D. H. Kim thanks the Department of Bioengineering at the University of Washington for the new faculty startup fund. D. H. Kim is also supported by the Perkins Coie Award for Discovery, the Wallace H. Coulter Foundation Translational Research Partnership Award, the Washington State Life Science Discovery Fund, and the American Heart Association Scientist Development Grant (13SDG14560076). J. Macadangdang and A. Jiao thank the support from the NIH Bioengineering Cardiovascular Training Grant Fellowship. &#160;Additional support for this work comes from the National Institutes of Health (NIH) grant R01HL111197 to M. Regnier.

وتجرى جميع الإجراءات في درجة حرارة الغرفة (~ 23 درجة مئوية) ما لم يذكر خلاف ذلك. 1. تلفيق سيليكون ماستر <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> رقاقة السيليكون نظيفة مع الإيثانول بنسبة 100٪ أو الزيلين والجافة تحت O 2/2 N الغاز. </li><li style=";text-align:right;direction:rtl"> وضع رقاقة السيليكون في المغطي تدور بسرعات دوران 2،000-4،000 دورة في الدقيقة لإنتاج فيلم سميكة 0.3-0.5 ميكرومتر. </li><li style=";text-align:right;direction:rtl"> نمط الفيلم مقاوم الضوء مع أبعاد الصحيح باستخدام نظام ضوئيه </li><li style=";text-align:right;direction:rtl"> تزج تماما رقائق السليكون مغلفة مقاومة للضوء منقوشة في حجم مناسب من حل مقاوم الضوء النامية. </li><li style=";text-align:right;direction:rtl"> شطف رقائق السليكون مغلفة مقاومة للضوء وضعها مع الماء منزوع الأيونات. </li><li style=";text-align:right;direction:rtl"> لتشكيل صفائف ميكرون الفرعي التلال النطاق مع قرب الجدران الجانبية العمودي، عميق رد الفعل ايون حفر السيليكون يتعرض باستخدام نظام الحفر. </li><li style=";text-align:right;direction:rtl"> إزالة مقاوم الضوء المتبقية عن طريق وضع رقاقة السيليكون في نظام آشر البلازما. </li><li style=";text-align:right;direction:rtl"> قطع السيليكون ثAFERS مع قطع الماس ذات الرؤوس في الماجستير السيليكون بحجم مناسب لاحقة صب متماثلة. </li></ol> 2. تلفيق بوا قالب من السيليكون ماستر ملاحظة: سوف حجم تضاف إلى سيد السيليكون لnanofabrication تختلف تبعا للمنطقة للسيد nanopatterned لتكرارها وكذلك لزوجة اكريليت البولي يوريثين (بوا) حل. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> نظيفة سطح سيد السيليكون مع الإيثانول بنسبة 100٪ أو الزيلين والجافة تحت O 2/2 N الغاز. </li><li style=";text-align:right;direction:rtl"> وضع نمط الماجستير السيليكون الجانب حتى في طبق بتري. </li><li style=";text-align:right;direction:rtl"> للسيد السيليكون مع 2 سم × 2 سم نمط السطح، ماصة 40 ميكرولتر من PUA إلى سطح النمط. </li><li style=";text-align:right;direction:rtl"> وضع ورقة من 4 سم × 4 سم شفافة البوليستر (PET) الفيلم على الاستغناء بوا. </li><li style=";text-align:right;direction:rtl"> اضغط لأسفل على ورقة PET ونشر بوا تحت ورقة عبر مواجهة نمط باستخدام الأسطوانة أو سطح مستو ارتفع (مثل بطاقة) بحيث بأكملهتتناول نمط من prepolymer بوا. </li><li style=";text-align:right;direction:rtl"> وضع سيد السيليكون، prepolymer، وPET حوالي 10 سم تحت ضوء 20 واط (115 V) والأشعة فوق البنفسجية (λ = 365 نانومتر) لمدة 50 ثانية. لكي تكون فعالة، لا يمكن للضوء الأشعة فوق البنفسجية الطول الموجي يكون في أي مكان بين 310-400 نانومتر. شدة الضوء 10-15 ميغاواط / سم 2 على سطح الركيزة. </li><li style=";text-align:right;direction:rtl"> بعد علاج، إزالة الفيلم PET ببطء مع ملقط. بوا يجب نعلق على الفيلم PET مع السلبية للnanopattern الماجستير السيليكون. </li><li style=";text-align:right;direction:rtl"> علاج nanopatterns بوا / PET تحت الأشعة فوق البنفسجية لمدة 12 ساعة على الأقل قبل استخدامها. التعرض