وأيد هذا البحث في جزء من المعاهد الوطنية للصحة المدير الجديد المبتكر برنامج الجائزة ، وهو جزء من المعاهد الوطنية للصحة خارطة الطريق للبحوث الطبية ، من خلال عدد المنح - 1 - DP2 OD004309 - 01. SBS - N. أود أن أتوجه بالشكر إلى جبهة الخلاص الوطني للبحوث زمالة دراسات عليا.

<ol>
	<li>Seif-Naraghi, S. B., Salvatore, M. A., Magoffin-Schup, P. J., Hu, D. P., Christman, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+characterization+of+an+injectable+pericardial+matrix+gel:+A+potentially+autologous+scaffold+for+cardiac+tissue+engineering.">Design and characterization of an injectable pericardial matrix gel: A potentially autologous scaffold for cardiac tissue engineering.</a> Tissue Engineering. , (2009).</li><li>Freytes, D. O., Martin, J., Velankar, S. S., Lee, A. S., Badylak, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+rheological+characterization+of+a+gel+form+of+the+porcine+urinary+bladder+matrix.">Preparation and rheological characterization of a gel form of the porcine urinary bladder matrix.</a> Biomaterials. 29, 1630-1630 (2008).</li><li>Gilbert, T. W., Sellaro, T. L., Badylak, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decellularization+of+tissues+and+organs.">Decellularization of tissues and organs.</a> Biomaterials. 27, 3675-3675 (2006).</li><li>Liao, J., Joyce, E. M., Sacks, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+decellularization+on+the+mechanical+and+structural+properties+of+the+porcine+aortic+valve+leaflet.">Effects of decellularization on the mechanical and structural properties of the porcine aortic valve leaflet.</a> Biomaterials. 29, 1065-1065 (2008).</li><li>Singelyn, J. M., DeQuach, J. A., Seif-Naraghi, S. B., Littlefield, R. B., Schup-Magoffin, P. J., Christman, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Naturally+derived+myocardial+matrix+as+an+injectable+scaffold+for+cardiac+tissue+engineering.">Naturally derived myocardial matrix as an injectable scaffold for cardiac tissue engineering.</a> Biomaterials. 30, 5409-5409 (2009).</li><li>Christman, K. L., Vardanian, A. J., Fang, Q., Sievers, R. E., Fok, H. H., Lee, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Injectable+fibrin+scaffold+improves+cell+transplant+survival,+reduces+infarct+expansion,+and+induces+neovasculature+formation+in+ischemic+myocardium.">Injectable fibrin scaffold improves cell transplant survival, reduces infarct expansion, and induces neovasculature formation in ischemic myocardium.</a> J Am Coll Cardiol. 44, 654-654 (2004).</li><li>Christman, K. L., Fok, H. H., Sievers, R. E., Fang, Q., Lee, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrin+glue+alone+and+skeletal+myoblasts+in+a+fibrin+scaffold+preserve+cardiac+function+after+myocardial+infarction.">Fibrin glue alone and skeletal myoblasts in a fibrin scaffold preserve cardiac function after myocardial infarction.</a> Tissue Eng. 10, 403-410 (2004).</li><li>Huang, N. F., Sievers, R. E., Park, J. S., Fang, Q., Li, S., Lee, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rodent+model+of+myocardial+infarction+for+testing+the+efficacy+of+cells+and+polymers+for+myocardial+reconstruction.">A rodent model of myocardial infarction for testing the efficacy of cells and polymers for myocardial reconstruction.</a> Nat Protoc. (1), 1596-1609 (2006).</li><li>Ott, H. C., Matthiesen, T. S., Goh, S. K., Black, L. D., Kren, S. M., Netoff, T. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perfusion-decellularized+matrix:+using+nature's+platform+to+engineer+a+bioartificial+heart.">Perfusion-decellularized matrix: using nature's platform to engineer a bioartificial heart.</a> Nat Med. 14, 213-221 (2008).</li><li>Badylak, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+extracellular+matrix+as+a+biologic+scaffold+material.">The extracellular matrix as a biologic scaffold material.</a> Biomaterials. 28, 3587-3593 (2007).</li></ol>

استخدم الحيوان : تم تنفيذ جميع التجارب في هذه الدراسة وفقا للمبادئ التوجيهية التي نشرتها رعاية الحيوان واللجنة المؤسسية استخدم في جامعة كاليفورنيا ، سان دييغو والرابطة الأمريكية لاعتماد المختبرات رعاية الحيوان.

