bioprinting

Several studies have explored both indirect and direct EB techniques in fabricating intricate vascular network to date.In indirect printing,tissue-specific,cell-incorporated hydrogels are used to encapsulate a prefabricated sacrificial vascular network,followed by the removal of the sacrificial ink using an appropriate are then seeded around the capillary channels using an injection/microfluidic perfusion of the capillary network with media or blood forms a monolayer of ECs around the capillaries within a couple of days that facilitates diffusional mass transfer and eventual remodeling into blood vessels.

Cytocompatible sacrificial templates have been explored to avoid the use of cytotoxic organic solvents or processing conditions to eliminate the sacrificial filaments from engineered constructs[20](Fig.1).In one study,carbohydrate glass filaments were dispensed at 110°C using a 3D printer to form a patterned 3D network,and then the network was encapsulated in an agarose polymer loaded with primary rat hepatocytes and fibroblast soluble sacrificial f i laments were removed by the cell culture media,the vascular lumen then seeded with human umbilical vein endothelial cells(HUVECs),and the vascular network perfused with blood in vascularized construct supported the metabolic function of primary rat hepatocytes,which maintained higher albumin secretion and urea synthesis than in gels without channels[21].However,complexities related to high temperature dispensing,random distribution of multiple cell types,and hygroscopic behavior of carbohydrate filaments limit vascularization number of studies have investigated the efficacy of sacrificial networks with respect to generating than random incorporation of multiple types of tissue-or organ-specific cells in the matrix,capillary vessels and various types of cells were printed separately side-by-side to mimic the native tissue or organ.In one study,a 3D bioprinting technique was used to co-print fibroblast-laden GelMA,fugitive ink,and human neonatal dermal fibroblast-loaded GelMA strands layer vascular network fabricated in this way was encapsulated into GelMA,and then the fugitive ink(Pluronic?F127)removed by liquifying at 4°C.When the HUVECs were injected in the fugitive ink-removed lumen followed by gentle rocking,greater than 95%viability was achieved with a confluent EC layer identified 48h after seeding[22].

Fig.1.(A)Carbohydrate glass lattice as the sacrificial structure for the creation of vascular architecture,(B)a single carbohydrate glass fiber encapsulated in a fibrin gel,(C)cross-section image of unlabeled HUVEC and 10T1/2 co-cultures(not expressing enhanced greenfluorescent protein(EGFP))encapsulated in the interstitial space of fibrin gel with perfusable channels,and(D)cross-section of cell-incorporated biomaterials(scale bars=200μm)(reproduced with permission from[21]).

Extrusion-based systems featuring coaxial or shell/core nozzles have been used to directly print hollow fibers or filaments with a microfluidic channel to form a vascular network[23,24].In particular,Fig.2 shows that the flow of ionic crosslinker()is maintained in the core side of a coaxial nozzle while the flow of hydrogel is maintained in the shell side[25].The flow rheology of the hydrogel precursor and crosslinker as well as the hydrogel percentage significantly affects the geometry of the core diameter,wall thickness,and microfluidic 3D microfluidic networks can be encapsulated into hydrogels containing multiple cell types.In general,microfluidic channels fabricated with a coaxial nozzle-extrusion system are seeded with ECs,and then perfused with culture media or blood to promote capillary blood vessel of shell/core size significantly affects the ultimate strength,compressive strength,and Young's modulus of the hollow fibers[26].In addition to seeding,ECs can also be encapsulated in the shell of the hollow channel during fabrication to generate high cell densities[27].In some studies,tissue spheroids composed of tissue-specific cells were dispensed simultaneously in the space between two successive hollow filaments using multiple robotic arms[28].Several studies report the efficacy of microfluidic channels fabricated with a coaxial nozzle for promoting human bone marrow stromal cells(hBMSCs)were seeded on the inner walls of hollow alginatepoly(vinyl alcohol)(PVA) fibers extruded from a coaxial nozzle,they showed excellent attachment and spreading after 14 days of culture[26].Similarly,cartilage progenitor cell(CPC)-encapsulated sodium alginate has been extruded from a coaxial nozzle to form the shell of a microfluidic ,CPC viability decreased due to the dispensing pressure,coaxial nozzle geometry,and alginate concentration,but improved significantly over 7 days of incubation[27].

To date,a wide range of natural,synthetic,and hybrid biomaterials have been used in bioprinting;however,none are free from biomaterials show uncontrolled degradation,immunogenicity,inflammation,and cytotoxicity in in vivo or in vitro some cases,biomaterials inhibit ECM secretion,distribution,and organization as well as cell-cell address these issues,researchers have investigated self-assemble approaches in which scaffold-free multicellular spheroids or filaments are extruded using a such systems,sacrificial spheroids and multicellular tissue spheroids are concurrently printed layer-by-layer as per the CAD incubation in a bioreactor,the multicellular spheroids fuse together to form single-or double-layered microvascular fusion process is time-consuming and causes non-uniform tubular surfaces,and the fabrication of long vascular tubes is a slow process and demonstrates poor spatial tackle these issues,RP technology has been used to bioprint cylindrical multicellular building blocks using collagen gel as a ,vascular cells eventually integrate with the collagen rod in the fusion process,which causes complexities in the removal of the collagen gel from the vascular cell-fused hollow inert agarose rods as a molding template solved the issues related to the collagen gel.When human umbilical vein smooth muscle cells and dermal fibroblasts were dispensed as a multicellular cylinder according to the CAD design,double-layered vascular walls formed 3 days after fusion[30].

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