printing of scaffolds fabricated by an extrusion-based technique:(A)crosslinker diffusion while the biomaterial is extruding through the outer tube,(B)crosslinking of biomaterial,(C)deposition of numerous stands,(D–F)printing layers of scaffolds and immersing in crosslinker,and characterization of the fusion phenomenon between adjacent alginate hollow filaments:(G)macroscopic image of a cuboid scaffold containing six layers of hollow strands,(H)inverted microscopic image of longitudinal section of the scaffold,(I)macroscopic image of the scaffold cross-section,(J–L),confocal microscopic images at different magnifications showing the crosssection of the scaffold,(M)SEM image of fused filaments,with fibroblasts encapsulated in hollow alginate filaments,(N and O)microscopic images showing the lumen and wall of the hollow strands(white light),(P and Q)laser confocal images showing the lumen and wall of the hollow strands,and the live and dead cells as fluorescent green and fluorescent red,respectively,and(R)laser confocal image revealing the fused structure with channels(reproduced with permission from[29]).

3D printing

Laser-based bioprinting,particularly laser-guided direct writing(LGDW)and matrix-assisted pulsed laser evaporation direct writing(MAPLE DW),have been explored in different studies for 2D and 3D cell patterning[31].This printing technique has some attractive features,including no nozzle clogging and the ability to print cells at high resolution and accuracy with high-viscosity to laser-induced forward transfer(LIFT),the MAPLE DW technique uses a lower powered pulsed laser to deposit multiple cell types.In this technique,laser pulse-induced bubbles create shock waves that compel cells to move toward the collector number of studies have used laser-based bioprinting to print patterned structures with vascular cells and observed capillary vessel example,a study using the LIFT-based cell printing technique to print HUVECs and human mesenchymal stem cells(hMSCs)in a defined pattern on a cardiac patch reported increased capillary vessel density and functional improvement of infarcted hearts[32].Researchers have also used LGDW to print a 3D vascular network by stacking cell aggregates layer-by-layer,with a hydrogel layer placed on top of each deposited cell 3D patterned HUVEC on Matrigel?formed elongated and tube-like structures in vitro[33].However,shortcomings such as long fabrication time,laser-induced cell damage,and low scalability limit the application of the techniques in tissue vascularization.

Stereolithography,a maskless photolithography,has been investigated to generate complex 3D vascular patterns with photosensitive materials[34].In particular,digital light projection(DLP)and laser-based stereolithography have been used to print intricate architectures based on designs developed from CAD software,computer tomographic,and magnetic resonance imaging(MRI)scanned information[35].In a DLP system,a digital mirror device containing several million tiny mirrors regulates the movement of the mirrors via a digital rotation of mirrors causes a two-dimensional pixel-pattern that is projected on the photo-curable biomaterial to obtain intricate 3D a study,a DLP chip was used to generate active and reflective dynamic photomasks as per the CAD the cross-sectional images of the 3D microstructure were reproduced from photomasks and the images were projected onto the methacrylate(GelMA)solution using an ultraviolet(UV)light the 3D intricate pattern was seeded with HUVECs,the HUVECs formed a confluent monolayer and maintained their phenotype for 4 days following dynamic seeding[36].Similarly,another study reported that HUVECs formed cord-like structures after 4 days of culture in a scaffold fabricated with a DLP system[37].While this technique can print 3D structures quickly with high resolution,shortcomings such as high costs,less detailed printing for large constructs,and cytotoxicity limit the application of the DLP technique.

Laser-based stereolithography(LS)was developed to eliminate the requirement of a photomask and assembly of multiple 2D planar surfaces to form 3D vascular LS is suitable for printing large and detailed vascular constructs,the lower printing speed of LS compared to the DLP technique needs to be improved[38].In this technique,a computer-controlled ultraviolet laser beam generates a pattern on a photosensitive material as per the CAD design[39],as shown in Fig.3.A number of researchers have printed complicated structures with LS and reported outstanding results with respect to forming seeding of ECs in the LS-printed scaffold showed improved viability,whereas incorporated cells in the photosensitive hydrogel demonstrated low viability due to laser(short wavelength)-induced cell avoid short wavelengths,researchers have employed two-or multi-photon laser systems to print intricate 3D structures with micro-or nano-scale precision[40].Although the two-and multi-photon laser systems maintain a less harsh environment than LS during printing,use of a photoinitiator in the gelation process significantly decreases cell viability[38].To address this issue,a multi-photon printing technique was used to fabricate 3D multi-scale patterns in soft silk protein hydrogels without using a photo-initiator;the 3D features supported the growth and penetration of human mesenchymal stem cells deep into the gel[41].Several studies have investigated the efficacy of vascular patterns/networks printed with the LS technique with respect to promoting vascular one study,polytetrahydrofuran diacrylate resin-based macro-scale vascular tubes and micro-scale bifurcating tubes were printed using LS and two‐photon polymerization(2PP)techniques, vascular vessels printed using this method could be seeded with ECs to form vasculature,because the grafts demonstrated good cytocompatibility and mechanical properties similar to native capillaries[42].Similarly,when granulosa cells were seeded on an epoxy-based acellular microcapillary vascular tree printed with the 2PP technique,improved cell growth and sustained cell-cell junctions were identified in vitro[43].These studies demonstrate that EC monolayer formation is possible in an LS-printed vascular pattern.

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