المفرط ليست قضية. </li><li style=";text-align:right;direction:rtl"> لتنظيف سادة السيليكون، ووضع فيلم آخر من الدائرة على رأس سيد بدون إضافة بوا وفضح لضوء الأشعة فوق البنفسجية (λ = 365 نانومتر) لمدة 50 ثانية وإزالة الفيلم PET. سيؤدي هذا إلى إزالة أي مونومرات غير المتفاعل. </li><li style=";text-align:right;direction:rtl"> شطف سيد السيليكون مع الإيثانول بنسبة 100٪ أو الزيلين والجافة تحت O 2/2 N الغاز. </li></ol> 3A. Nanopatterning من مادة البولي يوريثين البوليمر </ع&gt; <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إعداد 25 مم الشرائح الزجاجية الدائرية عن طريق وضع في غرفة العلاج بالأوزون لمدة 10 دقيقة. </li><li style=";text-align:right;direction:rtl"> وضع الشرائح الزجاجية المعالجة الأوزون على كتلة صغيرة PDMS لسهولة التعامل. </li><li style=";text-align:right;direction:rtl"> تطبيق طبقة رقيقة من سطح التصاق المروج مع الفرشاة على الشرائح الزجاجية. الهواء الشرائح الزجاجية الجافة لمدة 30 دقيقة. </li><li style=";text-align:right;direction:rtl"> وضع شريحة زجاجية على قطعة من ورق الطابعة. </li><li style=";text-align:right;direction:rtl"> إسقاط الاستغناء 10 ميكرولتر من البولي يوريثان (PU) قبل البوليمر (NOA 76) إلى وسط شريحة زجاجية. تأكد من عدم وجود فقاعات موجودة بعد إضافة. </li><li style=";text-align:right;direction:rtl"> المكان بوا العفن، ونمط وجهه لأسفل، على شريحة زجاجية. تفريق بو موحد في جميع أنحاء سطح شريحة زجاجية من قبل المتداول الأسطوانة الاسطوانة المطاطية على طول العفن بوا. ورقة الطابعة استيعاب البوليمر تجاوز. </li><li style=";text-align:right;direction:rtl"> واط مصباح الأشعة فوق البنفسجية. الوقت البلمرة من بو يعتمد على قوة مصدر للأشعة فوق البنفسجية. </li><li style=";text-align:right;direction:rtl"> إزالة عينة من مصدر ضوء الأشعة فوق البنفسجية وقشر بعناية القالب بوا بو المغلفة من شريحة زجاجية. يعتبر البلمرة من بو جomplete عند القشور العفن بوا نظيفة بعيدا عن العينة والزجاج الشريحة بو له مظهر براق. </li><li style=";text-align:right;direction:rtl"> وضع العينات في الانتهاء مجفف للتخزين لفترة طويلة كما في الشهر. </li></ol> 3B. Nanopatterning بولي (Lactide المشارك-Glycolide) هيدروجيل <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إنشاء قالب PDMS شقة عن طريق خلط بقوة سيليكون قاعدة المطاط الصناعي والمطاط الصناعي سيليكون كيل علاج في نسبة 10:1. </li><li style=";text-align:right;direction:rtl"> صب PDMS مختلطة السلائف الحل في طبق بتري بحيث السلائف PDMS تصل إلى 5 مم حتى حافة الطبق (أي بحيث PDMS هو 5 مم). </li><li style=";text-align:right;direction:rtl"> وضع طبق بتري وPDMS السلائف في مجفف لمدة 1 ساعة لديغا. </li><li style=";text-align:right;direction:rtl"> نقل طبق بتري وPDMS السلائف في الفرن 65 درجة مئوية لمدة 2 ساعة على الأقل لعلاج. </li><li style=";text-align:right;direction:rtl"> بعد الشفاء PDMS، استخدم شفرة حلاقة لقطع PDMS شقة في 3 سم × 3 سم أقسام مربع. وسوف تكون هذه القوالب PDMS شقة تستخدم لاحقا في عملية الزخرفة. </li><li style=";text-align:right;direction:rtl"> نظيفة 25 مم الشرائح الزجاجية الدائرية التي كتبها plالتفوق في ايزوبروبيل لمدة 30 دقيقة في sonicator المياه. </li><li style=";text-align:right;direction:rtl"> الجافة الشرائح الزجاجية تنظيفها تحت O 2/2 N الغاز. </li><li style=";text-align:right;direction:rtl"> إسقاط الاستغناء 100 ميكرولتر من حل PLGA (15٪ ث / ت PLGA في الكلوروفورم) على شريحة زجاجية. </li><li style=";text-align:right;direction:rtl"> وضع قالب PDMS شقة على أعلى PLGA الاستغناء لامتصاص المذيبات والحصول على سطح مستو PLGA. تطبيق الضغط الخفيف (~ 10 كيلو باسكال) عن طريق وضع 200 غرام من الوزن على الجزء العلوي من PDMS لمدة 5 