هذا الأسلوب يسمح لجيل من السقالات ، والمستمدة من الناحية البيولوجية لهندسة الأنسجة حقن عضلة القلب. وقد وضعت في البداية على الرغم من هذه الطرق لصنع واختبار في الجسم الحي من هلام مصفوفة عضلة القلب وقدمت هنا مع هلام تأموري مصفوفة ، ويمكن تكييف هذا البروتوكول للاست?...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Reagents:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Pepsin</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>p6887-1G</td>
<td>Lyophilized </td>
</tr>
<tr>
<td>Biotin</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>21217</td>
<td>&nbsp;</td>
<tr>
<td>Neutravidin-HRP</td>
<td>&nbsp;</td>
<td>Thomas Scientific</td>
<td>21130</td>
<td>&nbsp;</td>
<tr>
<td>Equipment: </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Wiley Mini Mill</td>
<td>&nbsp;</td>
<td>Thomas Scientific</td>
<td>3383L10</td>
<td>&nbsp;</td>
<tr>
<td>Labconco Lyophilizer</td>
<td>&nbsp;</td>
<td>Labconco, Inc</td>
<td>7670520</td>
<td>&nbsp;</td>
<tr>
<td>Surgical supplies:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Betadine</td>
<td>&nbsp;</td>
<td>Purdue Products, L.P.</td>
<td>67618-154-16</td>
<td>&nbsp;</td>
<tr>
<td>Lactated Ringers Solution</td>
<td>&nbsp;</td>
<td>MWI</td>
<td>003966</td>
<td>&nbsp;</td>
<tr>
<td>KY Jelly</td>
<td>&nbsp;</td>
<td>MWI</td>
<td>28658</td>
<td>&nbsp;</td>
<tr>
<td>Lidocaine, 2%</td>
<td>&nbsp;</td>
<td>MWI</td>
<td>17767</td>
<td>&nbsp;</td>
<tr>
<td>Buprenorphine hydrochloride</td>
<td>&nbsp;</td>
<td>Reckitt Benckiser Healthcare (UK) Ltd.</td>
<td>12496-0757-1</td>
<td>&nbsp;</td>
<tr>
<td>Artificial tear ointment</td>
<td>&nbsp;</td>
<td>Fisher </td>
<td>NC9860843</td>
<td>&nbsp;</td>
<tr>
<td>Triple antibiotic ointment</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>19082795</td>
<td>&nbsp;</td>
<tr>
<td>Isoflurane</td>
<td>&nbsp;</td>
<td>MWI</td>
<td>60307-120-25</td>
<td>&nbsp;</td>
<tr>
<td>Otoscope </td>
<td>&nbsp;</td>
<td>MWI</td>
<td>008699</td>
<td>&nbsp;</td>
<tr>
<td>Stop cock</td>
<td>&nbsp;</td>
<td>MWI</td>
<td>006245</td>
<td>&nbsp;</td>
<tr>
<td>3-0 Vicrile suture</td>
<td>&nbsp;</td>
<td>MWI</td>
<td>J327H</td>
<td>&nbsp;</td>
<tr>
<td>5-0 Proline suture</td>
<td>&nbsp;</td>
<td>MWI</td>
<td>s-1173</td>
<td>&nbsp;</td>
<tr>
<td>Reverse cutting (RC) needle</td>
<td>&nbsp;</td>
<td>Ethicon</td>
<td>8684G</td>
<td>&nbsp;</td>
<tr>
<td>Microhemostats </td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>13013-14</td>
<td>&nbsp;</td>
<tr>
<td>Rat tooth microforceps</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>11084-07</td>
<td>&nbsp;</td>
<tr>
<td>No. 10 scalpel</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>10110-01</td>
<td>&nbsp;</td>
<tr>
<td>Blunt scissors</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>14108-09</td>
<td>&nbsp;</td>
<tr>
<td>Sharp, curved scissors</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>14085-08</td>
<td>&nbsp;</td>
<tr>
<td>Large, serrated forceps</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>1106-12</td>
<td>&nbsp;</td>
<tr>
<td>PE160 suction tubing</td>
<td>&nbsp;</td>
<td>BD</td>
<td>427430 </td>
<td>&nbsp;</td>
<tr>
<td>Clippers </td>
<td>&nbsp;</td>
<td>MWI</td>
<td>21608</td>
<td>&nbsp;</td>
<tr>
<td>Skin staples/stapler </td>
<td>&nbsp;</td>
<td>Ethicon</td>
<td>PRR35</td>
<td>&nbsp;</td>
<tr>
<td>General supplies:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Stir plates</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>0.1 M HCl</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>1 M NaOH</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>10x PBS</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>1x PBS</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>70% Ethanol</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>0.1 mL syringes</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>10 mL syringe</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Q-tips</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Surgical glue</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Surgical drape</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Towel clamps</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Small hand-held vacuum </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

