المؤلف يشكر الدكتور جين قاو لتخليق PGS ، والدكتور بيتر Crapo للمناقشة متعمقة لإعداد مفاعل حيوي ، الدكاترة. محمد Ezzelarab ووو وى لقرد البابون explanting الشرايين السباتية. وأيد هذه الدراسة عن طريق منحة من المعاهد القومية للصحة (R01 HL089658).

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	<li>The American Heart Association Statistics Committee Stroke Statistics Subcommittee. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=American+Heart+Association:+Heart+disease+and+stroke+statistics-2009+update.">American Heart Association: Heart disease and stroke statistics-2009 update.</a> Circulation. 119, 480-486 (2009).</li><li>Isenberg, B. C., Williams, C., Tranquillo, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small-diameter+artificial+arteries+engineered+in+vitro.">Small-diameter artificial arteries engineered in vitro.</a> Circ Res. 98, 25-35 (2006).</li><li>Conte, M. 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M., Gao, J., Wang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seamless+tubular+poly(glycerol+sebacate)+scaffolds:+high-yield+fabrication+and+potential+applications.">Seamless tubular poly(glycerol sebacate) scaffolds: high-yield fabrication and potential applications.</a> J Biomed Mater Res A. 86, 354-363 (2008).</li><li>Gao, J., Ensley, A. E., Nerem, R. M., Wang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poly(glycerol+sebacate)+supports+the+proliferation+and+phenotypic+protein+expression+of+primary+baboon+vascular+cells.">Poly(glycerol sebacate) supports the proliferation and phenotypic protein expression of primary baboon vascular cells.</a> J Biomed Mater Res A. 83, 1070-1075 (2007).</li><li>Murphy, W. L., Dennis, R. G., Kileny, J. L., Mooney, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salt+fusion:+an+approach+to+improve+pore+interconnectivity+within+tissue+engineering+scaffolds.">Salt fusion: an approach to improve pore interconnectivity within tissue engineering scaffolds.</a> Tissue Eng. 8, 43-52 (2002).</li><li>Gao, J., Crapo, P. M., Wang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macroporous+elastomeric+scaffolds+with+extensive+micropores+for+soft+tissue+engineering.">Macroporous elastomeric scaffolds with extensive micropores for soft tissue engineering.</a> Tissue Eng. 12, 917-925 (2006).</li><li>Wilson, E., Mai, Q., Sudhir, K., Weiss, R. H., Ives, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+strain+induces+growth+of+vascular+smooth+muscle+cells+via+autocrine+action+of+PDGF.">Mechanical strain induces growth of vascular smooth muscle cells via autocrine action of PDGF.</a> J Cell Biol. 123, 741-747 (1993).</li><li>Leung, D. Y., Glagov, S., Mathews, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclic+stretching+stimulates+synthesis+of+matrix+components+by+arterial+smooth+muscle+cells+in+vitro.">Cyclic stretching stimulates synthesis of matrix components by arterial smooth muscle cells in vitro.</a> Science. 191, 475-477 (1976).</li><li>Kim, B. S., Nikolovski, J., Bonadio, J., Mooney, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclic+mechanical+strain+regulates+the+development+of+engineered+smooth+muscle+tissue.">Cyclic mechanical strain regulates the development of engineered smooth muscle tissue.</a> Nat Biotechnol. 17, 979-983 (1999).</li><li>Yang, J., Motlagh, D., Webb, A. R., Ameer, 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=Novel+biphasic+elastomeric+scaffold+for+small-diameter+blood+vessel+tissue+engineering.">Novel biphasic elastomeric scaffold for small-diameter blood vessel tissue engineering.</a> Tissue Eng. 11, 1876-1886 (2005).</li><li>Stankus, J. J., Soletti, L., Fujimoto, K., Hong, Y., Vorp, D. A., Wagner, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+cell+microintegrated+blood+vessel+constructs+through+electrohydrodynamic+atomization.">Fabrication of cell microintegrated blood vessel constructs through electrohydrodynamic atomization.</a> Biomaterials. 28, 2738-2746 (2007).</li><li>Nieponice, A., Soletti, L., Guan, J., Deasy, B. M., Huard, J., Wagner, W. R., Vorp, D. A. <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+a+tissue-engineered+vascular+graft+combining+a+biodegradable+scaffold,+muscle-derived+stem+cells+and+a+rotational+vacuum+seeding+technique.">Development of a tissue-engineered vascular graft combining a biodegradable scaffold, muscle-derived stem cells and a rotational vacuum seeding technique.</a> Biomaterials. 29, 825-833 (2008).</li></ol>

الإعلان عن أي تضارب في المصالح.

