وقد تم تمويل هذا المشروع من قبل جمعية البحوث الألمانية (DFG، وسعادة 4578/3-1).

A. الغضروف خزعة (غرفة الجراحة؛ الخطوات 1-5 في غرفة تحضير غير معقمة) <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إجراء مراقبة الوزن النهائي للأرنب (نيوزيلندا الأرنب الأبيض، أنثى، 3،5-4،0 كجم من وزن الجسم، 6 أشهر من العمر) من أجل أن تكون قادرة على المخدرات جرعة بشكل صحيح ومراقبة الوزن اللاحقة لعملية جراحية. </li><li style=";text-align:right;direction:rtl"> لحث على التخدير لأرنب عن طريق الحقن في الوريد من 10 مغ / كغ البروبوفول. </li><li style=";text-align:right;direction:rtl"> بعد التنبيب، والحفاظ على التخدير مع 1.5 ملغ / كغ / دقيقة البروبوفول و 0.05 مغ / كغ الفنتانيل عن طريق الوريد. رصد التخدير باستخدام capnography، التأكسج النبض ومعدل النبض. </li><li style=";text-align:right;direction:rtl"> يحلق في الركبة وسيتم تشغيلها على مع المقص الكهربائي والفراغ الفراء. </li><li style=";text-align:right;direction:rtl"> تطهير الركبة حليق تماما، وتغطي بقية الأرانب بضمادة معقمة. </li><li style=";text-align:right;direction:rtl"> جس الرضفة وإجراء شق الجلد بشكل إعلامي من الرضفة. </li><li style=";text-align:right;direction:rtl"> فتح مفصل الركبة قبل بضع المفصل المحيط بالرضفة الإنسي تحت ظروف معقمة. في محاولة لتجنب قطع أي سو الصغيرةالأوعية الدموية perficial. </li><li style=";text-align:right;direction:rtl"> تهجير الرضفة أفقيا. </li><li style=";text-align:right;direction:rtl"> فحص مفصل الركبة لأي الشذوذ والآفات العيانية الغضروف. </li><li style=";text-align:right;direction:rtl"> بعناية قشر قطعة الغضروف الصغيرة (2-3 ملم) من بكرة عظم الفخذ مع مشرط معقم (الشكل 1). </li><li style=";text-align:right;direction:rtl"> وضع على الفور هذه الخزعات غضروف في أنبوب مل 50 مع المتوسطة كاملة (DMEM + 10٪ مصل العجل الجنين (FCS) + 1٪ البنسلين / الستربتوميسين (القلم / بكتيريا)) إلى مواصلة ثقافة في المختبر مباشرة بعد العملية. </li><li style=";text-align:right;direction:rtl"> تنظيف العيوب وشطف لهم مع ملحي معقم. </li><li style=";text-align:right;direction:rtl"> إعادة الرضفة داخل أخدود البكرة. </li><li style=";text-align:right;direction:rtl"> إغلاق الجرح في طبقات مع الغرز زر واحد (VICRYL 4-0) وخياطة الجلدي المستمرة (Monocryl 4-0). استخدام مواد خياطة الجروح للامتصاص. </li><li style=";text-align:right;direction:rtl"> وأخيرا، ختم الجرح مع رذاذ خلع الملابس نفاذا إلى بخار الماء. </li></ol> B. في المختبر الثقافة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> معالجة الخزعة جقطعة artilage في أقرب وقت ممكن بعد العملية في تدفق الصفحي غطاء نسيج الثقافة العقيمة. </li><li style=";text-align:right;direction:rtl"> غسل العقيمة المقطوع الغضروف 2X في برنامج تلفزيوني مع الطرد المركزي في 500 x ج لاحقة لمدة 3 دقائق على RT. </li><li style=";text-align:right;direction:rtl"> قطع الغضروف في قطعة من 1 مم 3، ونقل إلى 50 مل فالكون وهضمها على شاكر لمدة 30 دقيقة مع 10 مل التربسين EDTA-(0.25٪)، و 10 مل PBS لمدة 30 دقيقة </li><li style=";text-align:right;direction:rtl"> بعد 30 دقيقة، ووقف trypsinization بإضافة المتوسطة كاملة. ثم، يهز قطعة غضروف لآخر لمدة 12 ساعة مع كولاجيناز A (0.21 U / ملغ) في المتوسط ​​مجانا المصل (DMEM + 1٪ القلم / بكتيريا). </li><li style=";text-align:right;direction:rtl"> أجهزة الطرد المركزي في الحل الناتج في 170 x ج لمدة 3 دقائق على RT. </li><li style=";text-align:right;direction:rtl"> Resuspend الكريات خلية في 5 مل المتوسطة كاملة. </li><li style=";text-align:right;direction:rtl"> البذور غضروفية المعزولة إلى 25 سم 2 قوارير زراعة الأنسجة في 5 مل المتوسطة كاملة والبقاء على ثقافة الأنسجة ترطيب حاضنة عند 37 درجة مئوية، 5٪ CO 2. </li><li