fabrication of biologically derived injectable materials for myocardial tissue engineering

هذا البروتوكول يوفر أساليب للإعداد للحقن هلام خارج الخلية (ECM) مصفوفة لعضلة القلب تطبيقات هندسة الأنسجة. باختصار ، هو مجفد decellularized الأنسجة ، والمضروب ، إنزيمي هضمها ، وتقديمهم إلى درجة الحموضة ثم الفسيولوجية. وlyophilization يزيل كل محتوى الماء من الأنسجة ، مما أدى إلى ECM الجافة التي يمكن أن تكون الأرض إلى مسحوق ناعم مع طاحونة صغيرة. بعد الطحن ، يتم هضمها ومسحوق ECM مع البيبسين لتشكيل مصفوفة تعطى عن طريق الحقن. بعد تعديل درجة الحموضة 7.4 ، يمكن حقن مادة سائلة مصفوفة في عضلة القلب. وقد أظهرت نتائج فحوصات توصيف المواد الهلامية المصفوفة السابقة التي تنتج من نسيج عضلة القلب والتامور decellularized الاحتفاظ مكونات ECM الأصلية ، بما في ذلك البروتينات المختلفة ، والببتيدات الجليكوزامينوجليكان. نظرا لاستخدام هذه المواد للهندسة الأنسجة ، في توصيف فيفو يكون مفيدا بشكل خاص ، وهنا ، وهي طريقة لأداء حقنة جماعية في البطين الأيسر (LV) وتقدم مجانا الجدار كوسيلة لتحليل استجابة المضيف للهلام في مصفوفة نموذج حيوان صغير. وتمكنت من الوصول إلى تجويف الصدر من خلال الحجاب الحاجز ويتم حقن قليلا فوق قمة في الجدار الحر LV. يمكن للسقالة مشتقة بيولوجيا يمكن تصور وضع العلامات التي البيوتين قبل الحقن وتلوين ثم أقسام الأنسجة مع الفجل البيروكسيداز - مترافق neutravidin ووضع تصور عن طريق تلوين (DAB) diaminobenzidine. ويمكن أيضا تحليل للمنطقة الحقن يمكن القيام به مع تلوين النسيجية والمناعية. في هذه الطريقة ، وأظهرت دراسة سبق الهلام مصفوفة التامور وعضلة القلب لتشكيل ليفي ، وتعزيز شبكات مسامية تشكيل سفينة داخل منطقة الحقن.

Watch this Scientific Journal Video about Fabrication of Biologically Derived Injectable Materials for Myocardial Tissue Engineering at JoVE.com

Fabrication of Biologically Derived Injectable Materials for Myocardial Tissue Engineering

طرق لإعداد جيل من مصفوفة حقن الأنسجة decellularized وحقنه في الفئران عضلة القلب<em&gt; في الجسم الحي</em&gt; موصوفة.

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.  ...

fabrication-biologically-derived-injectable-materials-for-myocardial

Research

JoVE Journal

Bioengineering

15.0K Views.  University of California, San Diego. طرق لإعداد جيل من مصفوفة حقن الأنسجة decellularized وحقنه في الفئران عضلة القلب في الجسم الحي موصوفة.