دقائق. </li><li style=";text-align:right;direction:rtl"> قشر ببطء بعيدا العفن PDMS شقة ووضع غطاء زجاجي على طبق مسخن (120 درجة مئوية) لمدة 5 دقائق لإزالة المذيبات المتبقية وزيادة التصاق بين PLGA وغطاء زجاجي. </li><li style=";text-align:right;direction:rtl"> وضع القالب NP بوا على رأس PLGA مسطحة وتطبيق الضغط المستمر (~ 100 كيلو باسكال) والحرارة (120 درجة مئوية) من خلال وضع 1 كجم الوزن على الجزء العلوي من العفن بوا بينما على لوحة التسخين لمدة 15 دقيقة. </li><li style=";text-align:right;direction:rtl"> إزالة الوزن من PLGA غطاء الشريحة والسماح للالطبقات التحتية لتبريد إلى درجة حرارة الغرفة. لا تقم بإزالة العفن بوا حتى كانت الطبقات التحتيةتبريده. </li><li style=";text-align:right;direction:rtl"> بمجرد أن يبرد الطبقات التحتية بما فيه الكفاية، قشر بعناية بعيدا عن العفن NP بوا، وكشف عن التحتية NP PLGA. يجب أن يكون قوام NP PLGA مظهر براق. </li><li style=";text-align:right;direction:rtl"> وضع العينات في الانتهاء مجفف للتخزين لفترة طويلة كما في الشهر. </li></ol> 4. البذر الخليوي والثقافة ملاحظة: هذا البروتوكول يصف ثقافة الأطفال حديثي الولادة myocytes البطين الفئران (NRVMs) والعضلية H7 الإنسان الجذعية الجنينية المستمدة من الخلايا (HESC-CMS) ولكن مصادر خلية أخرى يمكن استخدامها. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> نعلق لل coverslips ANFS (PU أو PLGA) إلى 35 ملم زراعة الأنسجة طبق البوليسترين. ماصة 20 ميكرولتر من نورلاند البصرية لاصق (NOA83H) إلى الجزء السفلي من الطبق وبلطف ضع ساترة ANFS على رأس NOA. تسمح الغراء لنشر وتغطية كامل أسفل ساترة. علاج NOA من خلال تعريض طبق للأشعة فوق البنفسجية لمدة 10 دقيقة. </li><li style=";text-align:right;direction:rtl"> تعقيم ANFS قبل الشطف مع 2 مل من محلول الإيثانول 70٪ مائي لمدة 5 دقائق، مرتين. إزالة البريدthanol بواسطة الشفط. السماح لANFS تماما الجافة الهواء ل~ 1 ساعة تحت مصباح الأشعة فوق البنفسجية التعقيم (λ = 200-290 نانومتر) في خزانة السلامة البيولوجية. </li><li style=";text-align:right;direction:rtl"> ومما يعزز التصاق الخلوية بواسطة طلاء ANFS في فبرونيكتين بين عشية وضحاها. تمييع فبرونيكتين في الماء DI إلى 5 ميكروغرام / مل. ماصة 2 مل من محلول فبرونيكتين في الطبق. وضع في حاضنة عند 37 درجة مئوية و 5٪ CO 2 بين عشية وضحاها (لا يقل عن 6 ساعة). </li><li style=";text-align:right;direction:rtl"> الحصول NRVMs، HESC-CMS، أو خلايا القلب وغيرها من الفائدة وفقا لبروتوكولات السابقة 22. </li><li style=";text-align:right;direction:rtl"> عينة خلية الطرد المركزي في 1000 دورة في الدقيقة لمدة 3 دقائق لخلايا بيليه. </li><li style=";text-align:right;direction:rtl"> إزالة بعناية طاف بواسطة الطموح. تأكد من عدم تعكير صفو بيليه. </li><li style=";text-align:right;direction:rtl"> الخلايا resuspend في وسائل الإعلام ثقافة مناسبة لتركيز 4.6 × 10 6 خلية / مل. </li><li style=";text-align:right;direction:rtl"> ماصة بعناية 200 ميكرولتر من تعليق خلية على ANFS تعقيمها. تأكد من يبقى تعليق الخلية على ساترة. </li><li style=";text-align:right;direction:rtl"> وضع الخلايا في الحاضنة عند 37 درجة مئوية و 5٪CO 2 لمدة 4 ساعة للسماح للخلايا لنعلق على ANFS. </li><li style=";text-align:right;direction:rtl"> إضافة 2 مل من وسائل الإعلام ثقافة الحارة إلى طبق واستبدال الخلايا في الحاضنة تحت نفس الظروف. </li><li style=";text-align:right;direction:rtl"> بعد 24 ساعة، وإزالة وسائل الإعلام، ويغسل مع 2 مل من DPBS مرتين لإزالة الخلايا الزائدة. </li><li style=";text-align:right;direction:rtl"> إضافة 2 مل من وسائل الإعلام ثقافة الحارة إلى طبق واستبدال الخلايا في الحاضنة تحت نفس الظروف. ثقافة الخلايا لنقطة التقاء. استبدال وسائل الإعلام كل يوم. </li></ol>

