Jeremiah Joyce

Fabrication of capillary-like structures with Pluronic F127® and Kerria lacca resin (shellac) in biocompatible tissue-engineered constructs#

The fabrication of large cellular tissue‐engineered constructs is currently limited by an inability to manufacture internal vasculature that can be anastomosed to the host circulatory system. Creation of synthetic tissues with microvascular networks that adequately mimic the size and density of in vivo capillaries remains one of the foremost challenges within tissue engineering, as cells must reside within 200–300 μm of vasculature for long‐term survival. In our previous work, we used a sacrificial microfibre technique whereby Pluronic® F127 fibres were embedded and then sacrificed within a collagen matrix, leaving behind a patent channel, which was subsequently seeded with endothelial and smooth muscle cells, forming a neointima and neomedia. We now have extended our technique and describe two approaches to synthesize a biocompatible tissue‐engineered construct with macro‐inlet and ‐outlet vessels, bridged by a dense network of cellularized microvessels, recapitulating the hierarchical organization of an arteriole, venule and capillary bed, respectively. Copyright

Abstract 162: in vivo microanastomosis of microvessel containing tissue-engineered constructs: the final frontier.

PurPose: Although autologous tissue transfer has been established as a reliable approach to the reconstruction of complex defects, there are associated consequences including donor site pain, functional loss, paresthesias, dysthesthia, and scarring. The ability to synthesize vascularized constructs for the management of these complex wounds would represent a quantum leap in the field of tissue engineering. In previous work we synthesized and performed an in vivo microvascular anastomosis of a collagen construct containing an unseeded internal longitudinal microchannel with inlet and outlet. Here we fabricate and microsurgically anastomose collagen constructs containing an internal endothelialized microchannel.

A Novel Three-Dimensional Platform to Investigate Neoangiogenesis, Transendothelial Migration, and Metastasis of MDAMB-231 Breast Cancer Cells.

Background: A crucial step in the progression of cancer involves the transendothelial migration of tumor cells into the bloodstream and invasion at distant sites. Most in vitro models of malignant cell behavior do not account for the presence of and interaction with vascular cells. Three-dimensional platforms to further explore the factors responsible for metastatic cellular behavior are under intensive investigation. Methods: Hydrogels with encapsulated MDAMB-231 breast cancer cells were fabricated with a central microchannel. The microchannel was lined with a co-culture of human umbilical vein endothelial cells and human aortic smooth muscle cells. For comparison, co-culture–seeded microchannels without breast cancer cells (MDAMB-negative) were fabricated. Results: After 7 and 14 days, the endoluminal lining of encapsulated MDAMB-231 co-culture–seeded microchannels demonstrated aberrant endothelial cell and smooth muscle cell organization and breast cancer cell transendothelial migration. MDAMB-231 cells performed matrix remodeling, forming tumor aggregates within the bulk, migrating preferentially toward the hydrogel “neovessel.” In contrast, MDAMB-negative constructs demonstrated maintenance of an intact endoluminal lining composed of endothelial cells and smooth muscle cells that organized into discrete layers. Furthermore, the thicknesses of the endoluminal lining of MDAMB-negative constructs were significantly greater than encapsulated MDAMB-231 co-culture–seeded constructs after 7 and 14 days (p = 0.012 and p < 0.001, respectively). Conclusion: The authors have created a powerful tool that may have tremendous impact on furthering our understanding of cancer recurrence and metastasis, shedding light on these poorly understood phenomena.

Abstract P29: Optimization of Functional, Perfusable Vascular Networks within Tissue Engineered Hydrogels

eleCtroPhysiology: SSEP negative peak amplitudes were significantly decreased (p<.05) at POD 14, 28, 37, and 79 when compared to baseline with significant recovery at POD 79. SSEP stimulation thresholds were significantly increased (p<.05) at POD 37 and 79 with significant recovery occurring at POD 79. Abductor pollicis brevis thresholds obtained from Tc-MEP stimulation were significantly increased at POD 37 and 79. CNAPs were first measured at POD 42 and confirmed with stimulation-response curve. Nerve conduction velocity was 40% baseline at POD 90 with significantly increased CNAP stimulation thresholds (p<.05).

Abstract 87: Beyond Cotton Candy: Fabrication of Capillary Networks within Biocompatible Tissue- Engineered Constructs from Kerria Lacca Resin (Shellac).