تقنية التصنيع باستخدام الاستومر القابلة للتحلل وصفها هنا العديد من الميزات. (1) استخدمنا حمض الهيالورونيك (ها) في بيان العفن. منذ ها هو ذوبان في الماء ، أفرج عن سقالة بسهولة من العفن الزجاج بعد أن تمرغ في المياه. في هذا التقرير ، كنا 1.0 بالوزن / المجلد ٪ من حل HA بسبب تركيز منخفض (&lt;0.5 بالوزن / المجلد ٪) من الحل وليس لزج يتدفق بسرعة عندما صب على رأس أنابيب زجاجية. إلى حل موحد HA معطف ، انقلبت لدينا أكثر من أنابيب الزجاج عندما حل طار أسفل الجزء السفلي من الأنابيب وكرر هذه الخطوة. هذا الطلاء ها هو إجراء حاسم لدينا تصنيع السقالات للإفراج النهائي. (2) استخدمنا الحرارة تقلص (HS) الأكمام للاحتفاظ الأملاح في أنابيب زجاجية. منذ الأملاح والمزدحمة بالسكان في المساحة الواقعة بين الجدار الداخلي للأنابيب الزجاج والأكمام HS ، احتفظ HS الأكمام بعد إزالة الأملاح مغزل وخاتم PTFE في الجزء السفلي من الأنابيب. يمكن أن نلغي HS الأكمام بسهولة عن طريق وضع القالب في الفرن على 120 درجة مئوية لمدة 5 دقائق ، ومن ثم الحصول على قوالب الملح أنبوبي. (3) وقد استخدمنا أسلوب الانصهار الملح. ومن المعروف جيدا أن الملح يمكن أن تحسن أسلوب الانصهار الترابط المسام والخواص الميكانيكية طريق تنويع وقت الانصهار 10. علاوة على ذلك ، منذ كنا PGS ، فقد تم إنتاج الكلي المسام من جزيئات الملح خلال عملية الترشيح ، في حين يحتمل أن تكون المسام الصغيرة التي تشكلت خلال بخار الجلسرين PGS المعالجة كما وصفناها سابقا 11. وبالتالي ، فإن هذا الأسلوب لديه القدرة على افتعال السقالات الأنبوبية التي يسهل اختراقها مع الاقتصاد الكلي والجزئي مختلف هياكل متفاوتة بواسطة جزيئات الملح ، وكذلك علاج PGS الشرط. قدمت تكييف الميكانيكية من مفاعل حيوي نابض نضح تدفق (الحد الأقصى يعني تدفق = 14 مل / دقيقة ، إجهاد القص أقصى = 15،3 داين / سم 2 ، والتردد = 0،5-1،7 هرتز) والضغط من الناحية الفسيولوجية ذات الصلة مع سقالة PGS ، مما أدى إلى SMC النمو والتوجه (الشكل 4). هذه النتائج متسقة مع الدراسات السابقة التي تمتد التقارير الدورية في هذا التردد وإجهاد القص يزيد انتشار SMC 12 و 13،14 ECM إنتاج البروتين. بالإضافة إلى النمو SMC والتوجيه ، وبناء الدعم PGS ECM إنتاج البروتين ، وخصوصا تنظيم circumferentially الألياف المرنة (الشكل رقم 5) في غضون 3 أسابيع الثقافة في مفاعل حيوي. وقد أظهرت بعض الدراسات باستخدام سقالة بناء المرنة باعتبارها ذات القطر الصغير الشرايين وضغط القوة الميكانيكية انفجار مماثل في الشرايين الأصلية 15 ، والاندماج السريع في السقالات SMC متوافقة باستخدام الدوار قارورة 16،17 ، في حين لم يتم العثور على الألياف المرنة في هذه البنى. نتائجنا تشير الى ان انتفاخ دوري شعاعي من مفاعل حيوي تحسين ميكانيكية نقل الإشارة بصورة أكثر فعالية لSMCs في سقالة PGS ، والتي ساهمت على الأرجح إلى الإيلاستين التوليف والتنظيم. منذ SMCs خلايا الأوعية الدموية هي الوحيدة المنتجة للبروتينات ECM في بطانة لدينا ، والنهج هادئة وتحسين القوة الميكانيكية اللازمة لتطوير ناجحة سريريا ذات القطر الصغير يبني الشرياني. وذكرت لنا أن الخلايا البطانية التعاون مع مثقف SMCs إنشاء المونولاير متكدسة وأيد بروتين تعبير النمط الظاهري في ظل ظروف ثقافتنا وتكييف الميكانيكية 9. لذا ، على أساس نهجنا وصفها هنا ، وتعديل شروط المشاركة في التجربة الثقافة ستكون الخطوة التالية لتحسين وظائف وتوليد التركيبات الناتجة nonthrombogenic ، قوية ، ومتوافقة مع الشرايين مماثلة لبناء الشرايين الأصلية.