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Authors have nothing to disclose.

أنسجة القلب ناضجة وظيفيا التي يفتقر إليها سواء في الجسم الحي والتطبيقات في المختبر من هندسة الأنسجة القلبية. الأساليب nanofabrication كفل الموصوفة هنا هي تقنيات قوية لتحقيق المواءمة الخلوية والتأثير على وظيفة الأنسجة العيانية نظرا لقابلية النظام. ويمكن بسهولة أ?...

أمراض القلب والأوعية الدموية هي السبب الرئيسي للمراضة والوفيات في العالم، وتقديم عبئا الاجتماعية والاقتصادية ذات الثقل على 1،17 النظام الصحي العالمي المتوترة بالفعل. هندسة الأنسجة القلبية لديه هدفين مختلفين: (1) لتجديد عضلة القلب التالفة بعد مرض نقص تروية عضلة القلب أو أو (2) لإنشاء نموذج عالي الدقة من القلب في المختبر لفحص المخدرات أو النمذجة المرض. القلب هو جهاز معقد التي يجب أن تعمل باستمرار لتزويد الدم للجسم. يتم ترتيب الهياكل الصفحي المكتظ العضلية والأنسجة الداعمة في أنماط حلزونية في جميع أنحاء جدار القلب 18،19. كما يقترن قلب الكتروميكانيكية 20 بطريقة منسقة للغاية لإخراج الدم بكفاءة إلى الجسم 21. لا تزال العديد من العقبات الرئيسية التي يتعين معالجتها، ومع ذلك، قبل أن موثوق تلخيصها تصميم الطبيعة معقدة في المختبر.أولا، على الرغم من أن وسائل قوية cardiomyocyte التمايز مواصلة تطوير 22، hPSC-مضادة يزال يحمل الظواهر غير ناضجة إلى حد ما. هذه الخصائص الكهروميكانيكية والتشكل أكثرها توافقا مع مستويات الجنين 23. الثانية، عندما يوضع في ظروف الثقافة التقليدية، وفشل كل الجذعية المشتقة الخلايا العضلية الأولية وإلى التجمع في وهياكل الأنسجة مثل الأم. بدلا من ذلك، تصبح الخلايا موجهة بشكل عشوائي ولا يحمل تجمعت على شكل قضيب ظهور عضلة القلب الكبار 24. وخارج الخلية المصفوفة (ECM) البيئة التي تتفاعل الخلايا تلعب دورا هاما في العديد من العمليات الخلوية 11،13،25. يتكون من ECM واضحة المعالم العظة معقدة، الجزيئية والطوبوغرافية التي تؤثر بشكل كبير على بنية ووظيفة خلايا 6،26. داخل القلب، والمحاذاة الخلوية تتابع عن كثب الألياف ECM مقياس متناهي الصغر الكامنة 2. تأثير هذه nanotopographالعظة كال على الخلايا والأنسجة وظيفة، ومع ذلك، لا يزال بعيدا عن المفهوم تماما. الدراسات الأولية للتفاعل خلايا بيولوجية مقياس متناهي الصغر تشير إلى الأهمية المحتملة وتأثير الفرعية ميكرون العظة الطبوغرافية للخلية مما يشير 27، التصاق 28-30، والنمو 31، والتمايز 32،33. ولكن نظرا لصعوبة في تطوير ركائز nanofabricated استنساخه وقابلة للتطوير، مثل هذه الدراسات لا يمكن أن تتكاثر آثار الخلوية متعددة على نطاق المجمع في بيئة ECM الجسم الحي. في هذا البروتوكول، وصفت تقنية nanofabrication واضحة وفعالة من حيث التكلفة لإنتاج السقالات خلية ثقافة محاكاة الأم محاذاة الألياف ECM القلب، مما يسمح لمجموعة واسعة من التحقيقات الرواية من التفاعلات cardiomyocyte مادة بيولوجية. يمكن فهم كيفية تفاعل العضلية مع البيئة ECM النانو تسمح القدرة على التحكم في السلوك الخلوية إلى أكثر من تقليد كثب ظائفه الأنسجة الأمنشوئها. علاوة على ذلك، الطبقات الوحيدة الخلية هي نظام تجريبية مبسطة مقارنة مع هياكل 3D ولكن لا يزال سلوكا متعددة الخلايا المعقدة للتحقيقات الثاقبة والفرز وظيفية 2،34-36. أخيرا، يمكن أن تستخدم مثل هذه السقالات لتحسين وظيفة الخلوية الكسب غير المشروع عندما زرعت في قلب لأغراض التجدد 37.

