Name: scaling of engineered vascular grafts using 3d printed guides and the ring stacking method
Uploaded: 2017-03-27T11:30:00.000+00:00
Duration: 9 min 38 s
Description: Watch this Scientific Journal Video about Scaling of Engineered Vascular Grafts Using 3D Printed Guides and the Ring Stacking Method at JoVE.com

The authors would like to thank our fellow Lam lab colleagues Ammar Chishti and Bijal Patel for their kind assistance with some of the histology and cell culture. Funding was provided by the Wayne State University Nanomedicine Fellowship (CBP), Start-Up Funds and Cardiovascular Research Institute Seed Grant (MTL).

1. Cell Culture Preparation
<ol>
	<li>Utilize human aortic smooth muscle cells purchased commercially.</li>
	<li>Maintain cells in smooth muscle cell growth media composed of 88.6% 231 media, 0.1% each of recombinant human insulin (rH-insulin), recombinant human fibroblast growth factor (rH-FGF), recombinant human epidermal growth factor (rH-FGF), and ascorbic acid; and 5% each of fetal bovine serum (FBS) and L-glutamine; and 1% antibiotic/antimycotic. 
	NOTE: Each growth factor, FBS and L-glutamine are purchased as a vascular media growth kit.</li>
	<li>Change media every 48 hours until the cells are about 90% confluent and ready for tissue seeding.</li>
	<li>Store cells in an incubator in between media changes for expansion.</li>
</ol>
2. Preparation of 3D Printed Inserts and Custom Silicone Molded Plates
<ol>
	<li>Use a commercial 3D printer (e.g., Replicator Mini) for 3D printing the plate inserts.
	<ol>
		<li>Use open source 3D design software such as Blender to create the 3D models of the printed outer-shells and posts.</li>
		<li>Export the model&#39;s driver file via a .stl format allowing for portability to the 3D printer&#39;s software.</li>
		<li>Produce printed posts and outer shells using poly(lactic acid) filament (PLA) loaded into the 3D printer.</li>
		<li>Following printing, perform a 30-minute soak in a 70%-100% ethanol solution to sterilize each insert.</li>
	</ol>
	</li>
	<li>Prepare a 1:10 curing agent to base polymer mixture of poly(dimethylsiloxane) (PDMS) silicone polymer and allow the mixture to degas at room temperature for 10 min.
	<ol>
		<li>Define petri dish sizes used as small (35 mm), intermediate (60 mm), and large (100 mm).</li>
		<li>Add 2 mL, 4 mL and 6 mL of uncured silicone to each small, intermediate and large plate, respectively, and create a thin layer across the entire bottom of the petri dish.</li>
		<li>Create posts for the small plate by pouring PDMS into a 100 mm plate to a height of 7 mm and allow to cure on a hot plate at 60 &#176;C for about 2-3 hours. Then use a 5 mm biopsy punch to punch out cylindrical posts. Use a small amount of uncured PDMS to secure each PDMS cylinder and to the center of each small plate.</li>
		<li>For the intermediate and large plates, prior to complete curing of the PDMS at the bottom of the plate, place the 3D printed posts 10 mm and 20 mm in diameter centrally into each intermediate and large plates, respectively. For the large plates, additionally place a 3D printed outer shell about 66.7 mm in diameter equidistance from the post.
		<ol>
			<li>Allow each dish to cure in open air on a hot plate at 60 &#176;C for about 2-3 hours, allowing 18 hours for degassing of the polymer. Fix printed components to the plate in the proper region and orientation as seen in Figure 1.</li>
		</ol>
		</li>
		<li>Add a solution of 70% ethanol with 30% distilled water for 30 minutes to the inside of all plates for sterilization and then cover each plate.</li>
		<li>Carefully aspirate the ethanol from each plate and allow to air dry.</li>
		<li>Arrange each plate in a biological safety cabinet (BSC) next to its lid, face-up. Expose plates and lids to UV light under the BSC for 30 minutes to complete sterilization. Sterile technique is performed with every step after UV exposure.</li>
	</ol>
	</li>
</ol>
3. Preparation of Fibrin Hydrogel, Seeding with Smooth Muscle Cells and Maintenance of Plates
<ol>
	<li>Mix a solution of fibrin gel containing growth media + 0.01% TGF-&#946;1 in the amounts of 0.5 mL, 1.1 mL and 1.81 mL for small, intermediate and large plate sizes, respectively.
	<ol>
		<li>Add 40 &#181;L, 88.4 &#181;L and 145 &#956;L of thrombin, from a stock of 100 U/mL, to the media of each small, intermediate and large plate, respectively. Gently swirl each plate by hand to ensure that thrombin is evenly mixed within the media.</li>
		<li>Next, add 160 &#181;L, 354 &#181;L and 580 &#956;L fibrinogen, from a stock of 20 mg/mL, drop-wise circularly to the thrombin-media mixture to each small, intermediate and large plate, respectively. Gently swirl by hand to ensure mixing and distribution of the hydrogel into an even layer.</li>
		<li>Allow hydrogel to cure for 10-15 minutes at room temperature.</li>
	</ol>
	</li>
	<li>Trypsinize smooth muscle cells expanded in 150 mm cell culture plates and centrifuge according to standard protocols. The resulting pellet should be resuspended in 3 mL of differentiation media consisting of 98% - 231 media, 1% FBS and 1% antibiotic/antimycotic.