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><th> اسم كاشف </th><th> شركة </th><th> فهرس العدد </th></tr><tr><td> ملح حمض الهيالورونيك الصوديوم </td><td> سيغما الدريخ </td><td> H7630 </td></tr><tr><td> رباعي هيدرو الفوران </td><td> سيغما الدريخ </td><td> 401757 </td></tr><tr><td> MCDB 131 </td><td> Mediatech </td><td> 15-100 - CV </td></tr><tr><td> مصل بقري جنيني </td><td> Lonza </td><td> BW14 - 502F </td></tr><tr><td> L - الجلوتامين </td><td> Mediatech </td><td> 25-005 - CV </td></tr><tr><td> حمض الأسكوربيك </td><td> فيشر العلمية </td><td> A62 - 500 </td></tr><tr><td> مضاد فطري حل للمضادات الحيوية </td><td> Mediatech </td><td> 30-004 - CI </td></tr><tr><td> التخزين المؤقت المالحة الفوسفات (PBS) </td><td> Mediatech </td><td> 21-031 - CV </td></tr><tr><td> نسيج تيك درجة الحرارة المثلى مجمع القطع ، 4583 </td><td> ساكورا Finetek </td><td> 25608-930 </td></tr></tbody></table>

elastomeric pgs scaffolds in arterial tissue engineering

أمراض القلب والأوعية الدموية هي واحدة من الأسباب الرئيسية للوفيات في الولايات المتحدة وخصوصا أمراض الشرايين التاجية يزداد مع شيخوخة السكان وزيادة السمنة 1. حاليا ، سيخضع لعملية جراحية باستخدام السفن ذاتي ، allografts ، وزرع الانسجة الصناعية والمعروفة باسم استخداما ألف لبدائل الشرياني 2. ومع ذلك ، فإن هذه الطعوم لها تطبيقات محدودة عندما القطر الداخلي للشرايين هو أقل من 6 مم بسبب توافر منخفضة ، ومضاعفات الجلطات ، عدم تطابق الامتثال ، وفرط التنسج باطنة أواخر 3،4. للتغلب على هذه القيود ، وقد تم تطبيقها بنجاح هندسة الأنسجة باعتباره بديلا واعدا لتطوير بنيات صغيرة قطرها الشرايين التي nonthrombogenic وقوية ومتوافقة. وقد وضعت العديد من الدراسات السابقة ذات القطر الصغير يبني الشرياني مع ثلاثي صفاحي هيكل وخصائص ميكانيكية ممتازة وضغوط مماثلة لانفجار الشرايين الأصلية 5،6. في حين أن عالية الشد والضغط انفجر عن طريق زيادة إنتاج الكولاجين من مادة جامدة أو خلية سقالة ورقة ، وهذه التركيبات لا تزال منخفضة وكان إنتاج الإيلاستين والامتثال ، والتي تعتبر مشكلة رئيسية في قضية الكسب غير المشروع فشل بعد الزرع. النظر في هذه القضايا ، وافترضنا أن مادة بيولوجية elastometric جنبا إلى جنب مع تكييف الميكانيكية من شأنه أن يوفر المرونة وسلوك الإشارات الميكانيكية أكثر كفاءة للخلايا الأوعية الدموية ، والتي تزيد من إنتاج مصفوفة خارج الخلية ، ودعم التوجه الخلوية. الهدف من هذا التقرير هو لإدخال تقنية تصنيع السقالات الأنبوبية التي يسهل اختراقها وتكييف دينامية ميكانيكية لتطبيقها في هندسة الأنسجة في الشرايين. استخدمنا الاستومر القابلة للتحلل ، وبولي (sebacate الجلسرين) (PGS) (7) لافتعال السقالات الأنبوبية التي يسهل اختراقها من الملح طريقة الانصهار. وكانت المصنفة الكبار الخلايا الأولية البابون العضلات الملساء (SMCs) على لمعة من السقالات ، والتي صممت لدينا مثقف في مفاعل حيوي تدفق نابض لمدة 3 أسابيع. والسقالات PGS سمك ثابت وتوزيعها عشوائيا الكلي والجزئي المسام. تكييف الميكانيكية من مفاعل حيوي تدفق نابض دعم وتعزيز التوجه SMC ECM الإنتاج في السقالات. هذه النتائج تشير إلى أن السقالات المرنة الميكانيكية وتكييف الثقافة مفاعل حيوي قد يكون وسيلة واعدة لهندسة الأنسجة في الشرايين.