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			<td class="xl63" height="43" style="height:32.25pt;width:66pt" width="88">Name</td>
			<td class="xl63" style="width:66pt" width="88">Company</td>
			<td class="xl63" style="width:66pt" width="88">Catalogue number</td>
			<td class="xl64" style="width:66pt" width="88">Comments (optional)</td>
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			<td class="xl65" height="43" style="height:32.25pt;width:66pt" width="88">Fibronectin</td>
			<td class="xl65" style="width:66pt" width="88">BD Biosciences</td>
			<td class="xl65" style="width:66pt" width="88">354008</td>
			<td class="xl66" style="width:66pt" width="88"></td>
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			<td class="xl65" height="64" style="height:48.0pt;width:66pt" width="88">NOA 76</td>
			<td class="xl65" style="width:66pt" width="88">Norland Products, Inc.</td>
			<td class="xl65" style="width:66pt" width="88">7606B</td>
			<td class="xl66" style="width:66pt" width="88"></td>
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		<tr height="106" style="height:79.5pt">
			<td class="xl65" height="106" style="height:79.5pt;width:66pt" width="88">Surface Adhesion Promotor (Glass Primer)</td>
			<td class="xl65" style="width:66pt" width="88">Minuta Tech</td>
			<td class="xl65" style="width:66pt" width="88"></td>
			<td class="xl66" style="width:66pt" width="88"></td>
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		<tr height="43" style="height:32.25pt">
			<td class="xl65" height="43" style="height:32.25pt;width:66pt" width="88">PUA</td>
			<td class="xl65" style="width:66pt" width="88">Minuta Tech</td>
			<td class="xl65" style="width:66pt" width="88">MINS-311RM</td>
			<td class="xl66" style="width:66pt" width="88"></td>
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			<td class="xl65" style="width:66pt" width="88">NOVA Electronic Materials</td>
			<td class="xl65" style="width:66pt" width="88">FA01-9900</td>
			<td class="xl66" style="width:66pt" width="88"></td>
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			<td class="xl65" height="22" style="height:16.5pt;width:66pt" width="88">Photoresist</td>
			<td class="xl65" style="width:66pt" width="88">Shipley</td>
			<td class="xl65" style="width:66pt" width="88">SPRT510</td>
			<td class="xl66" style="width:66pt" width="88"></td>
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			<td class="xl65" height="43" style="height:32.25pt;width:66pt" width="88">Photoresist Developer</td>
			<td class="xl65" style="width:66pt" width="88">Shipley</td>
			<td class="xl65" style="width:66pt" width="88">MF320</td>
			<td class="xl66" style="width:66pt" width="88"></td>
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			<td class="xl65" height="85" style="height:63.75pt;width:66pt" width="88">Electron-Beam Lithography System</td>
			<td class="xl65" style="width:66pt" width="88">JEOL</td>
			<td class="xl65" style="width:66pt" width="88">JBX-9300FS</td>
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			<td class="xl65" height="64" style="height:48.0pt;width:66pt" width="88">Etching System</td>
			<td class="xl65" style="width:66pt" width="88">Surface Technology Systems</td>
			<td class="xl65" style="width:66pt" width="88">NP10 8UJ</td>
			<td class="xl66" style="width:66pt" width="88"></td>
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			<td class="xl65" height="64" style="height:48.0pt;width:66pt" width="88">Plasma Asher System</td>
			<td class="xl65" style="width:66pt" width="88">BMR Technology Co.</td>
			<td class="xl65" style="width:66pt" width="88">DSF-200</td>
			<td class="xl66" style="width:66pt" width="88"></td>
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			<td class="xl65" height="43" style="height:32.25pt;width:66pt" width="88">Ozone Cure System</td>
			<td class="xl65" style="width:66pt" width="88">Minuta Tech</td>
			<td class="xl65" style="width:66pt" width="88">MT-UV-O- 08</td>
			<td class="xl66" style="width:66pt" width="88"></td>
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			<td class="xl65" height="43" style="height:32.25pt;width:66pt" width="88">Fusion Cure System</td>
			<td class="xl65" style="width:66pt" width="88">Minuta Tech</td>
			<td class="xl65" style="width:66pt" width="88">MT-UV-A 11</td>
			<td class="xl66" style="width:66pt" width="88"></td>
		</tr>
		<tr height="64" style="height:48.0pt">
			<td class="xl65" height="64" style="height:48.0pt;width:66pt" width="88">NOA 83H</td>
			<td class="xl65" style="width:66pt" width="88">Norland Products, Inc.</td>
			<td class="xl65" style="width:66pt" width="88">8301</td>
			<td class="xl66" style="width:66pt" width="88"></td>
		</tr>
		<tr height="43" style="height:32.25pt">
			<td class="xl65" height="43" style="height:32.25pt;width:66pt" width="88">Spin Coater</td>
			<td class="xl65" style="width:66pt" width="88">Laurel Technology</td>
			<td class="xl65" style="width:66pt" width="88">WS-400-6NPP</td>
			<td class="xl66" style="width:66pt" width="88"></td>
		</tr>
		<tr height="43" style="height:32.25pt">
			<td class="xl65" height="43" style="height:32.25pt;width:66pt" width="88">Skyrol PET Film</td>
			<td class="xl65" style="width:66pt" width="88">SKC Co., Ltd.</td>
			<td class="xl65" style="width:66pt" width="88">23038-59-9</td>
			<td class="xl66" style="width:66pt" width="88"></td>
		</tr>
		<tr height="43" style="height:32.25pt">
			<td class="xl65" height="43" style="height:32.25pt;width:66pt" width="88">25mm Glass Slides</td>
			<td class="xl65" style="width:66pt" width="88">Corning</td>
			<td class="xl65" style="width:66pt" width="88">2948</td>
			<td class="xl66" style="width:66pt" width="88"></td>
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		<tr height="85" style="height:63.75pt">
			<td class="xl65" height="85" style="height:63.75pt;width:66pt" width="88">Sylgard 184 Silicone Elastomer Kit</td>
			<td class="xl65" style="width:66pt" width="88">Dow Corning</td>
			<td class="xl67" style="width:66pt" width="88">6/5/2553</td>
			<td class="xl66" style="width:66pt" width="88"></td>
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		<tr height="64" style="height:48.0pt">
			<td class="xl65" height="64" style="height:48.0pt;width:66pt" width="88">Poly(D,L-lactide-co-glycolide)</td>
			<td class="xl65" style="width:66pt" width="88">Sigma-Aldrich</td>
			<td class="xl65" style="width:66pt" width="88">P2191-1G</td>
			<td class="xl66" style="width:66pt" width="88"></td>
		</tr>
		<tr height="43" style="height:32.25pt">
			<td class="xl65" height="43" style="height:32.25pt;width:66pt" width="88">Chloroform</td>
			<td class="xl65" style="width:66pt" width="88">Sigma-Aldrich</td>
			<td class="xl65" style="width:66pt" width="88">372978-1L</td>
			<td class="xl66" style="width:66pt" width="88"></td>
		</tr>
		<tr height="43" style="height:32.25pt">
			<td class="xl65" height="43" style="height:32.25pt;width:66pt" width="88">500g Weights</td>
			<td class="xl65" style="width:66pt" width="88">Global Insustrial</td>
			<td class="xl65" style="width:66pt" width="88">T9FB503120</td>
			<td class="xl66" style="width:66pt" width="88"></td>
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		<tr height="43" style="height:32.25pt">
			<td class="xl65" height="43" style="height:32.25pt;width:66pt" width="88">Isopropyl Alcohol</td>
			<td class="xl65" style="width:66pt" width="88">EMD Millipore</td>
			<td class="xl65" style="width:66pt" width="88">PX1835-2</td>
			<td class="xl66" style="width:66pt" width="88"></td>
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		<tr height="22" style="height:16.5pt">
			<td class="xl65" height="22" style="height:16.5pt;width:66pt" width="88">Hot Plate</td>
			<td class="xl65" style="width:66pt" width="88">Corning</td>
			<td class="xl65" style="width:66pt" width="88">PC-420D</td>
			<td class="xl66" style="width:66pt" width="88"></td>
		</tr>
		<tr height="22" style="height:16.5pt">
			<td class="xl65" height="22" style="height:16.5pt;width:66pt" width="88">Sonicator</td>
			<td class="xl65" style="width:66pt" width="88">Branson</td>
			<td class="xl65" style="width:66pt" width="88">B2510MTH</td>
			<td class="xl66" style="width:66pt" width="88"></td>
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capillary force lithography for cardiac tissue engineering