	<ol>
		<li>Vigorously mix cells by titrating up and down with a 2 mL pipette to break up any cell clumps. Count cells with a hemocytometer and create a cell suspension of 2 x 106 cells/mL, 1.0 x 107 cells/mL and 1.4 x 107 cells/mL for small, intermediate and large plates, respectively.</li>
		<li>Add 1 mL of each cell suspension into a corresponding 50 mL conical labelled small, intermediate and large. Set up an additional 50 mL conical in this manner for each additional tissue ring desired.</li>
		<li>Add differentiation media to each conical to obtain final seeding volumes of 2 mL, 4 mL and 5 mL for each small, intermediate and large vessel, respectively. Then, carefully pipet the cell solution drop-wise on top of the prepared hydrogel in each corresponding plate.</li>
		<li>Place plates into the incubator at 37 &#176;C and 5% CO2.</li>
	</ol>
	</li>
	<li>Change differentiation media every 48-72 hours for each plate. In the case of the larger plate, change media initially after 24 hours, then change it every 48-24 hours to compensate for the large cell seeding density.
	<ol>
		<li>After 2-4 days as the rings will have completely contracted in towards the post, add 10 &#181;L, 20 &#181;L and 35 &#956;L of TGF-&#946;1 to each small, intermediate and large ring, respectively. After exposure to TGF-&#946;1 for at least 24 hours, rings are ready to be handled.</li>
	</ol>
	</li>
</ol>
4. Assembly of Vascular Construct and Maintenance
<ol>
	<li>Before the fabrication of the final vascular construct, a specialized container is created to hold the completed vessel.
	<ol>
		<li>For the small vessel, create a tall plate for ring stacking by cutting a 2-inch section off the top of a 50 mL polycarbonate conical tube, and then PDMS glue the cut edge into a 35 mm plate. Use the conical lid as the plate lid.</li>
		<li>For the intermediate and large vessel tall ring stacking plates, cut a 1.75-inch diameter polycarbonate tube into 2.5 inch sections lengthwise to serve as the tall plate walls. For the tall plate bottoms, cut a 0.125-inch-thick polycarbonate sheet into 2-inch diameter circle pieces. Using acrylic solvent cement, bind the polycarbonate tube section to the circular cut piece. Use the lid from a 60 mm petri dish as the lid for the tall plate.</li>
		<li>3D print posts 5, 10 and 20 mm in diameter and 50 mm in length.</li>
		<li>Add 10 mL of uncured silicone to each container. Prior to the complete curing of the PDMS, centrally place each post created in step 4.1.3 into each small, intermediate, and large container.</li>
		<li>Allow to cure on a hot plate set to 60 &#176;C for 2-3 hours.</li>
		<li>Sterilize with a solution of 70% ethanol with 30% distilled water for 30 minutes.</li>
		<li>Carefully aspirate the ethanol from each container and then allow to air dry in the BSC. Next, arrange the containers in the hood with each plate placed next to its lid, face-up. Expose containers to UV light under the BSC for an additional 30 minutes for further sterilization. Use sterile technique with every step after UV exposure.</li>
	</ol>
	</li>
	<li>Using very fine forceps, carefully remove each tightly rolled smooth muscle hydrogel ring from its post and transfer to its corresponding larger container with the tall posts.
	<ol>
		<li>Use a pair of forceps in each hand and lift one side of the ring from the post, and then the other. Be careful to protect and maintain the lumen.</li>
		<li>Perform the transfer with this two handed method, sliding first one side, then the other side of the ring onto the tall post. Using gentle, gradual movements, and working circumferentially, slowly push the ring down onto the tall post. Subsequently stack tissue rings until the desired vessel length has been obtained, with each ring adding approximately 1-2 mm of length to the completed construct.</li>
	</ol>
	</li>
	<li>With the ring stack positioned on the tall 3D printed posts, turn the plate so that the post is parallel with the working surface.
	<ol>
		<li>Using a micropipette, add 40, 80 and 160 &#956;L of thrombin at a concentration of 100 U/mL gently to the outer surface of each small, intermediate and large vessel, respectively. While adding the thrombin, slowly rotate the plate to ensure even coverage of all surfaces of the construct. This will be the base for the fibrin glue utilized to secure the ring stack construct in the initial days after construction.</li>
		<li>Next, add 40, 80 and 160 &#956;L of fibrinogen at a concentration of 20 mg/mL to each small, intermediate, and large construct, respectively, using a micropipette while rotating the construct quickly. The thrombin and fibrinogen quickly set into a firm gel once mixed. Due to the short curing time, apply the fibrinogen as quickly and evenly as possible.</li>
	</ol>
	</li>
	<li>Add 20 mL of differentiation media to each container holding the construct. Place vessels into a 37 &#176;C incubator until needed. Change media every 3-5 days.</li>
</ol>