Watch this Scientific Journal Video about Elastomeric PGS Scaffolds in Arterial Tissue Engineering at JoVE.com

Elastomeric PGS Scaffolds in Arterial Tissue Engineering

قد PGS السقالات المرنة مع خلايا العضلات الملساء الوعائية المستزرعة في مفاعل حيوي يؤدي إلى تدفق نابض بنيات صغيرة قطرها الشرياني واعدة مع انتاج ECM الأم في فترة قصيرة نسبيا الثقافة.

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 ...

elastomeric-pgs-scaffolds-in-arterial-tissue-engineering

Research

JoVE Journal

Bioengineering

15.4K Views.  University of Pittsburgh. قد PGS السقالات المرنة مع خلايا العضلات الملساء الوعائية المستزرعة في مفاعل حيوي يؤدي إلى تدفق نابض بنيات صغيرة قطرها الشرياني واعدة مع انتاج ECM الأم في فترة قصيرة نسبيا الثقافة.

Video: Elastomeric PGS Scaffolds in Arterial Tissue Engineering

Tissue Engineering of the Intestine in a Murine Model

Tissue-engineered small intestine (TESI) has successfully been used to rescue Lewis rats after massive small bowel resection, resulting in return to preoperative weights within 40 days.1 In humans, massive small bowel resection can result in short bowel syndrome, a functional malabsorptive state that confers significant morbidity, mortality, and healthcare costs including parenteral nutrition dependence, liver failure and cirrhosis, and the need for multivisceral organ transplantation.2 In this paper, we describe and document our protocol for creating tissue-engineered intestine in a mouse model with a multicellular organoid units-on-scaffold approach. Organoid units are multicellular aggregates derived from the intestine that contain both mucosal and mesenchymal elements,3 the relationship between which preserves the intestinal stem cell niche.4 In ongoing and future research, the transition of our technique into the mouse will allow for investigation of the processes involved during TESI formation by utilizing the transgenic tools available in this species.5The availability of immunocompromised mouse strains will also permit us to apply the technique to human intestinal tissue and optimize the formation of human TESI as a mouse xenograft before its transition into humans. Our method employs good manufacturing practice (GMP) reagents and materials that have already been approved for use in human patients, and therefore offers a significant advantage over approaches that rely upon decellularized animal tissues. The ultimate goal of this method is its translation to humans as a regenerative medicine therapeutic strategy for short bowel syndrome.

This article and the accompanying video present our protocol for generating tissue-engineered intestine in the mouse, using an organoid units-on-scaffold approach.

هندسة الأنسجة من الأمعاء في نموذج الفئران

Tissue-engineered small intestine (TESI) has successfully been used to rescue Lewis rats after massive small bowel resection, resulting in return to ...

Density Gradient Multilayered Polymerization (DGMP): A Novel Technique for Creating Multi-compartment, Customizable Scaffolds for Tissue Engineering

