関マトリックスを介した組織再生の程度との間tionsは、組織学的に決定され、膀胱内圧測定により評価機能のコンプライアンスと容量は、このモデル11,12で示された。これらの結果は、したがって、齧歯類膀胱補強システムにおける生体材料の構成の機能評価は足場のプロパティを評価し、大規模な動物実験と臨床展開する前に、in vivoで実現可能で確立するための有用なフォーマットであることが示唆された。現在の研究では、絹の足場を使用して、マウスとラットの両方で、膀胱の拡張の様々な手術の段階を提示し、覚醒や麻酔膀胱内圧測定するためのテクニックのデモンストレーションを行います。

Watch this Scientific Journal Video about Evaluation of Biomaterials for Bladder Augmentation using Cystometric Analyses in Various Rodent Models at JoVE.com

Evaluation of Biomaterials for Bladder Augmentation using Cystometric Analyses in Various Rodent Models

膀胱拡大の外科的段階は、マウスおよびラットモデルでの3​​-Dの足場を使用して記述されています。膀胱拡大で使用するための生体材料の構成の有効性をテストするには、目を覚ましと麻酔の両方膀胱内圧測定するためのテクニックを紹介しています。

各種齧歯類モデルで内圧分析を用いた膀胱補強のためのバイオマテリアルの評価

Renal function and continence of urine are critically dependent on the proper function of the urinary bladder, which stores urine at low pressure and ...

evaluation-biomaterials-for-bladder-augmentation-using-cystometric

Research

JoVE Journal

Bioengineering

19.1K ビュー.  Harvard Medical School. 膀胱拡大の外科的段階は、マウスおよびラットモデルでの3​​-Dの足場を使用して記述されています。膀胱拡大で使用するための生体材料の構成の有効性をテストするには、目を覚ましと麻酔の両方膀胱内圧測定するためのテクニックを紹介しています。

ビデオ: Evaluation of Biomaterials for Bladder Augmentation using Cystometric Analyses in Various Rodent Models

Fabrication of Micro-tissues using Modules of Collagen Gel Containing Cells

This protocol describes the fabrication of a type of micro-tissues called modules.  The module approach generates uniform, scalable and vascularized tissues.  The modules can be made of collagen as well as other gelable or crosslinkable materials.  They are approximately 2 mm in length and 0.7 mm in diameter upon fabrication but shrink in size with embedded cells or when the modules are coated with endothelial cells.  The modules individually are small enough that the embedded cells are within the diffusion limit of oxygen and other nutrients but modules can be packed together to form larger tissues that are perfusable.  These tissues are modular in construction because different cell types can be embedded in or coated on the modules before they are packed together to form complex tissues.  There are three main steps to making the modules: (1) neutralizing the collagen and embedding cells in it, (2) gelling the collagen in the tube and cutting the modules and (3) coating the modules with endothelial cells.

Creation of micro-tissues using cylindrical collagen gels, called modules, that contain embedded cells and which surface is coated with endothelial cells.

コラーゲンゲル含有する細胞のモジュールを使用してマイクロ組織の作製

This protocol describes the fabrication of a type of micro-tissues called modules.  The module approach generates uniform, scalable and vascularized ...

生物工学

Polymer Microarrays for High Throughput Discovery of Biomaterials

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a combinatorial library of materials to be screened in a parallel, high throughput format1. Herein is described the formation and characterization of a polymer microarray using an on-chip photopolymerization technique 2. This involves mixing monomers at varied ratios to produce a library of monomer solutions, transferring the solution to a glass slide format using a robotic printing device and curing with UV irradiation. This format is readily amenable to many biological assays, including stem cell attachment and proliferation, cell sorting and low bacterial adhesion, allowing the ready identification of 'hit' materials that fulfill a specific biological criterion3-5. Furthermore, the use of high throughput surface characterization (HTSC) allows the biological performance to be correlated with physio-chemical properties, hence elucidating the biological-material interaction6. HTSC makes use of water contact angle (WCA) measurements, atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). In particular, ToF-SIMS provides a chemically rich analysis of the sample that can be used to correlate the cell response with a molecular moiety. In some cases, the biological performance can be predicted from the ToF-SIMS spectra, demonstrating the chemical dependence of a biological-material interaction, and informing the development of hit materials5,3.

A description of the formation of a polymer microarray using an on-chip photopolymerization technique. The high throughput surface characterization using atomic force microscopy, water contact angle measurements, X-ray photoelectron spectroscopy and time of flight secondary ion mass spectrometry and a cell attachment assay is also described.

