School of Medicine and Health Sciences Poster Presentations

Title

Plug ‘N Play: Induction of Biomimetic and Hierarchical Angiogenesis Under Stimulation with S1P

Poster Number

114

Document Type

Poster

Status

Medical Student

Abstract Category

Basic Biomedical Sciences

Keywords

Tissue Engineering, Cell Culture, Endothelial Cells, Reconstructive Surgery, Plastic Surgery

Publication Date

Spring 2018

Abstract

Introduction:

The field of tissue engineering has long held the promise of eventual production of an "off-the-shelf" vascularized free flap as a means of true dermal replacement. Our current state of dermal replacement utilizes an acellular dermal matrix that, while promising and effective, is severely limited by the condition of the wound bed. A tissue engineered dermal replacement that includes an inherent vascular network would obviate this limitation. Our previous experiments have demonstrated reliable endothelial cell sprouting from a monolayer under stimulation by a gradient of S1P, VEGF, and FGF, and we have even demonstrated the self-assembly of endothelial cells within a collagen matrix. The task remains to anastomose this microsurgically relevant monolayer with the capillary-like self-assembled vessels. It is our hypothesis that the induction of such sprouts into a self-assembled endothelial cell network will anastomose to the network, thereby achieving hierarchical vascular organization that can be utilized in the ultimate production of tissue-engineered pre-vascularized free flaps.

Methods:

Invasivity assays were created by injecting Type 1 Collagen impregnated with 1 mM S1P into well plates. A monolayer of GFP-tagged Human Umbilical Vein Endothelial Cells (HUVECs) were topically seeded, covered with Endothelial Cell media enhanced with VEGF and FGF, and cultured for 1, 3, and 5 Days. Self-assembled network assays were created by seeding a monolayer of GFP-tagged HUVECs as above, followed by injecting a second layer of collagen above the monolayer and coverage with growth factor enhanced media. After optimization, combination cultures were created by layering a collagen base layer, a YFP-Tagged HUVEC network second layer, an S1P impregnated collagen third layer, and a topically seeded GFP-Tagged HUVEC monolayer. All constructs were fixed and confocal imaged for analysis.

Results:

Invasivity assays demonstrated robust sprouting with an average of 46.89 ± 2.54 by Day 1, 131.4 ± 10.34 by Day 3, and 321.3 ± 39.31 by Day 5 per 0.6 mm². [AA1] Self-assembled network assays demonstrated biologically appropriate inter-capillary distance less than 50mm by Day 5. Combination cultures demonstrated robust sprouting from both the network and monolayer and anastomosis as evidenced by YFP and GFP multi-fluorescent channels, however quantification of anastomoses are still in progress.

Conclusions:

In this project we have successfully demonstrated the ability of endothelial cells to form a hierarchical vascular network that mimics that of normal human tissue in a way that is readily scalable and easily reproducible. Future iterations of this project will focus on a straight channel design and maintenance of the channel and network with flow.

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Creative Commons License
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Plug ‘N Play: Induction of Biomimetic and Hierarchical Angiogenesis Under Stimulation with S1P

Introduction:

The field of tissue engineering has long held the promise of eventual production of an "off-the-shelf" vascularized free flap as a means of true dermal replacement. Our current state of dermal replacement utilizes an acellular dermal matrix that, while promising and effective, is severely limited by the condition of the wound bed. A tissue engineered dermal replacement that includes an inherent vascular network would obviate this limitation. Our previous experiments have demonstrated reliable endothelial cell sprouting from a monolayer under stimulation by a gradient of S1P, VEGF, and FGF, and we have even demonstrated the self-assembly of endothelial cells within a collagen matrix. The task remains to anastomose this microsurgically relevant monolayer with the capillary-like self-assembled vessels. It is our hypothesis that the induction of such sprouts into a self-assembled endothelial cell network will anastomose to the network, thereby achieving hierarchical vascular organization that can be utilized in the ultimate production of tissue-engineered pre-vascularized free flaps.

Methods:

Invasivity assays were created by injecting Type 1 Collagen impregnated with 1 mM S1P into well plates. A monolayer of GFP-tagged Human Umbilical Vein Endothelial Cells (HUVECs) were topically seeded, covered with Endothelial Cell media enhanced with VEGF and FGF, and cultured for 1, 3, and 5 Days. Self-assembled network assays were created by seeding a monolayer of GFP-tagged HUVECs as above, followed by injecting a second layer of collagen above the monolayer and coverage with growth factor enhanced media. After optimization, combination cultures were created by layering a collagen base layer, a YFP-Tagged HUVEC network second layer, an S1P impregnated collagen third layer, and a topically seeded GFP-Tagged HUVEC monolayer. All constructs were fixed and confocal imaged for analysis.

Results:

Invasivity assays demonstrated robust sprouting with an average of 46.89 ± 2.54 by Day 1, 131.4 ± 10.34 by Day 3, and 321.3 ± 39.31 by Day 5 per 0.6 mm². [AA1] Self-assembled network assays demonstrated biologically appropriate inter-capillary distance less than 50mm by Day 5. Combination cultures demonstrated robust sprouting from both the network and monolayer and anastomosis as evidenced by YFP and GFP multi-fluorescent channels, however quantification of anastomoses are still in progress.

Conclusions:

In this project we have successfully demonstrated the ability of endothelial cells to form a hierarchical vascular network that mimics that of normal human tissue in a way that is readily scalable and easily reproducible. Future iterations of this project will focus on a straight channel design and maintenance of the channel and network with flow.