Name: production of nanofibrillar patterned collagen for tissue engineering
Uploaded: 2024-09-20T13:40:00.000+00:00
Duration: 7 min 34 s
Description: Regenerative biomaterials are designed to facilitate cell-material interactions to guide the repair of damaged tissues and organs. These materials are ...

<meta charset="utf8"></head><body>Fabricação de colágeno nanofibrilar projetado com padrão direcionado

<ol>
	<li>Padhi, A., Nain, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ECM+in+differentiation:+A+review+of+matrix+structure,+composition+and+mechanical+properties.">ECM in differentiation: A review of matrix structure, composition and mechanical properties.</a> Ann Biomed Eng. 48 (3), 1071-1089 (2020).</li><li>Schlie-Wolter, S., Ngezahayo, A., Chichkov, B. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+selective+role+of+ECM+components+on+cell+adhesion,+morphology,+proliferation+and+communication+in+vitro.">The selective role of ECM components on cell adhesion, morphology, proliferation and communication in vitro.</a> Exp Cell Res. 319 (10), 1553-1561 (2013).</li><li>Ricard-Blum, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+collagen+family.">The collagen family.</a> Cold Spring Harb Perspect Biol. 3 (1), a004978 (2011).</li><li>Shoulders, M. D., Raines, 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=Collagen+structure+and+stability.">Collagen structure and stability.</a> Annu Rev Biochem. 78, 929-958 (2009).</li><li>Gillies, A. R., Lieber, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+function+of+the+skeletal+muscle+extracellular+matrix.">Structure and function of the skeletal muscle extracellular matrix.</a> Muscle Nerve. 44 (3), 318-331 (2011).</li><li>Silver, F. H., Freeman, J. W., Seehra, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagen+self-assembly+and+the+development+of+tendon+mechanical+properties.">Collagen self-assembly and the development of tendon mechanical properties.</a> J Biomech. 36 (10), 1529-1553 (2003).</li><li>Friess, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagen-biomaterial+for+drug+delivery.">Collagen-biomaterial for drug delivery.</a> Eur J Pharm Biopharm. 45 (2), 113-136 (1998).</li><li>Lee, C. H., Singla, A., Lee, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomedical+applications+of+collagen.">Biomedical applications of collagen.</a> Int J Pharm. 221 (1-2), 1-22 (2001).</li><li>Liu, X., Zheng, C., Luo, X., Wang, X., Jiang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+of+collagen-based+biomaterials:+Multi-hierarchical+structure,+modification+and+biomedical+applications.">Recent advances of collagen-based biomaterials: Multi-hierarchical structure, modification and biomedical applications.</a> Mater Sci Eng C. 99, 1509-1522 (2019).</li><li>Harris, J. R., Reiber, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+saline+and+pH+on+collagen+type+I+fibrillogenesis+in+vitro:+Fibril+polymorphism+and+colloidal+gold+labelling.">Influence of saline and pH on collagen type I fibrillogenesis in vitro: Fibril polymorphism and colloidal gold labelling.</a> Micron. 38 (5), 513-521 (2007).</li><li>Williams, B. R., Gelman, R. A., Poppke, D. C., Piez, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagen+fibril+formation.+Optimal+in+vitro+conditions+and+preliminary+kinetic+results.">Collagen fibril formation. Optimal in vitro conditions and preliminary kinetic results.</a> J Biol Chem. 253 (18), 6578-6585 (1978).</li><li>Zhong, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+aligned+nanofibrous+collagen+scaffold+by+electrospinning+and+its+effects+on+in+vitro+fibroblast+culture.">An aligned nanofibrous collagen scaffold by electrospinning and its effects on in vitro fibroblast culture.</a> J Biomed Mater Res A. 79A (3), 456-463 (2006).</li><li>Malladi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+formation+of+ultrathin,+strong+collagen+sheets+with+tunable+anisotropy+and+compaction.">Continuous formation of ultrathin, strong collagen sheets with tunable anisotropy and compaction.</a> ACS Biomater Sci Eng. 6 (7), 4236-4246 (2020).</li><li>Lowe, C. J., Reucroft, I. M., Grota, M. C., Shreiber, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+highly+aligned+collagen+scaffolds+by+freeze-drying+of+self-assembled,+fibrillar+collagen+gels.">Production of highly aligned collagen scaffolds by freeze-drying of self-assembled, fibrillar collagen gels.</a> ACS Biomater Sci Eng. 2 (4), 643-651 (2016).</li><li>Torbet, J., Ronzi&#232;re, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+alignment+of+collagen+during+self-assembly.">Magnetic alignment of collagen during self-assembly.</a> Biochem J. 219 (3), 1057-1059 (1984).</li><li>Provenzano, P. P., Eliceiri, K. W., Inman, D. R., Keely, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+three-dimensional+collagen+matrices+to+provide+contact+guidance+during+3d+cell+migration.">Engineering three-dimensional collagen matrices to provide contact guidance during 3d cell migration.