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N., 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=Effectiveness+of+haemodialysis+access+with+an+autologous+tissue-engineered+vascular+graft:+a+multicentre+cohort+study.">Effectiveness of haemodialysis access with an autologous tissue-engineered vascular graft: a multicentre cohort study.</a> Lancet. 373 (9673), 1440-1446 (2009).</li><li>Wystrychowski, W., 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=First+human+use+of+an+allogeneic+tissue-engineered+vascular+graft+for+hemodialysis+access.">First human use of an allogeneic tissue-engineered vascular graft for hemodialysis access.</a> J Vasc Surg. 60 (5), 1353-1357 (2014).</li><li>Konig, G., 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=Mechanical+properties+of+completely+autologous+human+tissue+engineered+blood+vessels+compared+to+human+saphenous+vein+and+mammary+artery.">Mechanical properties of completely autologous human tissue engineered blood vessels compared to human saphenous vein and mammary artery.</a> Biomaterials. 30 (8), 1542-1550 (2009).</li><li>Gui, L., 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=Construction+of+tissue-engineered+small-diameter+vascular+grafts+in+fibrin+scaffolds+in+30+days.">Construction of tissue-engineered small-diameter vascular grafts in fibrin scaffolds in 30 days.</a> Tissue Eng Part A. 20 (9-10), 1499-1507 (2014).</li><li>Sundaram, S., Echter, A., Sivarapatna, A., Qiu, C., Niklason, L. <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+vascular+graft+engineered+using+human+embryonic+stem+cell-derived+mesenchymal+cells.">Small-diameter vascular graft engineered using human embryonic stem cell-derived mesenchymal cells.</a> Tissue Eng Part A. 20 (3-4), 740-750 (2014).</li><li>Quint, C., Arief, M., Muto, A., Dardik, A., Niklason, L. 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A., Lahti, M. T., Johnson, S. L., 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=Implantation+of+completely+biological+engineered+grafts+following+decellularization+into+the+sheep+femoral+artery.">Implantation of completely biological engineered grafts following decellularization into the sheep femoral artery.</a> Tissue Eng Part A. 20 (11-12), 1726-1734 (2014).</li><li>Syedain, Z. H., Meier, L. A., Bjork, J. W., Lee, A., Tranquillo, R. 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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=Engineered+vascular+tissue+fabricated+from+aggregated+smooth+muscle+cells.">Engineered vascular tissue fabricated from aggregated smooth muscle cells.</a> Cells Tissues Organs. 194 (1), 13-24 (2011).</li><li>Fearon, W. 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The authors have nothing to disclose.