Video: Fabrication of Biologically Derived Injectable Materials for Myocardial Tissue Engineering

Directed Cellular Self-Assembly to Fabricate Cell-Derived Tissue Rings for Biomechanical Analysis and Tissue Engineering

Each year, hundreds of thousands of patients undergo coronary artery bypass surgery in the United States.1 Approximately one third of these patients do not have suitable autologous donor vessels due to disease progression or previous harvest. The aim of vascular tissue engineering is to develop a suitable alternative source for these bypass grafts. In addition, engineered vascular tissue may prove valuable as living vascular models to study cardiovascular diseases. Several promising approaches to engineering blood vessels have been explored, with many recent studies focusing on development and analysis of cell-based methods.2-5 Herein, we present a method to rapidly self-assemble cells into 3D tissue rings that can be used in vitro to model vascular tissues.
To do this, suspensions of smooth muscle cells are seeded into round-bottomed annular agarose wells. The non-adhesive properties of the agarose allow the cells to settle, aggregate and contract around a post at the center of the well to form a cohesive tissue ring.6,7 These rings can be cultured for several days prior to harvesting for mechanical, physiological, biochemical, or histological analysis. We have shown that these cell-derived tissue rings yield at 100-500 kPa ultimate tensile strength8 which exceeds the value reported for other tissue engineered vascular constructs cultured for similar durations (&lt;30 kPa).9,10 Our results demonstrate that robust cell-derived vascular tissue ring generation can be achieved within a short time period, and offers the opportunity for direct and quantitative assessment of the contributions of cells and cell-derived matrix (CDM) to vascular tissue structure and function.

This article outlines a versatile method to create cell-derived tissue rings by cellular self-assembly. Smooth muscle cells seeded into ring-shaped agarose wells aggregate and contract to form robust three-dimensional (3D) tissues within 7 days. Millimeter-scale tissue rings are conducive to mechanical testing and serve as building blocks for tissue assembly.

توجه الخلوي الذاتي الجمعية الى افتعال خواتم نسيج الخلايا المستمدة من النشاط الحيوي لتحليل وهندسة الأنسجة

Each year, hundreds of thousands of patients undergo coronary artery bypass surgery in the United States.1 Approximately one third of these patients do ...

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 ...

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 ...

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. ...

Design and Construction of Artificial Extracellular Matrix (aECM) Proteins from Escherichia coli for Skin Tissue Engineering

Recombinant technology is a versatile platform to create novel artificial proteins with tunable properties. For the last decade, many artificial proteins that have incorporated functional domains derived from nature (or created de novo) have been reported. In particular, artificial extracellular matrix (aECM) proteins have been developed; these aECM proteins consist of biological domains taken from fibronectin, laminins and collagens and are combined with structural domains including elastin-like repeats, silk and collagen repeats. To date, aECM proteins have been widely investigated for applications in tissue engineering and wound repair. Recently, Tjin and coworkers developed integrin-specific aECM proteins designed for promoting human skin keratinocyte attachment and propagation. In their work, the aECM proteins incorporate cell binding domains taken from fibronectin, laminin-5 and collagen IV, as well as flanking elastin-like repeats. They demonstrated that the aECM proteins developed in their work were promising candidates for use as substrates in artificial skin. Here, we outline the design and construction of such aECM proteins as well as their purification process using the thermo-responsive characteristics of elastin.

Design and Construction of Artificial Extracellular Matrix aECM Proteins from Escherichia coli for Skin Tissue Engineering

Recombinant technologies have enabled material designers to create novel artificial proteins with customized functionalities for tissue engineering applications. For example, artificial extracellular matrix proteins can be designed to incorporate structural and biological domains derived from native ECMs. Here, we describe the construction and purification of aECM proteins containing elastin-like repeats.

تصميم وبناء الاصطناعي خارج الخلية مصفوفة (aECM) والبروتينات من<em&gt; الإشريكية القولونية</em&gt; للبشرة هندسة الأنسجة

Recombinant technology is a versatile platform to create novel artificial proteins with tunable properties. For the last decade, many artificial proteins ...