Complex tissue culture matrices, in which types and concentrations of biological stimuli (e.g. growth factors, inhibitors, or small molecules) or matrix structure (e.g. composition, concentration, or stiffness of the matrix) vary over space, would enable a wide range of investigations concerning how these variables affect cell differentiation, migration, and other phenomena. The major challenge in creating layered matrices is maintaining the structural integrity of layer interfaces without diffusion of individual components from each layer1. Current methodologies to achieve this include photopatterning2-3, lithography4, sequential functionalization5, freeze drying6, microfluidics7, or centrifugation8, many of which require sophisticated instrumentation and technical skills. Others rely on sequential attachment of individual layers, which may lead to delamination of layers9. 
DGMP overcomes these issues by using an inert density modifier such as iodixanol to create layers of varying densities10. Since the density modifier can be mixed with any prepolymer or bioactive molecule, DGMP allows each scaffold layer to be customized. Simply varying the concentration of the density modifier prevents mixing of adjacent layers while they remain aqueous. Subsequent single step polymerization gives rise to a structurally continuous multilayered scaffold, in which each layer has distinct chemical and mechanical properties. The density modifier can be easily removed with sufficient rinsing without perturbation of the individual layers or their components. This technique is therefore well suited for creating hydrogels of various sizes, shapes, and materials.
A protocol for fabricating a 2D-polyethylene glycol (PEG) gel, in which alternating layers incorporate RGDS-350, is outlined below. We use PEG because it is biocompatible and inert. RGDS, a cell adhesion peptide11, is used to demonstrate spatial restriction of a biological cue, and the conjugation of a fluorophore (Alexa Fluor 350) enables us to visually distinguish various layers. This procedure can be adapted for other materials (e.g. collagen, hyaluronan, etc.) and can be extended to fabricate 3D gels with some modifications10.

Density Gradient Multilayered Polymerization DGMP: A Novel Technique for Creating Multi-compartment, Customizable Scaffolds for Tissue Engineering

Here we describe a unique strategy for creating biocompatible, layered matrices with continuous interfaces between distinct layers for tissue engineering. Such a scaffold could provide an ideal customizable environment to modulate cell behavior by various biological, chemical or mechanical cues

التدرج الكثافة متعدد الطبقات البلمرة (DGMP): تقنية جديدة لإنشاء موضوع متعدد المقصورة، السقالات للهندسة الأنسجة تخصيص

Complex tissue culture matrices, in which types and concentrations of biological stimuli (e.g. growth factors, inhibitors, or small molecules) or matrix ...

Capillary Force Lithography for Cardiac Tissue Engineering

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical discoveries with the aims of developing functional tissues for cardiac regeneration as well as in vitro screening assays. However, the ability to create high-fidelity models of heart tissue has proven difficult. The heart&#8217;s extracellular matrix (ECM) is a complex structure consisting of both biochemical and biomechanical signals ranging from the micro- to the nanometer scale2. Local mechanical loading conditions and cell-ECM interactions have recently been recognized as vital components in cardiac tissue engineering3-5.
A large portion of the cardiac ECM is composed of aligned collagen fibers with nano-scale diameters that significantly influences tissue architecture and electromechanical coupling2. Unfortunately, few methods have been able to mimic the organization of ECM fibers down to the nanometer scale. Recent advancements in nanofabrication techniques, however, have enabled the design and fabrication of scalable scaffolds that mimic the in vivo structural and substrate stiffness cues of the ECM in the heart6-9.
Here we present the development of two reproducible, cost-effective, and scalable nanopatterning processes for the functional alignment of cardiac cells using the biocompatible polymer poly(lactide-co-glycolide) (PLGA)8 and a polyurethane (PU) based polymer. These anisotropically nanofabricated substrata (ANFS) mimic the underlying ECM of well-organized, aligned tissues and can be used to investigate the role of nanotopography on cell morphology and function10-14.
Using a nanopatterned (NP) silicon master as a template, a polyurethane acrylate (PUA) mold is fabricated. This PUA mold is then used to pattern the PU or PLGA hydrogel via UV-assisted or solvent-mediated capillary force lithography (CFL), respectively15,16. Briefly, PU or PLGA pre-polymer is drop dispensed onto a glass coverslip and the PUA mold is placed on top. For UV-assisted CFL, the PU is then exposed to UV radiation (&#955; = 250-400 nm) for curing. For solvent-mediated CFL, the PLGA is embossed using heat (120 &#176;C) and pressure (100 kPa). After curing, the PUA mold is peeled off, leaving behind an ANFS for cell culture. Primary cells, such as neonatal rat ventricular myocytes, as well as human pluripotent stem cell-derived cardiomyocytes, can be maintained on the ANFS2.

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 ...