The Ring Stacking Method presents multiple advantages over current vascular tissue engineered construct techniques. The RSM can be adapted to create human vessels of any size by simply customizing the post and outer shell dimensions. Our method allows for development of polymer-free engineered vessels composed solely of human cells and rapidly degrading support material found in the body&#39;s natural wound healing process. Polymer grafts are known to cause restenosis in the clinic and could become problematic if contain...

In treatment of coronary artery disease (CAD), a patient&#39;s own blood vessels are harvested as graft material for bypass surgery. However, oftentimes, ill patients do not have viable vessels to donate to themselves, and in cases where they do, the donor site causes considerable additional harm and has a serious risk for infection.1 Engineered vascular grafts could fill this need. Scalability is of utmost importance for engineering vessels in order to meet the wide range of patient vessel size requirements. However, present methods for engineering vessels are not easily scalable, and typically require remanufacture of complex molds or polymer scaffolds. Most engineered grafts either utilize a polymer tubular scaffold that is seeded with vascular fibroblasts, smooth muscle, or endothelial cells; or rolling a cell sheet around a mandrel to create a tissue tube. Two engineered vascular grafts in clinical trials are based on a decellularized polymer-ECM platform.2,3,4 Polymer grafts available for use in vascular repair are already known to have issues with patency, which could arise as a major issue with long-term application of a graft with a sustained polymer presence. Tubular molds have been used to fabricate completely cellular vessels,5,6,7,8,9,10,11,12,13 which procedures would require additional design and tool manufacturing for custom molds in order to produce vessels in a variety of sizes.
The method described herein encompasses a novel technique for creating easily scalable engineered vascular grafts using customizable 3D printed inserts and traditional culture plates.14 Cells are seeded into plates with inserts of a central post and outer shell. The post controls lumen diameter and allows the cell monolayer to self-assemble into a ring of tissue. The outer shell controls thickness of the ring, and thus wall thickness of the final vessel. Completed tissue rings are then stacked to form a tubular, vascular graft. The advantage to this method, termed the &#34;Ring Stacking Method,&#34; is that any adherent cell type can be seeded into the plate setup and tissue rings or tubes of any size needed for the desired application can be generated by simply modifying guide inserts. Comparative techniques in tissue engineering creating rings of tissue remain difficult to scale,15,16 requiring remanufacture of molds for each desired size. Additionally, vascular grafts made using this method can be produced in 2-3 weeks, several weeks faster compared to other engineered vessels.6 For the clinic, this time discrepancy can make a significant difference in the treatment of a deteriorating patient.

<table><tbody><tr><td>Human Aortic Smooth Muscle Cells&amp;nbsp;</td><td>ATCC</td><td>PCS-100-012</td><td>vascular smooth muscle cells</td></tr><tr><td>Medium 231</td><td>Gibco (Life Technologies&amp;nbsp;</td><td>M-231-500</td><td>media specific to vascular smooth muscle cells</td></tr><tr><td>Human Aortic Smooth Muscle Cell Growth Kit&amp;nbsp;</td><td>ATCC</td><td>PSC-100-042</td><td>growth factors for maintaining vascular smooth muscle cell viability</td></tr><tr><td>Replicator Mini 3D printer&amp;nbsp;</td><td>MakerBot&amp;nbsp;</td><td>N/A</td><td>3D printer</td></tr><tr><td>Poly(lactic acid) 3D ink (PLA)</td><td>MakerBot&amp;nbsp;</td><td>N/A</td><td>3D printer filament</td></tr><tr><td>Poly(dimethlysiloxane) (PDMS)</td><td>Ellworth Adhesives&amp;nbsp;</td><td>3097358-1004</td><td>polymer for gluing plate parts</td></tr><tr><td>Fibrinogen</td><td>Hyclone Labratories, Inc.</td><td>SH30256.01</td><td>fibrin gel component</td></tr><tr><td>Thrombin&amp;nbsp;</td><td>Sigma Life Sciences</td><td>F3879-5G</td><td>fibrin gel component</td></tr><tr><td>Tranforming Growth Factor-Beta 1&amp;nbsp;</td><td>PeproTech</td><td>100-21</td><td>growth factor for stimulating collagen production</td></tr><tr><td>Hemocytometer&amp;nbsp;</td><td>Hausser Scientific Co.</td><td>3200</td><td>for cell counting</td></tr><tr><td>Polycarbonate tubing&amp;nbsp;</td><td>US Plastics&amp;nbsp;</td><td>PCTUB1.750X1.625</td><td>material for making tall, ring stacking plates</td></tr><tr><td>Polycarbonate sheet&amp;nbsp;</td><td>Home Depot</td><td>409497</td><td>material for making tall, ring stacking plates</td></tr><tr><td>Adhesive polymer solvent&amp;nbsp;</td><td>SCIGRIP</td><td>10799</td><td>material for making tall, ring stacking plates</td></tr><tr><td>Instron 5940</td><td>Instron</td><td>N/A</td><td>tensile testing machine</td></tr><tr><td>U-Stretch</td><td>Cell Scale</td><td>N/A</td><td>tensile testing machine</td></tr><tr><td>Smooth Muscle Actin&amp;nbsp;</td><td>MA5-11547</td><td>Thermo Fisher</td><td>antibody</td></tr><tr><td>Tropomyosin</td><td>MA5-11783</td><td>Thermo Fisher</td><td>antibody</td></tr></tbody></table>