</a> Curr Protoc Cell Biol. Chpater 10 (Unit 10.17), (2010).</li><li>Saeidi, N., Sander, E. A., Zareian, R., Ruberti, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+highly+aligned+collagen+lamellae+by+combining+shear+force+and+thin+film+confinement.">Production of highly aligned collagen lamellae by combining shear force and thin film confinement.</a> Acta Biomater. 7 (6), 2437-2447 (2011).</li><li>Paten, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow-induced+crystallization+of+collagen:+A+potentially+critical+mechanism+in+early+tissue+formation.">Flow-induced crystallization of collagen: A potentially critical mechanism in early tissue formation.</a> ACS Nano. 10 (5), 5027-5040 (2016).</li><li>Yunoki, S., Hatayama, H., Ebisawa, M., Kondo, E., Yasuda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+method+for+continuous+formation+of+cord-like+collagen+gels+to+fabricate+durable+fibers+in+which+collagen+fibrils+are+longitudinally+aligned.">A novel method for continuous formation of cord-like collagen gels to fabricate durable fibers in which collagen fibrils are longitudinally aligned.</a> J Biomed Mater Res B Appl Biomater. 107 (4), 1011-1023 (2019).</li><li>Vader, D., Kabla, A., Weitz, D., Mahadevan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strain-induced+alignment+in+collagen+gels.">Strain-induced alignment in collagen gels.</a> PLoS One. 4 (6), e5902 (2009).</li><li>Ahmed, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+extensional+flows+promote+long-range+fiber+alignment+in+3d+collagen+hydrogels.">Local extensional flows promote long-range fiber alignment in 3d collagen hydrogels.</a> Biofabrication. 14 (3), (2022).</li><li>Su, C. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+a+3D+collective+cancer+invasion+model+with+control+over+collagen+fiber+alignment.">Engineering a 3D collective cancer invasion model with control over collagen fiber alignment.</a> Biomaterials. 275, 120922 (2021).</li><li>Nakayama, K. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bilayered+vascular+graft+derived+from+human+induced+pluripotent+stem+cells+with+biomimetic+structure+and+function.">Bilayered vascular graft derived from human induced pluripotent stem cells with biomimetic structure and function.</a> Regen Med. 10 (6), 745-755 (2015).</li><li>Nakayama, K. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoscale+patterning+of+extracellular+matrix+alters+endothelial+function+under+shear+stress.">Nanoscale patterning of extracellular matrix alters endothelial function under shear stress.</a> Nano Lett. 16 (1), 410-419 (2016).</li><li>Nakayama, K. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rehabilitative+exercise+and+spatially+patterned+nanofibrillar+scaffolds+enhance+vascularization+and+innervation+following+volumetric+muscle+loss.">Rehabilitative exercise and spatially patterned nanofibrillar scaffolds enhance vascularization and innervation following volumetric muscle loss.</a> NPJ Regen Med. 3, 16 (2018).</li><li>Nakayama, K. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+of+volumetric+muscle+loss+in+mice+using+nanofibrillar+scaffolds+enhances+vascular+organization+and+integration.">Treatment of volumetric muscle loss in mice using nanofibrillar scaffolds enhances vascular organization and integration.</a> Commun Biol. 2 (1), 170 (2019).</li><li>Alcazar, C. A., Hu, C., Rando, T. A., Huang, N. F., Nakayama, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+insulin-like+growth+factor-1+laden+scaffolds+combined+with+exercise+promotes+neuroregeneration+and+angiogenesis+in+a+preclinical+muscle+injury+model.">Transplantation of insulin-like growth factor-1 laden scaffolds combined with exercise promotes neuroregeneration and angiogenesis in a preclinical muscle injury model.</a> Biomater Sci. 8 (19), 5376-5389 (2020).</li><li>Lai, E. S., Huang, N. F., Cooke, J. P., Fuller, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aligned+nanofibrillar+collagen+regulates+endothelial+organization+and+migration.">Aligned nanofibrillar collagen regulates endothelial organization and migration.</a> Regen Med. 7 (5), 649-661 (2012).</li><li>Huang, N. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+patterning+of+endothelium+modulates+cell+morphology,+adhesiveness+and+transcriptional+signature.">Spatial patterning of endothelium modulates cell morphology, adhesiveness and transcriptional signature.</a> Biomaterials. 34 (12), 2928-2937 (2013).</li><li>Weadock, K. S., Miller, E. J., Bellincampi, L. D., Zawadsky, J. P., Dunn, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physical+crosslinking+of+collagen+fibers:+Comparison+of+ultraviolet+irradiation+and+dehydrothermal+treatment.">Physical crosslinking of collagen fibers: Comparison of ultraviolet irradiation and dehydrothermal treatment.</a> J Biomed Mater Res. 29 (11), 1373-1379 (1995).</li></ol>

