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 © 2016 John Wiley & Sons, Ltd.

Introduction: Resurfacing complex full thickness wounds requires free tissue transfer which creates donor site morbidity. We describe a method to fabricate a skin flap equivalent with a hierarchical microvascular network. Materials & methods: We fabricated a flap of skin-like tissue containing a hierarchical vascular network by sacrificing Pluronic® F127 macrofibers and interwoven microfibers within collagen encapsulating human pericytes and fibroblasts. Channels were seeded with smooth muscle and endothelial cells. Constructs were topically seeded with keratinocytes. Results: After 28 days in culture, multiphoton microscopy revealed a hierarchical interconnected network of macro- and micro-vessels; larger vessels (>100 μm) were lined with a monolayer endothelial neointima and a subendothelial smooth muscle neomedia. Neoangiogenic sprouts formed in the collagen protodermis and pericytes self-assembled around both fabricated vessels and neoangiogenic sprouts. Conclusion: We fabricated a prevascularized scaffold containing a hierarchical 3D network of interconnected macro- and microchannels within a collagen protodermis subjacent to an overlying protoepidermis with the potential for recipient microvascular anastomosis.

No abstract

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.Methods: Pluronic F127 microfibers were sacrificed in neutralized type I collagen with 1 x10 6 cells/mL MDA-MB231 breast cancer cells suspended in the bulk of the hydrogel creating a central loop microchannel, 1.5 mm in diameter. A 5 x10 6 cells/mL cell suspension of human umbilical vein endothelial cells (HUVEC) or HUVEC and human aortic smooth muscle cells (HASMC) was seeded into the microchannel. Scaffolds without microchannel seeding served as controls. Following 7, 14, and 28 days of culture specimens were fixed and processed for histology. results: After 7, 14, and 28 days of culture, MDA-MB 231 cells had performed significant matrix remodeling and migrated extensively toward the surface of the hydrogel or "lumen" of HUVEC/HASMC seeded microchannels. At 14 and 28 days dense MBA-MB231 tumor nests were seen within the collagen hydrogel bulk of HUVEC and HUVEC/ HASMC-seeded microchannel constructs. After 7,14, and 28 days MDAMB demonstrated transendothelial migration into the central 'neovessel' of HUVEC-only seeded microchannels resulting in HUVEC apoptosis. After 7, 14, and 28 days, the endothelial lining of HUVEC/HASMC appeared thinner and somewhat destabilized, without the elaboration of additional matrix proteins as had been seen in previous constructs without breast cancer cells in the bulk. Immunohistochemical staining demonstrated adherence of both CD31 and VWF expressing endothelial cells and α-SMA positive-smooth muscle cells along co-culture seeded microchannels. Epithelial cell adhesion molecule (EpCAM) positive staining of MDA-MB231 cells confirmed tumor cell migration toward HUVEC-and HASMC/HUVEC-seeded microchannels as well as breast cancer tumor aggregates within the bulk after 14 and 28 days. MDA-MB231 cells demonstrated no distinct pattern of tumor formation or transmigration within the hydrogels containing unseeded microchannels. ConClusion:We have successfully created an in vitro 3D biomimetic platform to analyze the progression,transendothelial migration, and metastasis of MDA-MD231 breast cancer cells within tissue-engineered constructs containing endothelialized microchannels.Using our...

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. Methods:Using an oil emulsion technique, 1% collagen microspheres, ranging 50 to 150 μm in diameter, were created using neutralized type 1 collagen. 7 mm diameter microsphere scaffolds were fabricated by embedding 1% collagen microspheres in 0.3% type 1 collagen bulk. According to Kepler's conjecture of close-packed spheres, approximately 74% of the density of the scaffold was comprised of higher density microspheres, and the remaining density was taken up by the 0.3% collagen. Microsphere scaffolds underwent thermal gelation at 37° C for 1 hour. Non-microsphere-containing 1% collagen scaffolds and non-microsphere-containing 0.3% collagen scaffolds were also fabricated for comparison. Microsphere and non-microsphere-containing scaffolds were implanted subcutaneously in dorsa of WT C57bl/6 mice. Following 7 or 14 days of implantation, scaffolds were procured and subsequently processed for histological analysis.results: Histological analysis following procurement of microsphere scaffolds from mice dorsa after 7 days of implantation revealed substantial and uniform cellular invasion spanning the entire depth of the scaffold. After 14 days of implantation, immunohistochemical analysis identified CD31+ endothelial precursors within microsphere scaffolds, indicative of a progression in cellular invasion with the formation of neovasculature. Comparatively, even after 14 days, cells sporadically and only partially invaded the 0.3% collagen scaffolds and failed to invade the 1% collagen scaffolds, instead proliferating along the periphery of the scaffold. ConClusions:We have demonstrated that altering the mechanical and spatial cues within hydrogel scaffolds by creating interfaces of differential collagen densities significantly improves cellular invasion. In addition to optimizing the architectural and structural cues sensed by cells, microspheres may also be impregnated with chemical moieties to further promote cellular invasion. We believe this approach holds tremendous promise for creating the optimal wound scaffold.