scaling of engineered vascular grafts using 3d printed guides and the ring stacking method

Coronary artery disease remains a leading cause of death, affecting millions of Americans. With the lack of autologous vascular grafts available, engineered grafts offer great potential for patient treatment. However, engineered vascular grafts are generally not easily scalable, requiring manufacture of custom molds or polymer tubes in order to customize to different sizes, constituting a time-consuming and costly practice. Human arteries range in lumen diameter from about 2.0-38 mm and in wall thickness from about 0.5-2.5 mm. We have created a method, termed the "Ring Stacking Method," in which variable size rings of tissue of the desired cell type, demonstrated here with vascular smooth muscle cells (SMCs), can be created using guides of center posts to control lumen diameter and outer shells to dictate vessel wall thickness. These tissue rings are then stacked to create a tubular construct, mimicking the natural form of a blood vessel. The vessel length can be tailored by simply stacking the number of rings required to constitute the length needed. With our technique, tissues of tubular forms, similar to a blood vessel, can be readily manufactured in a variety of dimensions and lengths to meet the needs of the clinic and patient.

Demonstrated here is fabrication of 3 different engineered vascular graft sizes (Figure 1), showing that the Ring Stacking Method (RSM) is scalable. To prove applicability, the 3 different vessel sizes chosen correlate to actual human vessel size for the left anterior descending artery (small; lumen diameter = 4 mm)17, descending aorta (intermediate; lumen diameter = 10 mm) and ascending aorta (large; lumen diameter = 20 mm)18</su...

Watch this Scientific Journal Video about Scaling of Engineered Vascular Grafts Using 3D Printed Guides and the Ring Stacking Method at JoVE.com

Scaling of Engineered Vascular Grafts Using 3D Printed Guides and the Ring Stacking Method

Scalable engineered blood vessels would improve clinical applicability. Using easily sizable 3D-printed guides, rings of vascular smooth muscle were created and stacked into a tubular form, forming a vascular graft. Grafts can be sized to meet the range of human coronary artery dimensions by simply changing the 3D-printed guide size.

Coronary artery disease remains a leading cause of death, affecting millions of Americans. With the lack of autologous vascular grafts available, ...

scaling-engineered-vascular-grafts-using-3d-printed-guides-ring

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Bioengineering

8.3K Views.  Wayne State University. Scalable engineered blood vessels would improve clinical applicability. Using easily sizable 3D-printed guides, rings of vascular smooth muscle were created and stacked into a tubular form, forming a vascular graft. Grafts can be sized to meet the range of human coronary artery dimensions by simply changing the 3D-printed guide size.

Video: Scaling of Engineered Vascular Grafts Using 3D Printed Guides and the Ring Stacking Method