バイオマテリアルのハイスループット検出用高分子マイクロアレイ

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a ...

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to evaluate new strategies that aim at improving vascularization, reliable methods are required to make the in-growth of new blood vessels into bio-artificial scaffolds visible and quantify the results. Over the past couple of years, our group has introduced a full skin defect model that enables the direct visualization of blood vessels by transillumination and provides the possibility of quantification through digital segmentation. In this model, one surgically creates full skin defects in the back of mice and replaces them with the material tested. Molecules or cells of interest can also be incorporated in such materials to study their potential effect. After an observation time of one&#8217;s own choice, materials are explanted for evaluation. Bilateral wounds provide the possibility of making internal comparisons that minimize artifacts among individuals as well as of decreasing the number of animals needed for the study. In comparison to other approaches, our method offers a simple, reliable and cost effective analysis. We have implemented this model as a routine tool to perform high-resolution screening when testing vascularization of different biomaterials and bio-activation approaches.

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Vascularization is key to approaches in successful tissue engineering. Therefore, reliable technologies are required to evaluate the development of vascular networks in tissue-constructs. Here we present a simple and cost-effective method to visualize and quantify vascularization in vivo.

生体材料の血管新生を評価するために、完全な皮膚欠損モデル<em&gt;インビボ</em

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to ...

Sandwich-like Microenvironments to Harness Cell/Material Interactions

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. However in vivo, most of the cells are completely surrounded by the extracellular matrix (ECM), resulting in a three-dimensional (3D) distribution of receptors. This may trigger differences in the outside-in signaling pathways and thus in cell behavior.
This article shows that stimulating the dorsal receptors of cells already adhered to a 2D substrate by overlaying a film of a new material (a sandwich-like culture) triggers important changes with respect to standard 2D cultures. Furthermore, the simultaneous excitation of ventral and dorsal receptors shifts cell behavior closer to that found in 3D environments. Additionally, due to the nature of the system, a sandwich-like culture is a versatile tool that allows the study of different parameters in cell/material interactions, e.g., topography, stiffness and different protein coatings at both the ventral and dorsal sides. Finally, since sandwich-like cultures are based on 2D substrates, several analysis procedures already developed for standard 2D cultures can be used normally, overcoming more complex procedures needed for 3D systems.

The following protocol describes the procedure to assemble sandwich-like cultures to be used as an intermediate stage between bi-dimensional (2D) and three-dimensional (3D) cellular environments. The engineered systems can have applications in microscopy, biomechanics, biochemistry and cell biology assays.

サンドイッチ状の微小環境は、Cell /材料の相互作用を活用します

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. ...

Synthesis of Multi-walled Carbon Nanotubes Modified with Silver Nanoparticles and Evaluation of Their Antibacterial Activities and Cytotoxic Properties

In this study, multi-walled carbon nanotubes (MWCNTs) were treated with an aqueous sulfuric acid solution to form an oxygen-based functional group. Silver MWCNTs were prepared by the reductive deposition of silver from an aqueous solution of AgNO3 on the oxidized MWCNTs. Given the unique color of the CNTs, it was not possible to apply them to the minimum inhibitory concentration or mitochondrial toxicity assays to evaluate the toxicity and antibacterial properties, since they would interfere with the assays. The inhibition zone and minimum bactericidal concentration for the Ag-MWCNTs were measured and Live/Dead and Trypan Blue assays were used to measure the toxicity and antibacterial properties without interfering with the color of the CNTs.

In this study, antimicrobial nanomaterials were synthesized by acidic oxidation of multiwalled carbon nanotubes and subsequent reductive deposition of silver nanoparticles. Antimicrobial activity and cytotoxicity tests were performed with the as-prepared nanomaterials.

銀ナノ粒子とその抗菌活性と細胞毒性の評価変更多層カーボンナノ チューブの合成

In this study, multi-walled carbon nanotubes (MWCNTs) were treated with an aqueous sulfuric acid solution to form an oxygen-based functional group. Silver ...

