One promising approach is droplet-based laser bioprinting, though to date, only a few studies have reported fabrication of complex 3D patterns. Now, researchers from the University of Florida and Tulane University have demonstrated the feasibility of laser printing straight and Y-shaped tubes with and without living cells. Branched tubes are of particular importance as they provide the basic component for creating blood vessels, a major technological challenge when printing 3D human organs (Biofabrication 7 045011).

 "Due to its potential in the freeform fabrication of 3D heterogeneous structures, I think that 3D bioprinting will eventually enable the printing of entire organs, bringing significant societal impacts," said corresponding author Yong Huang from the University of Florida.

How to bioprint

During 3D laser bioprinting, structures are typically fabricated layer-by-layer, with each layer formed from numerous deposited droplets patterned in a pre-designed shape. After each layer is printed, a Z-platform moves it down a specified distance into a crosslinking solution (calcium chloride solution in this work), thereby gelling the newly deposited layer.

Huang and colleagues studied two bio-inks: 8% alginate solution and 2% alginate-based fibroblast suspension. They employed optimal printing conditions, identified as 2125 mJ/cm2 laser fluence, 100 mm/min substrate velocity and 10 Hz laser pulse repetition rate for alginate; and 1445 mJ/cm2 laser fluence, 80 mm/min substrate velocity and 10 Hz repetition rate for the cellular bio-ink. To optimize printing quality, the step size of the Z-platform must be adapted to the material being printed. For the alginate and cellular bio-inks, they identified optimal step sizes of 30 and 25 µm/layer, respectively.

The researchers first fabricated straight alginate tubes, printed with 170 layers to a height of 5.1 mm and a diameter of 5.0 mm (the mean diameter of blood vessel). The printing process took around 30 minutes. The wall thickness of the tubes was measured as 1.3 ± 0.1 mm. Images of a representative tube showed it to be well-defined, with a better surface finish, smaller wall thickness, and smoother tube ends than tubes printed under non-optimal conditions.

Next, they printed 5 mm diameter Y-shaped alginate tubes, which took around 120 minutes. Creating the Y-shapes requires printing of overhang and spanning structures – a previously unresolved manufacturing challenge. To do this, the researchers used the calcium chloride solution both as a crosslinking solution and also to provide a buoyant force to help support the printed structures.

The Y-shaped tubes had an average wall thickness of 1.4 ± 0.3 mm and a total height of around 9.5 mm. The two branches were successfully created with the designed 45° inclination angle and no residual alginate material between the two branches.

Cellular tubes

The team then used the cellular bio-ink to fabricate straight fibroblast tubes with 260 layers, which took around 45 minutes. A representative tube had a diameter of 5.0 mm, a height of 6.5 mm and a wall thickness of 2.3 ± 0.3 mm – almost double that of the alginate-only tubes.

The cell viability in these tubes was 63.8% immediately after printing and 68.2% after 24 hours incubation, an increase attributed to cell proliferation and recovery from cell injury. The authors note that this viability is reasonable for effective bioprinting and could be further improved by optimizing the printing conditions.

Finally, they printed Y-shaped cellular constructs with 5 mm diameter, which took around 180 minutes. The resulting tubes had a well-defined morphology, a total height of around 9.5 mm and wall thickness of 2.5 ± 0.3 mm. As seen in the straight tubes, the wall thickness of Y-shaped cellular constructs was almost double that of the alginate-only tubes, attributed to the non-ideal droplet formation process when printing cell-laden bio-inks.

The cell viability of the bifurcated constructs was 68.1% immediately after printing and 70.8% after 24 hours of incubation. The slightly higher post-printing viability seen for the Y-shaped over the straight construct was attributed to less impact force damage during overhang printing.

"This is the first example of laser bioprinting a cellular overhang construct," Huang told medicalphysicsweb. "We have also successfully printed cellular overhang constructs using inkjetting. However, laser printing is more versatile in printing highly viscous bio-inks, since it is an orifice-free technology, it has no concern of nozzle clogging."

Next, Huang and colleagues plan to focus on printing 3D heterogeneous multicellular constructs, modelling of printing-induced cell injury, post-printing tissue fusion and maturation, and clinical testing.

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