style=";text-align:right;direction:rtl"> تنمو غضروفية لكثافة 80٪. </li><li style=";text-align:right;direction:rtl"> الإفراج عن جمعة الخلاياالقوارير المضغوطة عن طريق الغسيل في برنامج تلفزيوني قبل تعريض 2X إلى 0.05٪ التربسين EDTA-لمدة 3 دقائق. مرة واحدة غضروفية فصل، ووقف trypsinization بإضافة من 10 ميكرولتر المتوسطة كاملة. </li><li style=";text-align:right;direction:rtl"> الطرد المركزي تعليق في 300 x ج (3 دقائق) وبيليه resuspend في المتوسط ​​كاملة. </li><li style=";text-align:right;direction:rtl"> خلايا البذور في قوارير زراعة الأنسجة (75 سم 2) والانقسام في نسبة 1:3 (~ 5،000 خلية / سم 2) كل يوم 5 أو عشر على 80٪ confluency. </li><li style=";text-align:right;direction:rtl"> تحديد أعداد الخلايا وقدرتها على البقاء من قبل التريبان الأزرق تلطيخ. </li></ol> C. الخلوي البذر من مصفوفة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> قبل أن يغسل زرع الخلايا والإفراج كما هو موضح أعلاه. تنفيذ عدد خلايا ونقل 5 × 10 4 خلايا في أنبوب microcentrifuge 1.5 مل. ثم الطرد المركزي في 500 x ج لمدة 5 دقائق على RT. </li><li style=";text-align:right;direction:rtl"> بيليه resuspend في 15 ميكرولتر المتوسطة كاملة لالتشبع الكامل للمصفوفة. </li><li style=";text-align:right;direction:rtl"> قطع طبقة ثنائية الكولاجين سقالة للأبعاد الدقيق للخلل البكرية بواسطة خزعة العقيمةلكمة. </li><li style=";text-align:right;direction:rtl"> مكان المصفوفة إلى الحجم المناسب الأنسجة صحن الثقافة (على سبيل المثال لوحات 24 أيضا). </li><li style=";text-align:right;direction:rtl"> خلايا البذور على مسامية، الجانب الخلية لاصقة من المصفوفة. هذا تردع الهجرة الخلية بعيدا عن المصفوفة. </li><li style=";text-align:right;direction:rtl"> ترك المصفوفات المصنفة الخلية دون مزيد من الخلايا المتوسطة الثقافة في حاضنة لمدة 1 ساعة للسماح للالتزام الكامل للغضروفية. </li><li style=";text-align:right;direction:rtl"> ثم، مكان الخلية مصفوفة يبني في حاويات زراعة (على سبيل المثال لوحات 24 أيضا) مع المتوسطة كاملة جديدة. والحرص على عدم غسل خارج أي خلايا قبالة مصفوفة على الرغم من الالتزام القوي من المتوقع. </li><li style=";text-align:right;direction:rtl"> الآن، يمكن أن تؤخذ خلية مصفوفة بنيات إلى غرفة الجراحة لإعادة التوطين. </li></ol> D. مصفوفة بمساعدة زرع خلية غضروفية ذاتي <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> أداء بضع المفصل المحيط بالرضفة إلى الركبة المقابل كما هو موضح أعلاه (A. 1-8). </li><li style=";text-align:right;direction:rtl"> إنشاء اثنين من العيوب غضروفي معزولة في الأخدود البكرية مع operatin الهواء العقيمز قوة الحفر (3.6 مليمتر في القطر). </li><li style=";text-align:right;direction:rtl"> تنظيف عيب وشطف مع ملحي معقم. </li><li style=";text-align:right;direction:rtl"> وينبغي إيلاء اهتمام لا لفتح تجويف نخاع في أي وقت. إذا لزم الأمر، وختم الجزء السفلي من العيوب مع مكواة الكهربائية ضد النزيف. </li><li style=";text-align:right;direction:rtl"> زرع المصفوفات المصنف مع غضروفية مثقف مع مسامية الجانب السلبي الوجه (الشكل 2). ثم الصحافة تنسجم مع الحفر وتدفق سطح المحيط. </li><li style=";text-align:right;direction:rtl"> ختم المصفوفات مزروع مع قليلا من الغراء الليفين (Tissucol الثنائي S) لضمان تثبيت آمن. </li><li style=";text-align:right;direction:rtl"> بعد تخثر الدم، انتقال الرضفة داخل أخدود البكرية وتطبيق مجموعة كاملة من الحركة لمفصل الركبة عدة مرات. </li><li style=";text-align:right;direction:rtl"> تهجير الرضفة أفقيا مرة أخرى والتحقق من أي حدث أو علامة على عدم استقرار تثبيت. يجب أن تعقد يزال المصفوفات في المكان. </li><li style=";text-align:right;direction:rtl"> استبدال الرضفة مرة أخرى، وإنهاء العملية مع إغلاق الجرح في طبقات ورذاذ خلع الملابس كما هو موضح أعلاه. </li></ol>