لا تزال أمراض القلب والشرايين السبب الرئيسي للوفاة في العالم 1. هندسة الأنسجة القلبية يحمل الكثير من الأمل لتقديم الاكتشافات الطبية الرائدة مع أهداف تطوير أنسجة وظيفية لتجديد القلب، وكذلك في فحوصات المختبر فحص. ومع ذلك، فقد ثبت القدرة على خلق نماذج عالية الدقة من أنسجة القلب صعبة. القلب المصفوفة خارج الخلية (ECM) هو بنية معقدة تتألف من إشارات البيوكيميائية والنشاط الحيوي بدءا من الدقيقة لمقياس متناهي الصغر 2. ظروف التحميل الميكانيكية والتفاعلات خلية ECM المحلية في الآونة الأخيرة تم الاعتراف بها باعتبارها مكونات حيوية في هندسة الأنسجة القلبية 3-5. ويتألف جزء كبير من ECM القلب من ألياف الكولاجين الانحياز بأقطار نانو النطاق الذي يؤثر بشكل كبير العمارة الأنسجة واقتران الكهروميكانيكية 2. للأسف، عدد قليل من الطرق هفه كان قادرا على تقليد تنظيم ألياف ECM وصولا الى مقياس متناهي الصغر. التطورات الأخيرة في تقنيات nanofabrication، ومع ذلك، مكنت تصميم وتصنيع السقالات تحجيم التي تحاكي في الجسم الحي العظة صلابة الهيكلية والركيزة للECM في قلب 6-9. هنا نقدم تطوير اثنين من استنساخه، والعمليات nanopatterning قابلة للمحاذاة الوظيفية للخلايا القلب باستخدام البوليمر بولي حيويا (lactide-CO-glycolide) (PLGA) 8 والبولي يوريثان (PU) البوليمر المستندة فعالة من حيث التكلفة، و. هذه الطبقات التحتية nanofabricated anisotropically (ANFS) تحاكي ECM الكامنة وراء والأنسجة الانحياز منظمة تنظيما جيدا، ويمكن استخدامها للتحقيق في دور nanotopography على مورفولوجيا الخلايا وظيفة 10-14. باستخدام nanopatterned (NP) سيد السيليكون كقالب، ولفق اكريليت البولي يوريثين (بوا) العفن. ثم يتم استخدام هذا القالب لبوا سنوياttern بو أو PLGA هيدروجيل عبر بمساعدة الأشعة فوق البنفسجية أو المذيبات بوساطة الطباعة الحجرية القوة الشعرية (كفل)، على التوالي 15،16. لفترة وجيزة، بو أو PLGA قبل البوليمر هو انخفاض الاستغناء على ساترة الزجاج ويوضع القالب بوا على القمة. لبمساعدة الأشعة فوق البنفسجية كفل، ومن ثم يتعرض بو للأشعة فوق البنفسجية (λ = 250-400 نانومتر) لعلاج. لبوساطة المذيبات كفل، هو منقوش على PLGA باستخدام الحرارة (120 درجة مئوية) وضغط (100 كيلو باسكال). بعد المعالجة، ومقشر القالب بوا قبالة، مخلفا وراءه ANFS للثقافة الخلية. الخلايا الأولية، مثل حديثي الولادة myocytes البطين الفئران، وكذلك الجذعية المشتقة من الخلايا العضلية المحفزة الإنسان، يمكن الحفاظ على ANFS 2.

الرقم 1 هو لمحة التخطيطي لعملية الإنتاج لأساليب تلفيق اثنين. بسبب حيود الضوء الناجم عن تضاريس النانو، ينبغي أن يؤدي nanopatterning في سطح قزحي الألوان إلى ANFS الشكل 2 يصور هذا السطح قزحي الألوان على منقوشة جيدا 25 ملم NP-PU ساترة (الشكل 2A) مع التلال 800 ن?...

Watch this Scientific Journal Video about Capillary Force Lithography for Cardiac Tissue Engineering at JoVE.com

Capillary Force Lithography for Cardiac Tissue Engineering

In this protocol, we demonstrate the fabrication of biomimetic cardiac cell culture substrata made from two distinct polymeric materials using capillary force lithography. The described methods provide a scalable, cost-effective technique to engineer the structure and function of macroscopic cardiac tissues for in vitro and in vivo applications.

الشعرية قوة الطباعة الحجرية لهندسة الأنسجة القلبية

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical ...

capillary-force-lithography-for-cardiac-tissue-engineering

Research

JoVE Journal

Bioengineering

12.3K Views.  University of Washington. In this protocol, we demonstrate the fabrication of biomimetic cardiac cell culture substrata made from two distinct polymeric materials using capillary force lithography. The described methods provide a scalable, cost-effective technique to engineer the structure and function of macroscopic cardiac tissues for in vitro and in vivo applications.

Video: Capillary Force Lithography for Cardiac Tissue Engineering

Fabrication of Biologically Derived Injectable Materials for Myocardial Tissue Engineering

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications. Briefly, decellularized tissue is lyophilized, milled, enzymatically digested, and then brought to physiological pH. The lyophilization removes all water content from the tissue, resulting in dry ECM that can be ground into a fine powder with a small mill. After milling, the ECM powder is digested with pepsin to form an injectable matrix. After adjustment to pH 7.4, the liquid matrix material can be injected into the myocardium. Results of previous characterization assays have shown that matrix gels produced from decellularized pericardial and myocardial tissue retain native ECM components, including diverse proteins, peptides and glycosaminoglycans. Given the use of this material for tissue engineering, in vivo characterization is especially useful; here, a method for performing an intramural injection into the left ventricular (LV) free wall is presented as a means of analyzing the host response to the matrix gel in a small animal model. Access to the chest cavity is gained through the diaphragm and the injection is made slightly above the apex in the LV free wall. The biologically derived scaffold can be visualized by biotin-labeling before injection and then staining tissue sections with a horse radish peroxidase-conjugated neutravidin and visualizing via diaminobenzidine (DAB) staining. Analysis of the injection region can also be done with histological and immunohistochemical staining. In this way, the previously examined pericardial and myocardial matrix gels were shown to form fibrous, porous networks and promote vessel formation within the injection region.

Methods for preparing an injectable matrix gel from decellularized tissue and injecting it into rat myocardium in vivo are described.

تصنيع مواد حقن المشتقة بيولوجيا للهندسة الأنسجة عضلة القلب

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications.  ...