Engineering Biological-Based Vascular Grafts Using a Pulsatile Bioreactor

Much effort has been devoted to develop and advance the methodology to regenerate functional small-diameter arterial bypasses. In the physiological environment, both mechanical and chemical stimulation are required to maintain the proper development and functionality of arterial vessels1,2.
Bioreactor culture systems developed by our group are designed to support vessel regeneration within a precisely controlled chemo-mechanical environment mimicking that of native vessels. Our bioreactor assembly and maintenance procedures are fairly simple and highly repeatable3,4. Smooth muscle cells (SMCs) are seeded onto a tubular polyglycolic acid (PGA) mesh that is threaded over compliant silicone tubing and cultured in the bioreactor with or without pulsatile stimulation for up to 12 weeks. There are four main attributes that distinguish our bioreactor from some predecessors. 1) Unlike other culture systems that simulate only the biochemical surrounding of native blood vessels, our bioreactor also creates a physiological pulsatile environment by applying cyclic radial strain to the vessels in culture. 2) Multiple engineered vessels can be cultured simultaneously under different mechanical conditions within a controlled chemical environment. 3) The bioreactor allows a mono layer of endothelial cells (EC) to be easily coated onto the luminal side of engineered vessels for animal implantation models. 4) Our bioreactor can also culture engineered vessels with different diameter size ranged from 1 mm to 3 mm, saving the effort to tailor each individual bioreactor to fit a specific diameter size. 
The engineered vessels cultured in our bioreactor resemble native blood vessels histologically to some degree. Cells in the vessel walls express mature SMC contractile markers such as smooth muscle myosin heavy chain (SMMHC)3. A substantial amount of collagen is deposited within the extracellular matrix, which is responsible for ultimate mechanical strength of the engineered vessels5. Biochemical analysis also indicates that collagen content of engineered vessels is comparable to that of native arteries6. Importantly, the pulsatile bioreactor has consistently regenerated vessels that exhibit mechanical properties that permit successful implantation experiments in animal models3,7. Additionally, this bioreactor can be further modified to allow real-time assessment and tracking of collagen remodeling over time, non-invasively, using a non-linear optical microscopy (NLOM)8. To conclude, this bioreactor should serve as an excellent platform to study the fundamental mechanisms that regulate the regeneration of functional small-diameter vascular grafts.

Our group has developed a bioreactor culture system that mimics the physiological pulsatile stresses of the cardiovascular system to regenerate implantable small-diameter vascular grafts.

Much effort has been devoted to develop and advance the methodology to regenerate functional small-diameter arterial bypasses. In the physiological ...

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.

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

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

The development of Tissue Engineered Vessels (TEVs) is advanced by the ability to routinely and effectively implant TEVs (4-5 mm in diameter) into a large animal model. A step by-step protocol for inter-positional placement of the TEV and real-time digital assessment of the TEV and native carotid arteries is described here. In vivo monitoring is made possible by the implantation of flow probes, catheters and ultrasonic crystals (capable of recording dynamic diameter changes of implanted TEVs and native carotid arteries) at the time of surgery. Once implanted, researchers can calculate arterial blood flow patterns, invasive blood pressure and artery diameter yielding parameters such as pulse wave velocity, augmentation index, pulse pressures and compliance. Data acquisition is accomplished using a single computer program for analysis throughout the duration of the experiment. Such invaluable data provides insight into TEV matrix remodeling, its resemblance to native/sham controls and overall TEV performance in vivo.

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

A step-by-step protocol for the inter-positional placement of Tissue Engineered Vessels (TEVs) into the carotid artery of a sheep using end-to-end anastomosis and real-time digital assessment in vivo until animal sacrifice.

The development of Tissue Engineered Vessels (TEVs) is advanced by the ability to routinely and effectively implant TEVs (4-5 mm in diameter) into a large ...

Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. The current protocol presents an efficient and reproducible strategy in which functional tissue engineered vessel grafts can be generated using partially induced pluripotent stem cell (PiPSC) from human fibroblasts. We designed a decellularized vessel scaffold bioreactor, which closely mimics the matrix protein structure and blood flow that exists within a native vessel, for seeding of PiPSC-endothelial cells or smooth muscle cells prior to grafting into mice. This approach was demonstrated to be advantageous because immune-deficient mice engrafted with the PiPSC-derived grafts presented with markedly increased survival rate 3 weeks after surgery. This protocol represents a valuable tool for regenerative medicine, tissue engineering and potentially patient-specific cell-therapy in the near future.

Here, we present a protocol to generate tissue engineered vessel grafts that are functional for grafting into mice by double seeding partially induced pluripotent stem cell (PiPSC) - derived smooth muscle cells and PiPSC - derived endothelial cells on a decellularized vessel scaffold bioreactor.

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. ...

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.