<ol>
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الكتاب ليس لديهم ما يكشف.

يوفر بروتوكول المعروضة على تأسيس 9،12،13 واستنساخه بسهولة تقنية لعزل غضروفية ذاتي لالانتشار اللاحقة وإعادة غرس في عيوب الغضروف مصطنع في الركبتين أرنب. استخدام غضروفية ذاتي لإعادة وإصلاح الآفات الغضروف المفصلي هو بالفعل في الاستخدام السريري تقديم نتائج مرضية ع?...

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

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

تعتبر عيوب الغضروف المفصلي مشكلة صحية كبيرة بسبب الغضروف المفصلي لديه قدرة محدودة على التجدد الذاتي 1. الآفات الغضروف دون علاج يؤدي إلى آلام مستمرة، وتؤثر سلبا على نوعية الحياة ويؤهب لالتهاب المفاصل. خلال العقود الماضية، وقد تم تطوير العديد من التقنيات الجراحية لعلاج هذه الآفات. ولكن، حتى الآن لم يكن من الممكن لتحقيق إصلاح كامل من حيث تغطية الخلل مع زجاجي الغضروف المفصلي أو توفير الانتعاش مرضية على المدى الطويل 2-4. ولذلك، لا تزال هناك إصابات الغضروف المفصلي هدفا رئيسيا لتقنيات التجدد مثل هندسة الأنسجة. وعلى النقيض من التقنيات الجراحية الأخرى، والتي غالبا ما تؤدي إلى تكوين أنسجة ليفية أو الليفية الغضروفية، ويهدف هندسة الأنسجة في استعادة كامل البنية المعقدة وخواص الغضروف المفصلي الأصلي باستخدام إمكانات مكون للغضروف من الخلايا المزروعة. Rفتحت التطورات ecent آفاقا واعدة لعلاج الغضروف التجدد. تم تنفيذ النهج القائم على الخلية الأولى لعلاج الغضروف كامل سمك أو آفات عظمي غضروفي في عام 1994 من قبل لارس بيترسون وماتس Brittberg الذي كان رائدا السريرية زرع خلية غضروفية ذاتي (ACI) 5. اليوم، يتم سريريا راسخة تقنية لعلاج عيوب غضروف زجاجي كبير في الركبة، والحفاظ على نتائج سريرية جيدة حتى بعد مرور 10 إلى 20 سنة غرس 6. في السنوات الأخيرة، وغرس غضروفية ذاتي خضع لعملية التقدم السريع. أصبح استخدام وسيلة ثلاثية الأبعاد الكولاجين مصفوفة الاصطناعية التي يتم زراعتها الخلايا بعد ذلك أكثر وأكثر شعبية 7-9. MACT تضم اثنين من العمليات الجراحية: أولا، من أجل جمع غضروفية، خزعة الغضروف يحتاج إلى أن يؤديها من منطقة الغضروف تحمل الوزن غير روقال انه مفصل الركبة. ثم، يتم استخراج غضروفية، وتنقيته وتوسيعها لعدد الخلايا في المختبر كافية. ثم هي المصنفة غضروفية على مصفوفة ثلاثية الأبعاد، ويمكن بعد ذلك يتم إعادة زرعها. عند إعداد زرع الأنسجة المهندسة، ومعدل الانتشار وقدرة التفريق الحاسم لتجديد الأنسجة ناجحة 10. ويعتقد أن استخدام مصفوفة ثلاثية الأبعاد باعتبارها الناقل الخليوي لدعم هذه الخصائص الخلوية 11. سوف بروتوكول التالية تلخيص ويبرهن على وجود تقنية لعزل غضروفية من الخزعات الغضروف، انتشارها في المختبر، وبذر بهم في الصعود إلى 3D مصفوفة (غضروفي جايد، المواد الحيوية Geistlich، Wollhusen، سويسرا). وأخيرا، سوف يمكن وصفها غرس للبنيات الخلية مصفوفة في عيوب غضروفي مصطنع من مفصل الركبة والأرنب. هذه التقنية يمكن استخدامها في بيئة تجريبيةلمزيد من التجارب من إصلاح الغضروف.