Elastomeric PGS Scaffolds in Arterial Tissue Engineering

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and increasing obesity1. Currently, bypass surgery using autologous vessels, allografts, and synthetic grafts are known as a commonly used for arterial substitutes2. However, these grafts have limited applications when an inner diameter of arteries is less than 6 mm due to low availability, thrombotic complications, compliance mismatch, and late intimal hyperplasia3,4. To overcome these limitations, tissue engineering has been successfully applied as a promising alternative to develop small-diameter arterial constructs that are nonthrombogenic, robust, and compliant. Several previous studies have developed small-diameter arterial constructs with tri-lamellar structure, excellent mechanical properties and burst pressure comparable to native arteries5,6. While high tensile strength and burst pressure by increasing collagen production from a rigid material or cell sheet scaffold, these constructs still had low elastin production and compliance, which is a major problem to cause graft failure after implantation. Considering these issues, we hypothesized that an elastometric biomaterial combined with mechanical conditioning would provide elasticity and conduct mechanical signals more efficiently to vascular cells, which increase extracellular matrix production and support cellular orientation.
The objective of this report is to introduce a fabrication technique of porous tubular scaffolds and a dynamic mechanical conditioning for applying them to arterial tissue engineering. We used a biodegradable elastomer, poly (glycerol sebacate) (PGS)7 for fabricating porous tubular scaffolds from the salt fusion method. Adult primary baboon smooth muscle cells (SMCs) were seeded on the lumen of scaffolds, which cultured in our designed pulsatile flow bioreactor for 3 weeks. PGS scaffolds had consistent thickness and randomly distributed macro- and micro-pores. Mechanical conditioning from pulsatile flow bioreactor supported SMC orientation and enhanced ECM production in scaffolds. These results suggest that elastomeric scaffolds and mechanical conditioning of bioreactor culture may be a promising method for arterial tissue engineering.

Elastomeric PGS scaffolds with vascular smooth muscle cells cultured in a pulsatile flow bioreactor may lead to promising small-diameter arterial constructs with native ECM production in a relatively short culture period.

PGS السقالات المرنة في هندسة الأنسجة الشرياني

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and ...

Plasma Lithography Surface Patterning for Creation of Cell Networks

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular phenomena. To address this requirement, we have developed a plasma lithography technique to manipulate the cellular microenvironment by creating a patterned surface with feature sizes ranging from 100 nm to millimeters. The goal of this technique is to be able to study, in a controlled way, the behaviors of individual cells as well as groups of cells and their interactions.
This plasma lithography method is based on selective modification of the surface chemistry on a substrate by means of shielding the contact of low-temperature plasma with a physical mold. This selective shielding leaves a chemical pattern which can guide cell attachment and movement. This pattern, or surface template, can then be used to create networks of cells whose structure can mimic that found in nature and produces a controllable environment for experimental investigations. The technique is well suited to studying biological phenomenon as it produces stable surface patterns on transparent polymeric substrates in a biocompatible manner. The surface patterns last for weeks to months and can thus guide interaction with cells for long time periods which facilitates the study of long-term cellular processes, such as differentiation and adaption. The modification to the surface is primarily chemical in nature and thus does not introduce topographical or physical interference for interpretation of results. It also does not involve any harsh or toxic substances to achieve patterning and is compatible for tissue culture. Furthermore, it can be applied to modify various types of polymeric substrates, which due to the ability to tune their properties are ideal for and are widely used in biological applications. The resolution achievable is also beneficial, as isolation of specific processes such as migration, adhesion, or binding allows for discrete, clear observations at the single to multicell level.
This method has been employed to form diverse networks of different cell types for investigations involving migration, signaling, tissue formation, and the behavior and interactions of neurons arraigned in a network.

A versatile plasma lithography technique has been developed to generate stable surface patterns for guiding cellular attachment. This technique can be applied to create cell networks including those that mimic natural tissues and has been used for studying several, distinct cell types.

البلازما الطباعة الحجرية الزخرفة السطحية لإنشاء شبكات الخليوي

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular ...

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.

تغليف الخلايا العضلية في هيدروجيل الفايبرين للهندسة الأنسجة القلبية

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

Postproduction Processing of Electrospun Fibres for Tissue Engineering

Electrospinning is a commonly used and versatile method to produce scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo undergo biaxial distension to varying extents such as skin, bladder, pelvic floor and even the hard palate as children grow. In producing scaffolds for these purposes there is a need to develop scaffolds of appropriate biomechanical properties (whether achieved without or with cells) and which are sterile for clinical use. The focus of this paper is not how to establish basic electrospinning parameters (as there is extensive literature on electrospinning) but on how to modify spun scaffolds post production to make them fit for tissue engineering purposes - here thickness, mechanical properties and sterilisation (required for clinical use) are considered and we also describe how cells can be cultured on scaffolds and subjected to biaxial strain to condition them for specific applications.
Electrospinning tends to produce thin sheets; as the electrospinning collector becomes coated with insulating fibres it becomes a poor conductor such that fibres no longer deposit on it. Hence we describe approaches to produce thicker structures by heat or vapour annealing increasing the strength of scaffolds but not necessarily the elasticity. Sequential spinning of scaffolds of different polymers to achieve complex scaffolds is also described. Sterilisation methodologies can adversely affect strength and elasticity of scaffolds. We compare three methods for their effects on the biomechanical properties on electrospun scaffolds of poly lactic-co-glycolic acid (PLGA).
Imaging of cells on scaffolds and assessment of production of extracellular matrix (ECM) proteins by cells on scaffolds is described. Culturing cells on scaffolds in vitro can improve scaffold strength and elasticity but the tissue engineering literature shows that cells often fail to produce appropriate ECM when cultured under static conditions. There are few commercial systems available that allow one to culture cells on scaffolds under dynamic conditioning regimes - one example is the Bose Electroforce 3100 which can be used to exert a conditioning programme on cells in scaffolds held using mechanical grips within a media filled chamber.4 An approach to a budget cell culture bioreactor for controlled distortion in 2 dimensions is described. We show that cells can be induced to produce elastin under these conditions. Finally assessment of the biomechanical properties of processed scaffolds cultured with or without cells is described.

