assembly

of a capillary bed-like structure mimicking native tissue:(A)fabrication of a micro-scale structure using a soft lithographic technique,(B)microprinting using conformational contact to form a pattern of ink on the surface,(C)microfluidic channels fabricated using micromolds(channels are used to form micro fibers of a sacrificial substance that is then removed to form hollow fibers),and(D)complex vascularized structures fabricated using an assembly of microgels(reproduced with permission from[53]).

of omnidirectional and electrostatic discharge fabrication of 3D microvascular networks:(A)extrusion of a fugitive ink into a gel in a hierarchical fashion,(B)migration of fluid from capping layer to the voids generated by nozzle translational speed,(C)photopolymerization of hydrogel matrix,(D and E)microvascular channels that are created by dissolving and removing the fugitive ink under a modest vacuum,(F) fluorescent image of a 3D microvascular network(scale bar=10 mm),(G)blue fooddye injected microvascular networks in an acrylic block with three fluidic access points(scale bar=1 cm),and(H)branched microvascular networks embedded in a molded PLA block incorporated with a hierarchy of microchannel diameters(scale bar=2 cm)(reproduced with permission from[62,63]).

The success of a macro-scale tissue engineered construct depends on several factors,including the availability of a mass diffusion network within 100–200μm from the cell population,uniform distribution of multiple cell types with reasonable density,and nonthrombogenic phenotype of ECs upon integration with the host vasculature[4,70].To address this issue,researchers have taken a bottom-up approach to fabricate macro-scale scaffolds,and found that self-assembled micro-tissues or-modules were a possible solution for fabricating large engineered constructs,as shown in such micro-modules are loaded into large tissue constructs,the micro-dimensions of these modules facilitate the diffusion of nutrients,oxygen,and essential biomolecules to the cell population embedded in the gels[71].To date,several methods have been investigated to generate microtissues molding and UV crosslinking,directed self-assembly,and gravity-enforced self-assembly approaches have frequently been used to build micro-scale modules[72–74].During preparation,tissue-specific cells are often encapsulated in the micro-modules,while a confluent layer of HUVECs is provided to coat their outer surface[75].In some studies,the outer surface was further coated with protein molecules before EC seeding to improve the micro-collagen modules implanted in mice formed more stable,mature,and perfused capillaries 14 days postoperatively compared to collagen modules without the fibronectin coating[76].Further,EC-coated microgels embedded with stem or progenitor cells showed impressive results with respect to stabilizing the newly formed capillary blood vessels relative to EC-coated modules;for example,implanted EC-coated collagen micro-modules containing BMSCs in the omental pouch in rats formed less leaky and more dense and mature capillaries[77].

A number of studies have used random packing,directed,or sequential assembly to accumulate cell-loaded micro-modules with different sizes and shapes to prepare macro-scale vascularized tissue constructs[78,79].In random packing,EC-coated modules are perfused with blood or culture medium that causes interconnected channel formation in the interstitial spaces of the directional assembly,shape-controlled microgels spontaneously form offset,linear,branched,or random aggregates depending on surfactant concentration,aspect ratio,agitation rate,and time[79].The sequential assembly approach was investigated in an effort to control the organization of the microgels having internal microchannels[80].Such microgels were prepared with photolithography and then assembled into a tubular construct where an interconnected and bifurcated structure resembling native vasculature was particular,SMCs and HUVECs were incorporated in the outer and inner layer of the microgel to form a biomimetic 3D including the thickness and diameter of the microgels,swiping speed of the needle,concentration of surfactants,and space between two successive microgels affected the length of the assembled 3D tubular sequential assembly is economical and applicable with respect to the formation of a complex vascular network,shortcomings associated with the fabrication of thick microgels(≥ 600μm)containing non-straight vertical cross-sections and with thin microgels(≤ 150μm)showing poor mechanical strength limit the application of this method[81].Overall,vascularized tissue formed with the modular approach shows poor tissue integration and capillary network formation in vivo.

assembly:(A)collagen solution loaded with human hepatoma(HepG2)cells was gelled into ethylene oxide tube at 37°C for 30 min,the tube was then segmented into 2-mm length,and the collagen modules were collected after rotating in a HepG2 cell loaded modules were seeded with HUVECs,accumulated into a larger tube,and perfused with medium or blood,(B)light micrographic image of a collagen–HepG2 module without HUVECs,(C)confocal microscopic image of vascular endothelial(VE)-cadherin-stained module showing a confluent layer of HUVECs around the outer surface of module after 7 days of culture,(D)perfusion of a modular construct in a large tube with phosphate buffered saline(PBS),(E)confocal microscopic image of a collagen–HepG2–HUVEC module after 7 days of culture with HepG2 cells labeled with a Vybrant?CFDA SE cell tracer kit,and(F)schematic diagram of the microgel assembly process(reproduced with permission from[75,79]).

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