PurPose: Creation of synthetic tissues with microvascular networks, which adequately mimics the size and density of capillaries found in vivo, remains one of the foremost challenges in the field of tissue engineering. In our previous work, we utilized a sacrificial microfiber technique whereby 1.5 mm Pluronic F127 microfibers were embedded within a collagen matrix, leaving a patent internal channel, which was subsequently seeded with endothelial and smooth muscle cells forming a neointima and neomedia respectively. Here we describe two approaches to synthesize a tissue-engineered construct with macro-inlet and outlet vessels, bridged by a dense network of microvessels, which recapitulates the hierarchical organization of an arteriole, venule, and capillary bed found in vivo.

Abstract 153: A Novel 3D Platform to Investigate Neoangiogenesis, Transendothelial Migration and Metastasis of Breast Cancer Cells.

PurPose: Breast cancer remains the most common cancer afflicting women and is the second leading cause of death from cancer. A crucial step in the progression of this disease is the transendothelial migration of tumor cells into the blood stream or lymphatic system. The factors guiding this process remain poorly understood. The development an in vitro biomimetic platform to further investigate these factors is under intensive investigation. In previous work we synthesized a tissue-engineered scaffold containing an endothelialized internal loop microchannel for microsurgical anastomosis and in vivo perfusion utilizing a sacrificial microfiber technique. Here we design a novel 3D platform to investigate tumor cell behavior in the presence of vascular cells in order to better understand the cell-cell and cell-matrix interactions that drive neoangiogenesis, invasion, metastasis and ultimately tumor progression.

Abstract P37: Optimizing Cellular Invasion into Hydrogel Scaffolds Using Microspheres to Create Interfaces of Differential Densities

PurPose: Although several acellular engineered tissue templates are available for clinical use, their success is limited to application within well-vascularized wound beds. In poorly vascularized wounds, such as those that have been irradiated or those with exposed hardware, bone or tendon, cellular and vascular invasion into tissue-engineered templates remains largely insufficient, leading to failure of incorporation or infection. Previous work in our lab demonstrated that cells preferentially invade scaffolds at the interface of differential densities, in some cases even more robustly than in scaffolds with well-defined microfeatures such as pores. As such, we fabricated a novel scaffold containing closely packed higher density collagen microspheres encased in a lower density collagen bulk, which created regularly spaced interfaces of differential densities so as to optimize cellular invasion and neovascularization.

Innovative 3D Collagen Microsphere Scaffold (MSS) Promotes Robust Cellular Invasion

METHODS: Utilizing Kepler’s conjecture of sphere packing, which states that the arrangement of spheres in a 3D space has a density of 74%, we fabricated 7 mm microsphere scaffolds (MSS) with a regular arrangement of density gradients. Type I collagen microspheres (1%, 0.6% or 0.4%), ranging 50 to 150 μm in diameter, were manufactured via an oil emulsion technique. MSS were fabricated by encasing microspheres of varying collagen density into collagen bulk of varying density (0.3%, 0.2% or 0.6%), so that 74% of the scaffold’s volume was comprised of microspheres and 26% of bulk collagen. MSS underwent thermal gelation at 37°C for 1 hour. Nonmicrosphere-containing 1% or 0.3% collagen scaffolds were fabricated as controls. Scaffolds were implanted subcutaneously in the dorsa of 8 week old wild-type mice and harvested for histological analysis after 7 or 14 days.

LOP09: Fabrication of three-dimensional vascular networks within tissue engineered hydrogel constructs

transmigration assay. To evaluate tube formation capacity LEC were plated on MatrigelTM. Cell sprouting was investigated in a 3D-assay. Results: LEC proliferation and migration could be elevated by MSC CM due to secretion of LEC stimulating factors in a higher extent than by stimulation with control medium or growth factors like VEGF-C and bFGF. MSC secreted factors furthermore enhanced LEC tube formation and sprouting directly. Ongoing experiments are focusing on loss-of-function and gain-of-function studies and aiming at identifying the unknown MSC-derived lymphangiogenic activity. Conclusions: The MSC-derived pro-lymphangiogenic activity could be demonstrated and provide the basis for current in vivo experiments to establish a lymphatic endothelial cell network in the nude rat AV-loop model. This model will allow deeper insights into lymphangiogenesis and subsequently be used for antilymphangiogenesis and metastasis research.