<table><tbody><tr><td>Collagen I, High Concentration, Rat Tail</td><td>Corning</td><td>CB354249</td><td></td></tr><tr><td>Eclipse TE-2000-U microscope</td><td>Nikon</td><td>TE-2000-U&amp;nbsp;</td><td>Inverted microscope</td></tr><tr><td>Extra Thick Microscope Slides</td><td>Fisher Scientific</td><td>22-267-005</td><td>Works well for collagen extrusion surface</td></tr><tr><td>FEI Helios G3 NanoLab DualBeam</td><td>Thermo Fisher Scientific</td><td>N/A</td><td>Scanning electron microscope</td></tr><tr><td>Glass Cutter Tool Set 2mm-20mm Pencil Style Oil Feed Carbide Tip</td><td>Amazon/MOARMOR</td><td>B07Y1D243H</td><td>Option for a glass cutter</td></tr><tr><td>Hamilton Kel-F Hub Blunt Point Needles (Luer Lock, 22 G)</td><td>Fisher Scientific</td><td>14-815-574</td><td>Needles for collagen extrusion</td></tr><tr><td>Nexterion Slide H 3-D</td><td>Schott</td><td>NC0782819</td><td>Hydrophobic slides</td></tr><tr><td>PBS Tablets</td><td>Thermo Fisher Scientific</td><td>18912014</td><td></td></tr><tr><td>Polyethylene Glycol 8000</td><td>Fisher BioReagents</td><td>BP233-1</td><td></td></tr><tr><td>Seamless Cellulose Dialysis Tubing&amp;nbsp;</td><td>Fisher Scientific</td><td>S25645G</td><td></td></tr><tr><td>SnakeSkin Dialysis Clips</td><td>Thermo Fisher Scientific</td><td>PI68011</td><td></td></tr><tr><td>Synthware Glass Cutter</td><td>Synthware</td><td>31-501-927</td><td>Option for a glass cutter</td></tr></tbody></table>

production of nanofibrillar patterned collagen for tissue engineering

<meta charset="utf8"></head><body>Produção de colágeno estampado nanofibrilar para engenharia de tecidos

Produção de colágeno estampado nanofibrilar para engenharia de tecidos

Regenerative biomaterials are designed to facilitate cell-material interactions to guide the repair of damaged tissues and organs. These materials are ...

production-of-nanofibrillar-patterned-collagen-for-tissue-engineering

Research

JoVE Journal

Bioengineering

Production of Nanofibrillar Patterned Collagen for Tissue Engineering

302 visualizações.  Oregon Health & Science University. O protocolo a seguir apresenta um método de extrusão baseado em cisalhamento para a fabricação de hidrogéis de colágeno com fibrilas padronizadas em nanoescala, que pode ser aplicado a uma ampla gama de aplicações de engenharia de tecidos.

Vídeo: Produção de colágeno estampado nanofibrilar para engenharia de tecidos

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.

Biologicamente fabricação de materiais derivados injetável para Engenharia de Tecidos do Miocárdio

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

Bioengenharia

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 elastomérico Andaimes em Engenharia de Tecidos Arterial

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

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.

Processamento de pós-produção de Fibras electrospun para Engenharia de Tecidos

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.

