A modular tissue engineering approach may have advantages over current therapies in providing rapid and sustained revascularization of ischemic tissue. In this study, modular protein microbeads were prepared from pure fibrin (FIB) and collagen-fibrin composites (COL-FIB) using a simple water-in-oil emulsification technique. Human endothelial cells and fibroblasts were embedded directly in the microbead matrix. The resulting microbeads were generally spheroidal with a diameter of 100–200 μm. Cell viability was high (75–80% viable) in microbeads, but was marginally lower than in bulk hydrogels of corresponding composition (85–90% viable). Cell proliferation was significantly greater in COL-FIB microbeads after two weeks in culture, compared to pure FIB microbeads. Upon embedding of microbeads in a surrounding fibrin hydrogel, endothelial cell networks formed inside the microbead matrix and extended into the surrounding matrix. The number of vessel segments, average segment length, and number of branch points was higher in FIB samples, compared to COL-FIB samples, resulting in significantly longer total vessel networks. Anastomosis of vessel networks from adjacent microbeads was also observed. These studies demonstrate that primitive vessel networks can be formed by modular protein microbeads containing embedded endothelial cells and fibroblasts. Such microbeads may find utility as prevascularized tissue modules that can be delivered minimally invasively as a therapy to restore blood flow to ischemic tissues.
The process of new blood vessel formation is critical in tissue development, remodeling and regeneration. Modular tissue engineering approaches have been developed to enable the bottom-up assembly of more complex tissues, including vascular networks. In this study, collagen-fibrin composite microbeads (100-300 μm in diameter) were fabricated using a water-in-oil emulsion technique. Human endothelial cells and human fibroblasts were embedded directly in the microbead matrix at the time of fabrication. Microbead populations were characterized and cultured for 14 days either as free-floating populations or embedded in a surrounding fibrin gel. The collagen-fibrin matrix efficiently entrapped cells and supported their viability and spreading. By 7 days in culture, endothelial cell networks were evident within microbeads, and these structures became more prominent by day 14. Fibroblasts co-localized with endothelial cells, suggesting a pericyte-like function, and laminin deposition indicated maturation of the vessel networks over time. Microbeads embedded in a fibrin gel immediately after fabrication showed the emergence of cells and the coalescence of vessel structures in the surrounding matrix by day 7. By day 14, inosculation of neighboring cords and prominent vessel structures were observed. Microbeads pre-cultured for 7 days prior to embedding in fibrin gave rise to vessel networks that emanated radially from the microbead by day 7, and developed into connected networks by day 14. Lumen formation in endothelial cell networks was confirmed using confocal sectioning. These data show that collagen-fibrin composite microbeads support vascular network formation. Microbeads embedded directly after fabrication emulated the process of vasculogenesis, while the branching and joining of vessels from pre-cultured microbeads resembled angiogenesis. This modular microtissue system has utility in studying the processes involved in new vessel formation, and may be developed into a therapy for the treatment of ischemic conditions.
Microvascular endothelial cells (MVEC) are a preferred cell source for autologous revascularization strategies, since they can be harvested and propagated from small tissue biopsies. Biomaterials-based strategies for therapeutic delivery of cells are aimed at tailoring the cellular microenvironment to enhance the delivery, engraftment, and tissue-specific function of transplanted cells. In the present study, we investigated a modular tissue engineering approach to therapeutic revascularization using fibrin-based microtissues containing embedded human MVEC and human fibroblasts (FB). Microtissues were formed using a water-in-oil emulsion process that produced populations of spheroidal tissue modules with a diameter of 100–200 µm. The formation of MVEC sprouts within a fibrin matrix over 7 days in culture was dependent on the presence of FB, with the most robust sprouting occurring at a 1:3 MVEC:FB ratio. Cell viability in microtissues was high (>90%) and significant FB cell proliferation was observed over time in culture. Robust sprouting from microtissues was evident, with larger vessels developing over time and FB acting as pericyte-like cells by enveloping endothelial tubes. These neovessels were shown to form an interconnected vascular plexus over 14 days of culture when microtissues were embedded in a surrounding fibrin hydrogel. Vessel networks exhibited branching and inosculation of sprouts from adjacent microtissues, resulting in MVEC-lined capillaries with hollow lumens. Microtissues maintained in suspension culture aggregated to form larger tissue masses (1–2 mm in diameter) over 7 days. Vessels formed within microtissue aggregates at a 1:1 MVEC:FB ratio were small and diffuse, whereas the 1:3 MVEC:FB ratio produced large and highly interconnected vessels by day 14. This study highlights the utility of human MVEC as a cell source for revascularization strategies, and suggests that the ratio of endothelial to support cells can be used to tailor vessel characteristics. The modular microtissue format may allow minimally invasive delivery of populations of prevascularized microtissues for therapeutic applications.
Revascularization of ischemic tissues is a major barrier to restoring tissue function in many pathologies. Delivery of pro-angiogenic factors has shown some benefit, but it is difficult to recapitulate the complex set of factors required to form stable vasculature. Cell-based therapies and pre-vascularized tissues have shown promise, but the former require time for vascular assembly in situ while the latter require invasive surgery to implant vascularized scaffolds. Here, we developed cell-laden fibrin microbeads that can be pre-cultured to form primitive vascular networks within the modular structures. These microbeads can be delivered in a minimally invasive manner and form functional microvasculature in vivo. Microbeads containing endothelial cells and stromal fibroblasts were pre-cultured for 3 days in vitro and then injected within a fibrin matrix into subcutaneous pockets on the dorsal flanks of SCID mice. Vessels deployed from these pre-cultured microbeads formed functional connections to host vasculature within 3 days and exhibited extensive, mature vessel coverage after 7 days in vivo. Cellular microbeads showed vascularization potential comparable to bulk cellular hydrogels in this pilot study. Furthermore, our findings highlight some potentially advantageous characteristics of pre-cultured microbeads, such as volume preservation and vascular network distribution, which may be beneficial for treating ischemic diseases.
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