Biological Compatibility Profile on Biomaterials for Bone Regeneration

Large non-union bone fractures are a significant challenge in orthopedic surgery. Although auto- and allogeneic bone grafts are excellent for healing such lesions, there are potential complications with their use. Thus, material scientists are developing synthetic, biocompatible biomaterials to overcome these problems. In this study, we present a multidisciplinary platform for evaluating biomaterials for bone repair. We combined expertise from bone biology and immunology to develop a platform including in vitro osteoclast (OC) and osteoblast (OB) assays and in vivo mouse models of bone repair, immunogenicity, and allergenicity. We demonstrate how to perform the experiments, summarize the results, and report on biomaterial biocompatibility. In particular, we tested OB viability, differentiation, and mineralization and OC viability and differentiation in the context of &#946;-tricalcium phosphate (&#946;-TCP) disks. We also tested a &#946;-TCP/Collagen (&#946;-TCP/C) foam which is a commercially available material used clinically for bone repair in a critical-sized calvarial bone defect mouse model to determine the effects on the early phase of bone healing. In parallel experiments, we evaluated immune and allergic responses in mice. Our approach generates a biological compatibility profile of a bone biomaterial with a range of parameters necessary for predicting the biocompatibility of biomaterials used for bone healing and repair in patients.

The number of novel biomaterials engineered for repairing large bone lesions is continuously expanding with the aim to enhance bone healing and overcome the complications associated with bone transplantation. Here, we present a multidisciplinary strategy for pre-clinical biocompatibility testing of biomaterials for bone repair.

骨再生のための生体材料の生物学的互換性プロファイル

Large non-union bone fractures are a significant challenge in orthopedic surgery. Although auto- and allogeneic bone grafts are excellent for healing such ...

Purification and Analytics of a Monoclonal Antibody from Chinese Hamster Ovary Cells Using an Automated Microbioreactor System

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using Chinese hamster ovary (CHO) cells, culture and process conditions must be optimized to maximize antibody titers and achieve target quality profiles. Typically, this optimization uses automated microscale bioreactors (15 mL) to screen multiple process conditions in parallel. Optimization criteria include culture performance and the critical quality attributes (CQAs) of the monoclonal antibody (mAb) product, which may impact its efficacy and safety. Culture performance metrics include cell growth and nutrient consumption, while the CQAs include the mAb's N-glycosylation and aggregation profiles, charge variants, and molecular weight. This detailed protocol describes how to purify and subsequently analyze HCCF samples produced by an automated microbioreactor system to gain valuable performance metrics and outputs. First, an automated protein A fast protein liquid chromatography (FPLC) method is used to purify the mAb from harvested cell culture samples. Once concentrated, the glycan profiles are analyzed by mass spectrometry using a specific platform (refer to the Table of Materials). Antibody molecular weights and aggregation profiles are determined using size exclusion chromatography-multiple angle light scattering (SEC-MALS), while charge variants are analyzed using microchip capillary zone electrophoresis (mCZE). In addition to the culture performance metrics captured during the bioreactor process (i.e., culture viability, cell counts, and common metabolites including glutamine, glucose, lactate, and ammonia), spent media is analyzed to identify limiting nutrients to improve the feeding strategies and overall process design. Therefore, a detailed protocol for the absolute quantification of amino acids by liquid chromatography-mass spectrometry (LC-MS) of spent media is also described. The methods used in this protocol take advantage of high-throughput platforms that are compatible for large numbers of small-volume samples.

A detailed protocol for the purification and subsequent analysis of a monoclonal antibody from harvested cell culture fluid (HCCF) of automated microbioreactors has been described. Use of analytics to determine critical quality attributes (CQAs) and maximizing limited sample volume to extract vital information is also presented.

自動化された微生物叢システムを用いた中国ハムスター卵巣細胞からのモノクローナル抗体の精製と分析

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using ...

Evaluation of Photosynthetic Behaviors by Simultaneous Measurements of Leaf Reflectance and Chlorophyll Fluorescence Analyses

Chlorophyll a fluorescence analysis is widely used to measure photosynthetic behaviors in intact plants, and has resulted in the development of many parameters that efficiently measure photosynthesis. Leaf reflectance analysis provides several vegetation indices in ecology and agriculture, including the photochemical reflectance index (PRI), which can be used as an indicator of thermal energy dissipation during photosynthesis because it correlates with non-photochemical quenching (NPQ). However, since NPQ is a composite parameter, its validation is required to understand the nature of the PRI parameter. To obtain physiological evidence for evaluation of the PRI parameter, we simultaneously measured chlorophyll fluorescence and leaf reflectance in xanthophyll cycle defective mutant (npq1) and wild-type Arabidopsis plants. Additionally, the qZ parameter, which likely reflects the xanthophyll cycle, was extracted from the results of chlorophyll fluorescence analysis by monitoring relaxation kinetics of NPQ after switching the light off. These simultaneous measurements were carried out using a pulse-amplitude modulation (PAM) chlorophyll fluorometer and a spectral radiometer. The fiber probes from both instruments were positioned close to each other to detect signals from the same leaf position. An external light source was used to activate photosynthesis, and the measuring lights and saturated light were provided from the PAM instrument. This experimental system enabled us to monitor light-dependent PRI in the intact plant and revealed that light-dependent changes in PRI differ significantly between the wild type and npq1 mutant. Furthermore, PRI was strongly correlated with qZ, meaning that qZ reflects the xanthophyll cycle. Together, these measurements demonstrated that simultaneous measurement of leaf reflectance and chlorophyll fluorescence is a valid approach for parameter evaluation.