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 ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

سقالات الإبلاغ الذاتي للخلية ثقافة 3 الأبعاد

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Electrospun Fibrous Scaffolds of Poly(glycerol-dodecanedioate) for Engineering Neural Tissues From Mouse Embryonic Stem Cells

For tissue engineering applications, the preparation of biodegradable and biocompatible scaffolds is the most desirable but challenging task. &#160;Among the various fabrication methods, electrospinning is the most attractive one due to its simplicity and versatility. Additionally, electrospun nanofibers mimic the size of natural extracellular matrix ensuring additional support for cell survival and growth. This study showed the viability of the fabrication of long fibers spanning a larger deposit area for a novel biodegradable and biocompatible polymer named&#160;poly(glycerol-dodecanoate) (PGD)1 by using a newly designed collector for electrospinning. PGD exhibits unique elastic properties with similar mechanical properties to nerve tissues, thus it is suitable for neural tissue engineering applications. The synthesis and fabrication set-up for making fibrous scaffolding materials was simple, highly reproducible, and inexpensive. In biocompatibility testing, cells derived from mouse embryonic stem cells could adhere to and grow on the electrospun PGD fibers. In summary, this protocol provided a versatile fabrication method for making PGD electrospun fibers to support the growth of mouse embryonic stem cell derived neural lineage cells.

Electrospun Fibrous Scaffolds of Polyglycerol-dodecanedioate for Engineering Neural Tissues From Mouse Embryonic Stem Cells

Synthesis and fabrication of electrospun long fibers spanning a larger deposit area via a newly designed collector from a novel biodegradable polymer named poly(glycerol-dodecanoate) (PGD) was reported. The fibers were able to support the growth of cells derived from mouse pluripotent stem cells.

Electrospun الليفية سقالات بولي (الجلسرين-dodecanedioate) للهندسة الأنسجة العصبية من الخلايا الجذعية الجنينية الماوس

For tissue engineering applications, the preparation of biodegradable and biocompatible scaffolds is the most desirable but challenging task. &#160;Among ...

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. ...

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.

Microenvironments-شطيرة مثل لتسخير خلية / المواد التفاعلات

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

Epithelial Cell Repopulation and Preparation of Rodent Extracellular Matrix Scaffolds for Renal Tissue Development

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model substrates for organ-scale tissue development. Sprague Dawley rat kidneys are cannulated by inserting a catheter into the renal artery and perfused with a series of low-concentration detergents (Triton X-100 and sodium dodecyl sulfate (SDS)) over 26 hr to derive intact, whole-kidney scaffolds with intact perfusable vasculature, glomeruli, and renal tubules. Following decellularization, the renal scaffold is placed inside a custom-designed perfusion bioreactor vessel, and the catheterized renal artery is connected to a perfusion circuit consisting of: a peristaltic pump; tubing; and optional probes for pH, dissolved oxygen, and pressure. After sterilizing the scaffold with peracetic acid and ethanol, and balancing the pH (7.4), the kidney scaffold is prepared for seeding via perfusion of culture medium within a large-capacity incubator maintained at 37 &#176;C and 5% CO2. Forty million renal cortical tubular epithelial (RCTE) cells are injected through the renal artery, and rapidly perfused through the scaffold under high flow (25 ml/min) and pressure (~230 mmHg) for 15 min before reducing the flow to a physiological rate (4 ml/min). RCTE cells primarily populate the tubular ECM niche within the renal cortex, proliferate, and form tubular epithelial structures over seven days of perfusion culture. A 44 &#181;M resazurin solution in culture medium is perfused through the kidney for 1 hr during medium exchanges to provide a fluorometric, redox-based metabolic assessment of cell viability and proliferation during tubulogenesis. The kidney perfusion bioreactor permits non-invasive sampling of medium for biochemical assessment, and multiple inlet ports allow alternative retrograde seeding through the renal vein or ureter. These protocols can be used to recellularize kidney scaffolds with a variety of cell types, including vascular endothelial, tubular epithelial, and stromal fibroblasts, for rapid evaluation within this system.

This protocol describes decellularization of Sprague Dawley rat kidneys by antegrade perfusion of detergents through the vasculature, producing acellular renal extracellular matrices that serve as templates for repopulation with human renal epithelial cells. Recellularization and use of the resazurin perfusion assay to monitor growth is performed within specially-designed perfusion bioreactors.

الظهارية إعادة تعمير خلية وإعداد القوارض خارج الخلية مصفوفة السقالات للتنمية نسيج الكلى

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model ...

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 ...