Fabrication of Mechanically Tunable and Bioactive Metal Scaffolds for Biomedical Applications

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness and low bioactivity of metals. In this study, we have developed a new method towards fabricating a new type of bioactive and mechanically reliable porous metal scaffolds-densified porous Ti scaffolds. The method consists of two fabrication processes, 1) the fabrication of porous Ti scaffolds by dynamic freeze casting, and 2) coating and densification of the porous scaffolds. The dynamic freeze casting method to fabricate porous Ti scaffolds allowed the densification of porous scaffolds by minimizing the chemical contamination and structural defects. The densification process is distinctive for three reasons. First, the densification process is simple, because it requires a control of only one parameter (degree of densification). Second, it is effective, as it achieves mechanical enhancement and sustainable release of biomolecules from porous scaffolds. Third, it has broad applications, as it is also applicable to the fabrication of functionally graded porous scaffolds by spatially varied strain during densification.

Bioactive and mechanically reliable metal scaffolds have been fabricated through a method which consists of two processes, dynamic freeze casting for the fabrication of porous Ti, and coating and densification of the Ti scaffolds. The densification process is simple, effective and applicable to the fabrication of functionally graded scaffolds.

تصنيع ميكانيكيا الانضباطي والحيويه المعادن سقالات للتطبيقات الطبية الحيوية

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness ...

Fabrication of Inverted Colloidal Crystal Poly(ethylene glycol) Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing drugs targeted at the liver, requires a platform in which cells receive proper biochemical and mechanical cues. Recent liver tissue engineering systems have employed three-dimensional (3D) scaffolds composed of synthetic or natural hydrogels, given their high water retention and their ability to provide the mechanical stimuli needed by the cells. There has been growing interest in the inverted colloidal crystal (ICC) scaffold, a recent development, which allows high spatial organization, homotypic and heterotypic cell interaction, as well as cell-extracellular matrix (ECM) interaction. Herein, we describe a protocol to fabricate the ICC scaffold using poly (ethylene glycol) diacrylate (PEGDA) and the particle leaching method. Briefly, a lattice is made from microsphere particles, after which a pre-polymer solution is added, properly polymerized, and the particles are then removed, or leached, using an organic solvent (e.g., tetrahydrofuran). The dissolution of the lattice results in a highly porous scaffold with controlled pore sizes and interconnectivities that allow media to reach cells more easily. This unique structure allows high surface area for the cells to adhere to as well as easy communication between pores, and the ability to coat the PEGDA ICC scaffold with proteins also shows a marked effect on cell performance. We analyze the morphology of the scaffold as well as the hepatocarcinoma cell (Huh-7.5) behavior in terms of viability and function to explore the effect of ICC structure and ECM coatings. Overall, this paper provides a detailed protocol of an emerging scaffold that has wide applications in tissue engineering, especially liver tissue engineering.

Fabrication of Inverted Colloidal Crystal Polyethylene glycol Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

This manuscript presents a detailed protocol for the fabrication of an emerging three-dimensional hepatocyte culture platform, the inverted colloidal crystal scaffold, and the concomitant techniques to assess hepatocyte behavior. The size-controllable pores, interconnectivity and ability to conjugate extracellular matrix proteins to the poly(ethylene glycol) (PEG) scaffold enhance Huh-7.5 cell performance.

تصنيع مقلوب الغروية كريستال بولي (جلايكول الإثيلين) سقالة: وثلاثي الأبعاد منصة الثقافة خلية للهندسة الأنسجة الكبد

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing ...

Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus of this article is on the methods for fabricating ECM-derived porous foams and microcarriers for use as biologically-relevant substrates in advanced 3D in vitro cell culture models or as pro-regenerative scaffolds and cell delivery systems for tissue engineering and regenerative medicine. Using decellularized tissues or purified insoluble collagen as a starting material, the techniques can be applied to synthesize a broad array of tissue-specific bioscaffolds with customizable geometries. The approach involves mechanical processing and mild enzymatic digestion to yield an ECM suspension that is used to fabricate the three-dimensional foams or microcarriers through controlled freezing and lyophilization procedures. These pure ECM-derived scaffolds are highly porous, yet stable without the need for chemical crosslinking agents or other additives that may negatively impact cell function. The scaffold properties can be tuned to some extent by varying factors such as the ECM suspension concentration, mechanical processing methods, or synthesis conditions. In general, the scaffolds are robust and easy to handle, and can be processed as tissues for most standard biological assays, providing a versatile and user-friendly 3D cell culture platform that mimics the native ECM composition. Overall, these straightforward methods for fabricating customized ECM-derived foams and microcarriers may be of interest to both biologists and biomedical engineers as tissue-specific cell-instructive platforms for in vitro and in vivo applications.