We describe a new technical approach to study photosynthetic responses in higher plants involving simultaneous measurements of chlorophyll a fluorescence and leaf reflectance using a PAM and a spectral radiometer for the detection of signals from the same leaf area in Arabidopsis.

葉反射率とクロロフィル蛍光解析の同時測定による光合成挙動の評価

Chlorophyll a fluorescence analysis is widely used to measure photosynthetic behaviors in intact plants, and has resulted in the development of many ...

Calvarial Model of Bone Augmentation in Rabbit for Assessment of Bone Growth and Neovascularization in Bone Substitution Materials

The basic principle of the rabbit calvarial model is to grow new bone tissue vertically on top of the cortical part of the skull. This model allows assessment of bone substitution materials for oral and craniofacial bone regeneration in terms of bone growth and neovascularization support. Once animals are anesthetized and ventilated (endotracheal intubation), four cylinders made of polyether ether ketone (PEEK) are screwed onto the skull, on both sides of the median and coronal sutures. Five intramedullary holes are drilled within the bone area delimited by each cylinder, allowing influx of bone marrow cells. The material samples are placed into the cylinders which are then closed. Finally, the surgical site is sutured, and animals are awaken. Bone growth may be assessed on live animals by using microtomography. Once animals are euthanized, bone growth and neovascularization may be evaluated by using microtomography, immune-histology and immunofluorescence. As the evaluation of a material requires maximum standardization and calibration, the calvarial model appears ideal. Access is very easy, calibration and standardization are facilitated by the use of defined cylinders and four samples may be assessed simultaneously. Furthermore, live tomography may be used and ultimately a large decrease in animals to be euthanized may be anticipated.

Here we present a surgical protocol in rabbits with the aim to assess bone substitution materials in terms of bone regeneration capacities. By using PEEK cylinders fixed onto rabbit skulls, osteoconduction, osteoinduction, osteogenesis and vasculogenesis induced by the materials may be evaluated either on live or euthanized animals.

骨置換材料における骨成長と新生血管の評価のためのウサギにおける骨増強の卵子モデル

The basic principle of the rabbit calvarial model is to grow new bone tissue vertically on top of the cortical part of the skull. This model allows ...

Primary Clarification of CHO Harvested Cell Culture Fluid using an Acoustic Separator

Primary clarification is an essential step in a biomanufacturing process for the initial removal of cells from therapeutic products within the harvested cell culture fluid. While traditional methods like centrifugation or filtration are widely implemented for cell removal, the equipment for these processes have large footprints and operation can involve contamination risks and filter fouling. Additionally, traditional methods may not be ideal for continuous bioprocessing schemes for primary clarification. Thus, an alternate application using acoustic (sound) waves was investigated to continuously separate cells from the cell culture fluid. Presented in this study is a detailed protocol for using a bench-scale acoustic wave separator (AWS) for the primary separation of culture fluid containing a monoclonal IgG1 antibody from a CHO cell bioreactor harvest. Representative data are presented from the AWS and demonstrate how to achieve effective cell clarification and product recovery. Finally, potential applications for AWS in continuous bioprocessing are discussed. Overall, this study provides a practical and general protocol for the implementation of AWS in primary clarification for CHO cell cultures and further describes its application potential in continuous bioprocessing.

Presented here is a protocol for the primary clarification of CHO cell culture using an acoustic separator. This protocol can be used for the primary clarification of shake flask cultures or bioreactor harvests and has the potential application for continuous clarification of the cell bleed material during perfusion bioreactor operations.

音響セパレータを用いたCHO採取細胞培養液の一次解明

Primary clarification is an essential step in a biomanufacturing process for the initial removal of cells from therapeutic products within the harvested ...