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

Fabrication of Custom Agarose Wells for Cell Seeding and Tissue Ring Self-assembly Using 3D-Printed Molds

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease modeling. Self-assembled tissues offer advantages over scaffold-based tissue engineering, such as enhanced matrix deposition, strength, and function. However, there are few available methods for fabricating 3D tissues without seeding cells on or within a supporting scaffold. Previously, we developed a system for fabricating self-assembled tissue rings by seeding cells into non-adhesive agarose wells. A polydimethylsiloxane (PDMS) negative was first cast in a machined polycarbonate mold, and then agarose was gelled in the PDMS negative to create ring-shaped cell seeding wells. However, the versatility of this approach was limited by the resolution of the tools available for machining the polycarbonate mold. Here, we demonstrate that 3D-printed plastic can be used as an alternative to machined polycarbonate for fabricating PDMS negatives. The 3D-printed mold and revised mold design is simpler to use, inexpensive to produce, and requires significantly less agarose and PDMS per cell seeding well. We have demonstrated that the resulting agarose wells can be used to create self-assembled tissue rings with customized diameters from a variety of different cell types. Rings can then be used for mechanical, functional, and histological analysis, or for fabricating larger and more complex tubular tissues.

This protocol describes a platform for fabricating self-assembled tissue rings in variable sizes using a customized 3D-printed plastic mold. PDMS negatives are cured in the 3D-printed mold; then agarose is cast in the cured PDMS negatives. Cells are seeded into the resulting agarose wells where they aggregate into tissue rings.

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease ...

Stepwise Cell Seeding on Tessellated Scaffolds to Study Sprouting Blood Vessels

The cardiovascular system is a key player in human physiology, providing nourishment to most tissues in the body; vessels are present in different sizes, structures, phenotypes, and performance depending on each specific perfused tissue. The field of tissue engineering, which aims to repair or replace damaged or missing body tissues, relies on controlled angiogenesis to create a proper vascularization within the engineered tissues. Without a vascular system, thick engineered constructs cannot be sufficiently nourished, which may result in cell death, poor engraftment, and ultimately failure. Thus, understanding and controlling the behavior of engineered blood vessels is an outstanding challenge in the field. This work presents a high-throughput system that allows for the creation of organized and repeatable vessel networks for studying vessel behavior in a 3D scaffold environment. This two-step seeding protocol shows that vessels within the system react to the scaffold topography, presenting distinctive sprouting behaviors depending on the compartment geometry in which the vessels reside. The obtained results and understanding from this high throughput system can be applied in order to inform better 3D bioprinted scaffold construct designs, wherein fabrication of various 3D geometries cannot be rapidly assessed when using 3D printing as the basis for cellularized biological environments. Furthermore, the understanding from this high throughput system may be utilized for the improvement of rapid drug screening, the rapid development of co-cultures models, and the investigation of mechanical stimuli on blood vessel formation to deepen the knowledge of the vascular system.

Engineered tissues heavily rely on proper vascular networks to provide vital nutrients and gases and remove metabolic waste. In this work, a stepwise seeding protocol of endothelial cells and support cells creates highly organized vascular networks in a high-throughput platform for studying developing vessel behavior in a controlled 3D environment.

The cardiovascular system is a key player in human physiology, providing nourishment to most tissues in the body; vessels are present in different sizes, ...

Standardized Technique of Aortic Valve Re-implantation for Valve-sparing Aortic Root Replacement

Despite the obvious advantages of the preservation of a normal aortic valve during aortic root replacement, the complexity of valve sparing procedures prevents a number of cardiac surgeons from incorporating them into their practice. The aim of this protocol is to describe a simplified and user-friendly technique of an aortic valve-sparing root replacement (VSRR) procedure by re-implantation of the aortic valve. Proper selection of patients and limitations of the technique are discussed.
In 54 consecutive patients, normal appearing aortic valves were re-implanted in a commercially available polyester prosthesis with pre-shaped sinuses by a simplified and standardized technique. Placement of the first row of the proximal suture line, choice of the prosthesis size, and adjustment of the height of the commissures of the patient to the fixed height of the sinus portion of the prosthesis were slightly modified from the reference techniques with the aim of increasing its feasibility for use by other cardiac surgeons. Early mortality and morbidity as well as 5-year survival, freedom from aortic valve reoperation, and freedom from recurrent moderate regurgitation were collected in all patients.
Thirty-day mortality, re-sternotomy for bleeding, re-sternotomy for mediastinitis, and the incidence of stroke were very low, 1.8% for each (1 of 54). No patient required permanent pace-maker implantation. At 5 years, survival, freedom from aortic valve reoperation, and freedom from recurrent moderate regurgitation were 97.5%, 95.2%, and 91.6%, respectively.
Mid-term results of our standardized technique of re-implantation of the aortic valve for valve-sparing aortic root replacement are very good and compare with more complex techniques reported by experienced surgeons. By following the present protocol of the standardized re-implantation technique, a greater number of cardiac surgeons can perform this procedure with comparable good results.