يسمح تقنية جراحية وصفت العزلة الناجحة وغرس غضروفية ذاتي إلى خلل غضروفي الاصطناعي. نتج عن الإعداد التجريبية في الاندماج الناجح للزرع في الغضروف المحيطة بها. بعد 12 أسبوعا في الجسم الحي، وقد شغل العيب غضروفي بواسطة إصلاح الأنس...

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

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

يوصف تقنية تجريبية لعلاج العيوب غضروفي في مفصل الركبة الأرنب. غرس غضروفية ذاتي المصنف على مصفوفة هو وسيلة مقبولة تماما لإعادة عرض وإصلاح الآفات الغضروف المفصلي تقديم نتائج مرضية على المدى الطويل. مصفوفة بمساعدة زرع خلية غضروفية ذاتي (MACT) يقدم طريقة زرع موحدة، وأنشأ سريريا.

مصفوفة بمساعدة زرع خلية غضروفية ذاتي لإعادة عرض وإصلاح العيوب غضروفي في نموذج الأرنب

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

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

Research

JoVE Journal

Medicine

14.3K Views.  Klinikum rechts der Isar der Technischen Universität München. يوصف تقنية تجريبية لعلاج العيوب غضروفي في مفصل الركبة الأرنب. غرس غضروفية ذاتي المصنف على مصفوفة هو وسيلة مقبولة تماما لإعادة عرض وإصلاح الآفات الغضروف المفصلي تقديم نتائج مرضية على المدى الطويل. مصفوفة بمساعدة زرع خلية غضروفية ذاتي (MACT) يقدم طريقة زرع موحدة، و?...

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

A Mouse Model of in Utero Transplantation

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

A Mouse Model of in Utero Transplantation

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

نموذج الفأر من في الرحم زراعة

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

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

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

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

تطوير النزلات الشعبية مسد في نموذج الفئران من زرع الرئة Orthotopic

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

A Mouse Fetal Skin Model of Scarless Wound Repair

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

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

نموذج الجلد ماوس الجنين من إصلاح الجرح Scarless

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

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

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

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

بالون إصابة الفئران السباتي نموذج لاختبار التداوي إعادة عرض لمكافحة الأوعية الدموية

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

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

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

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

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

العلاج الجيني بوساطة ناقلات لينتيفيرال من خلايا الكبد فيفو السابقين للزرع الذاتي في الخنازير

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

Transthoracic Echocardiographic Examination in the Rabbit Model

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

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

فحص صدى القلب عبر الصدر في نموذج الأرنب

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

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

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

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

زرع ظهارة صبغة الشبكية في نموذج الرئيسيات غير البشرية لأمراض الشبكية التنكسية

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

A Simplified Model for Heterotopic Heart Valve Transplantation in Rodents

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

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

نموذج مبسط لزرع صمام القلب غير المتجانس في القوارض

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

Left Lung Orthotopic Transplantation in a Juvenile Porcine Model for ESLP

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

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

زرع تقويم الرئة اليسرى في نموذج خنزير الأحداث ل ESLP

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

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

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

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

استبدال الصمام الرئوي عبر القسطرة من التامور الذاتي بدعامة نيتينول ذاتية التوسع في نموذج الأغنام البالغة

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