Electrospun scaffolds can be processed post production for tissue engineering applications. Here we describe methods for spinning complex scaffolds (by consecutive spinning), for making thicker scaffolds (by multi-layering using heat or vapour annealing), for achieving sterility (aseptic production or sterilisation post production) and for achieving appropriate biomechanical properties.

المعالجة بعد الإنتاج من الألياف Electrospun للهندسة الأنسجة

Electrospinning is a commonly used and versatile method to produce  scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo ...

Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.	
	Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).	
	Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.	
	In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

Tissue Engineering of a Human 3D in vitro Tumor Test System

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

هندسة الأنسجة ل3D الإنسان<em&gt; في المختبر</em&gt; نظام ورم اختبار

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

الهندسة 3D Cellularized الهلام الكولاجين لتجديد الأنسجة الوعائية

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Preparation of Thermoresponsive Nanostructured Surfaces for Tissue Engineering

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the Langmuir-Schaefer method. Amphiphilic block copolymers composed of polystyrene and PIPAAm (St-IPAAms) were synthesized by reversible addition-fragmentation chain transfer (RAFT) radical polymerization. A chloroform solution of St-IPAAm molecules was gently dropped into a Langmuir-trough apparatus, and both barriers of the apparatus were moved horizontally to compress the film to regulate its density. Then, the St-IPAAm Langmuir film was horizontally transferred onto a hydrophobically modified glass substrate by a surface-fixed device. Atomic force microscopy images clearly revealed nanoscale sea-island structures on the surface. The strength, rate, and quality of cell adhesion and detachment on the prepared surface were modulated by changes in temperature across the lower critical solution temperature range of PIPAAm molecules. In addition, a two-dimensional cell structure (cell sheet) was successfully recovered on the optimized surfaces. These unique PIPAAm surfaces may be useful for controlling the strength of cell adhesion and detachment.

Nanoscaled sea-island surfaces composed of thermoresponsive block copolymers were fabricated by the Langmuir-Schaefer method for controlling spontaneous cell adhesion and detachment. Both the preparation of the surface and the adhesion and detachment of cells on the surface were visualized.

إعداد Thermoresponsive ذات البنية النانومترية السطوح للهندسة الأنسجة

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the ...

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and trade one defect for another. Tissue engineering holds the promise to address this increasing demand. However, most tissue engineering strategies fail to generate stable and functional tissue substitutes because of poor vascularization. This paper focuses on an in vivo tissue engineering chamber model of intrinsic vascularization where a perfused artery and a vein either as an arteriovenous loop or a flow-through pedicle configuration is directed inside a protected hollow chamber. In this chamber-based system angiogenic sprouting occurs from the arteriovenous vessels and this system attracts ischemic and inflammatory driven endogenous cell migration which gradually fills the chamber space with fibro-vascular tissue. Exogenous cell/matrix implantation at the time of chamber construction enhances cell survival and determines specificity of the engineered tissues which develop. Our studies have shown that this chamber model can successfully generate different tissues such as fat, cardiac muscle, liver and others. However, modifications and refinements are required to ensure target tissue formation is consistent and reproducible. This article describes a standardized protocol for the fabrication of two different vascularized tissue engineering chamber models in vivo.

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling.

هندسة الأنسجة عن طريق الجوهرية الأوعية الدموية في<em&gt; في فيفو</em&gt; غرفة هندسة الأنسجة

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and ...

A Protocol for Decellularizing Mouse Cochleae for Inner Ear Tissue Engineering

In mammals, mechanosensory hair cells that facilitate hearing lack the ability to regenerate, which has limited treatments for hearing loss. Current regenerative medicine strategies have focused on transplanting stem cells or genetic manipulation of surrounding support cells in the inner ear to encourage replacement of damaged stem cells to correct hearing loss. Yet, the extracellular matrix (ECM) may play a vital role in inducing and maintaining function of hair cells, and has not been well investigated. Using the cochlear ECM as a scaffold to grow adult stem cells may provide unique insights into how the composition and architecture of the extracellular environment aids cells in sustaining hearing function. Here we present a method for isolating and decellularizing cochleae from mice to use as scaffolds accepting perfused adult stem cells. In the current protocol, cochleae are isolated from euthanized mice, decellularized, and decalcified. Afterward, human Wharton&#39;s jelly cells (hWJCs) that were isolated from the umbilical cord were carefully perfused into each cochlea. The cochleae were used as bioreactors, and cells were cultured for 30 days before undergoing processing for analysis. Decellularized cochleae retained identifiable extracellular structures, but did not reveal the presence of cells or noticeable fragments of DNA. Cells perfused into the cochlea invaded most of the interior and exterior of the cochlea and grew without incident over a duration of 30 days. Thus, the current method can be used to study how cochlear ECM affects cell development and behavior.

The goal of this protocol is to demonstrate an effective method to decellularize and decalcify mouse cochleae for utilization as scaffolds for tissue engineering applications.

بروتوكول ديسيلولاريزينج كوكليا الماوس لهندسة الأنسجة الإذن الداخلية

In mammals, mechanosensory hair cells that facilitate hearing lack the ability to regenerate, which has limited treatments for hearing loss. Current ...