Valve-sparing aortic root replacement has the advantage of preserving the patient&#39;s own aortic valve. The complexity of the reported techniques to date restricts their use to a limited number of cardiac surgeons. This protocol describes step-by-step a standardized technique reproducible by a greater number of cardiac surgeons.

Despite the obvious advantages of the preservation of a normal aortic valve during aortic root replacement, the complexity of valve sparing procedures ...

Medicine

Fabrication of Engineered Vascular Flaps Using 3D Printing Technologies

Engineering implantable, functional, thick tissues requires designing a hierarchical vascular network. 3D bioprinting is a technology used to create tissues by adding layer upon layer of printable biomaterials, termed bioinks, and cells in an orderly and automatic manner, which allows for creating highly intricate structures that traditional tissue engineering techniques cannot achieve. Thus, 3D bioprinting is an appealing in vitro approach to mimic the native vasculature complex structure, ranging from millimetric vessels to microvascular networks. 
Advances in 3D bioprinting in granular hydrogels enabled the high-resolution extrusion of low-viscosity extracellular matrix-based bioinks. This work presents a combined 3D bioprinting and sacrificial mold-based 3D printing approach for fabricating engineered vascularized tissue flaps. 3D bioprinting of endothelial and support cells using recombinant collagen-methacrylate bioink within a gelatin support bath is utilized for the fabrication of a self-assembled capillary network. This printed microvasculature is assembled around a mesoscale vessel-like porous scaffold, fabricated using a sacrificial 3D printed mold, and is seeded with endothelial cells. 
This assembly induces the endothelium of the mesoscale vessel to anastomose with the surrounding capillary network, establishing a hierarchical vascular network within an engineered tissue flap. The engineered flap is then directly implanted by surgical anastomosis to a rat femoral artery using a cuff technique. The described methods can be expanded for the fabrication of various vascularized tissue flaps for use in reconstruction surgery and vascularization studies.

Engineered flaps require an incorporated functional vascular network. In this protocol, we present a method of fabricating a 3D printed tissue flap containing a hierarchical vascular network and its direct microsurgical anastomoses to rat femoral artery.

Engineering implantable, functional, thick tissues requires designing a hierarchical vascular network. 3D bioprinting is a technology used to create ...

Three-Dimensional Printing of a Complex Aortic Anomaly

Complex congenital aortic anomalies include diverse types of malformations that may be clinically asymptomatic or present with respiratory or esophageal symptoms. These anomalies may be associated with other congenital heart diseases. It is hard to identify the accurate anatomic vessel location from two-dimensional imaging data, such as computed tomography. As an additive manufacturing method, three-dimensional (3-D) printing can covert the acquired imaging data into 3-D physical models. This protocol describes the procedure for modeling the volumetric DICOM imaging into 3-D data and printing it as an anatomically realistic 3-D model. Using this model, surgeons can identify the vessel location of complex aortic anomalies, which is helpful for pre-operative planning and intra-operative guidance.

Here, we present a protocol to use three dimensional printed models for pre-operative planning and intra-operative reorganization of complicated vascular locations when handling a congenital aortic anomaly.

Complex congenital aortic anomalies include diverse types of malformations that may be clinically asymptomatic or present with respiratory or esophageal ...

Scaling of Engineered Vascular Grafts Using 3D Printed Guides and the Ring Stacking Method

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Chapters in this video

Engineering Biological-Based Vascular Grafts Using a Pulsatile Bioreactor

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

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Fabrication of Custom Agarose Wells for Cell Seeding and Tissue Ring Self-assembly Using 3D-Printed Molds

Stepwise Cell Seeding on Tessellated Scaffolds to Study Sprouting Blood Vessels

Standardized Technique of Aortic Valve Re-implantation for Valve-sparing Aortic Root Replacement

Fabrication of Engineered Vascular Flaps Using 3D Printing Technologies

Three-Dimensional Printing of a Complex Aortic Anomaly