Capilar Força Litografia de Engenharia tecido cardíaco

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

Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

We designed a loading device that is capable of applying uniaxial or biaxial mechanical strain to a tissue engineered biocomposites fabricated for transplantation. While the device primarily functions as a bioreactor that mimics the native mechanical strains, it is also outfitted with a load cell for providing force feedback or mechanical testing of the constructs. The device subjects engineered cartilage constructs to biaxial mechanical loading with great precision of loading dose (amplitude and frequency) and is compact enough to fit inside a standard tissue culture incubator. It loads samples directly in a tissue culture plate, and multiple plate sizes are compatible with the system. The device has been designed using components manufactured for precision-guided laser applications. Bi-axial loading is accomplished by two orthogonal stages. The stages have a 50 mm travel range and are driven independently by stepper motor actuators, controlled by a closed-loop stepper motor driver that features micro-stepping capabilities, enabling step sizes of less than 50 nm. A polysulfone loading platen is coupled to the bi-axial moving platform. Movements of the stages are controlled by Thor-labs Advanced Positioning Technology (APT) software. The stepper motor driver is used with the software to adjust load parameters of frequency and amplitude of both shear and compression independently and simultaneously. Positional feedback is provided by linear optical encoders that have a bidirectional repeatability of 0.1 &mu;m and a resolution of 20 nm, translating to a positional accuracy of less than 3 &mu;m over the full 50 mm of travel. These encoders provide the necessary position feedback to the drive electronics to ensure true nanopositioning capabilities. In order to provide the force feedback to detect contact and evaluate loading responses, a precision miniature load cell is positioned between the loading platen and the moving platform. The load cell has high accuracies of 0.15% to 0.25% full scale.

We designed a novel mechanical loading bioreactor that can apply uniaxial or biaxial mechanical strain to a cartilage biocomposite prior to transplantation into an articular cartilage defect.

Projeto de um biorreator Carregando Mecânica Biaxial para Engenharia de Tecidos

We  designed a loading device that is capable of applying uniaxial or biaxial  mechanical strain to a tissue engineered biocomposites fabricated for  ...

Engineering Fibrin-based Tissue Constructs from Myofibroblasts and Application of Constraints and Strain to Induce Cell and Collagen Reorganization

Collagen content and organization in developing collagenous tissues can be influenced by local tissue strains and tissue constraint. Tissue engineers aim to use these principles to create tissues with predefined collagen architectures. A full understanding of the exact underlying processes of collagen remodeling to control the final tissue architecture, however, is lacking. In particular, little is known about the (re)orientation of collagen fibers in response to changes in tissue mechanical loading conditions. We developed an in&#160;vitro model system, consisting of biaxially-constrained myofibroblast-seeded fibrin constructs, to further elucidate collagen (re)orientation in response to i) reverting biaxial to uniaxial static loading conditions and ii) cyclic uniaxial loading of the biaxially-constrained constructs before and after a change in loading direction, with use of the Flexcell FX4000T loading device. Time-lapse confocal imaging is used to visualize collagen (re)orientation in a nondestructive manner.
Cell and collagen organization in the constructs can be visualized in real-time, and an internal reference system allows us to relocate cells and collagen structures for time-lapse analysis. Various aspects of the model system can be adjusted, like cell source or use of healthy and diseased cells. Additives can be used to further elucidate mechanisms underlying collagen remodeling, by for example adding MMPs or blocking integrins. Shape and size of the construct can be easily adapted to specific needs, resulting in a highly tunable model system to study cell and collagen (re)organization.

This model system starts from a myofibroblast-populated fibrin gel that can be used to study endogenous collagen (re)organization real-time in a nondestructive manner. The model system is very tunable, as it can be used with different cell sources, medium additives, and can be adapted easily to specific needs.

Engenharia baseada em fibrina construções de tecido de miofibroblastos e de aplicação de restrições e tensão para induzir a organização das células e colágeno (Re)

Collagen content and organization in developing collagenous tissues can be influenced by local tissue strains and tissue constraint. Tissue engineers aim ...

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.

Engenharia 3D celularizados Colágeno Gel para Vascular Regeneração Tecidual

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

Engineered 3D Silk-collagen-based Model of Polarized Neural Tissue

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a deeper comprehension of the neural system scientists aim to simplistically reconstruct the tissue by assembling it in vitro from basic building blocks using a tissue engineering approach. Our group developed a tissue-engineered silk and collagen-based 3D brain-like model resembling the white and gray matter of the cortex. The model consists of silk porous sponge, which is pre-seeded with rat brain-derived neurons, immersed in soft collagen matrix. Polarized neuronal outgrowth and network formation is observed with separate axonal and cell body localization. This compartmental architecture allows for the unique development of niches mimicking native neural tissue, thus enabling research on neuronal network assembly, axonal guidance, cell-cell and cell-matrix interactions and electrical functions.

Insight into the complex actions of the brain requires advanced research tools. Here we demonstrate a novel silk-collagen-based 3D engineered model of neural tissue resembling brain-like architecture. The model can be used to study neuronal network assembly, axonal guidance, cell-cell interactions and electrical activity.

Baseada em Silk-colágeno Engineered 3D Model of Tissue polarizada Neural

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a ...

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.

Preparação de Thermoresponsive nanoestruturados Surfaces para Engenharia de Tecidos

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.

Engenharia de Tecidos por Intrínseca Vascularização numa<em&gt; In Vivo</em&gt; Câmara Engenharia de Tecidos

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