The tissue-specific extracellular matrix (ECM) is a key mediator of cell function. This article describes methods for synthesizing pure ECM-derived foams and microcarriers that are stable in culture without the need for chemical crosslinking for applications in advanced 3D in vitro cell culture models or as pro-regenerative bioscaffolds.

تصنيع كسوات وناقل دقيق مستمد مصفوفة خارج الخلية حيث الثقافة خلية الأنسجة محددة ومنصات التوصيل

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus ...

Isolation of Murine Adipose Tissue-derived Microvascular Fragments as Vascularization Units for Tissue Engineering

A functional microvascular network is of pivotal importance for the survival and integration of engineered tissue constructs. For this purpose, several angiogenic and prevascularization strategies have been established. However, most cell-based approaches include time-consuming in vitro steps for the formation of a microvascular network. Hence, they are not suitable for intraoperative one-step procedures. Adipose tissue-derived microvascular fragments (ad-MVF) represent promising vascularization units. They can be easily isolated from fat tissue and exhibit a functional microvessel morphology. Moreover, they rapidly reassemble into new microvascular networks after in vivo implantation. In addition, ad-MVF have been shown to induce lymphangiogenesis. Finally, they are a rich source of mesenchymal stem cells, which may further contribute to their high vascularization potential. In previous studies we have demonstrated the remarkable vascularization capacity of ad-MVF in engineered bone and skin substitutes. In the present study, we report on a standardized protocol for the enzymatic isolation of ad-MVF from murine fat tissue.

We present a protocol to isolate adipose tissue-derived microvascular fragments that represent promising vascularization units. They can be rapidly isolated, do not require in vitro processing and, thus, may be used for one-step prevascularization in different fields of tissue engineering.

عزل الفئران الدهنية المشتقة من الأنسجة الاوعية الدموية الدقيقة شظايا كما وحدات الأوعية الدموية للهندسة الأنسجة

A functional microvascular network is of pivotal importance for the survival and integration of engineered tissue constructs. For this purpose, several ...

Scalable Fabrication of Stretchable, Dual Channel, Microfluidic Organ Chips

A significant number of lead compounds fail in the pharmaceutical pipeline because animal studies often fail to predict clinical responses in human patients. Human Organ-on-a-Chip (Organ Chip) microfluidic cell culture devices, which provide an experimental in vitro platform to assess efficacy, toxicity, and pharmacokinetic (PK) profiles in humans, may be better predictors of therapeutic efficacy and safety in the clinic compared to animal studies. These devices may be used to model the function of virtually any organ type and can be fluidically linked through common endothelium-lined microchannels to perform in vitro studies on human organ-level and whole body-level physiology without having to conduct experiments on people. These Organ Chips consist of two perfused microfluidic channels separated by a permeable elastomeric membrane with organ-specific parenchymal cells on one side and microvascular endothelium on the other, which can be cyclically stretched to provide organ-specific mechanical cues (e.g., breathing motions in lung). This protocol details the fabrication of flexible, dual channel, Organ Chips through casting of parts using 3D printed molds, enabling combination of multiple casting and post-processing steps. Porous poly (dimethyl siloxane) (PDMS) membranes are cast with micrometer sized through-holes using silicon pillar arrays under compression. Fabrication and assembly of Organ Chips involves equipment and steps that can be implemented outside of a traditional cleanroom. This protocol provides researchers with access to Organ Chip technology for in vitro organ- and body-level studies in drug discovery, safety and efficacy testing, as well as mechanistic studies of fundamental biological processes.

Here, we present a protocol that describes the fabrication of stretchable, dual channel, organ chip microfluidic cell culture devices for recapitulating organ-level functionality in vitro.

تصنيع قابلة المط ومزدوجة القناة، رقائق موائع جزيئية الجهاز

A significant number of lead compounds fail in the pharmaceutical pipeline because animal studies often fail to predict clinical responses in human ...