Biology printed in 3D

Almost 46 years ago, the world caught the first glimpse of the concept of three-dimensional (3D) printing. In October 1974, David Jones wrote in his weekly column in New Scientist about liquid monomers that can be polymerised into solid when exposed to ultraviolet or even visible light. He then fantasised how manipulating two beams of lasers can substantiate solid structure of any shape at their intersection, unveiling the unlimited potential to produce products previously impossible to mould.

3D printing today

Today, 3D printing is no longer uncommon owing to its ever-maturing technology and increasingly affordable price. While still catching up with the speed of conventional manufacturing, the versatility of 3D printing offers unchallenged advantage over other choices, especially in the wake of the COVID-19 pandemic. It certainly gained fame by rescuing some broken production lines of not only ventilator components desperately needed in intensive care units, but also millions of personal protective equipment (PPE) including face shields, masks and nasal swabs.

But 3D printing has been transforming the world long before these uncertain times. Several years ago, Brazilian paediatricians fulfilled the desire of parents-to-be who are visually impaired to follow the development of their child. With a 3D-printed sculpture based on ultrasound imaging, these patients could finally feel the features of their babies with touch.

Bioprinting in 3D has also been booming in the field of tissue engineering over the past decade. The main aim is to generate biomimetic tissues or whole organs to repair or replace the damaged sites due to injuries or diseases. Using a unique bio-gel, a research team in Newcastle University successfully printed human cornea in 3D containing surviving stem cells, which brings us a step closer to minimising the dependence on donors for those who suffer from vision loss.

Printing complex vascular systems

Two commonly used bioprinting technologies are stereolithography and extrusion-based printing. 

Two common technologies used in 3D bioprinting.
In stereolithography printing, dynamic photo-patterning is controlled by a digital micro-mirror device (DMD), which accurately projects the light source onto the bio-ink to initiate polymerisation on the x-y plane for polymerisation. As the elevator moves down the z-axis, a 3D product is printed layer-by-layer.
In extrusion-based printing, bio-ink is ejected from the extrusion nozzle in 3D space by either pneumatic pressure or mechanical force (piston or screw).
Diagram by Kelvin Kwok

Developed by Charles Hull in 1986, stereolithography is based on the very concept of photo-polymerisation of monomers proposed by David Jones just twelve years ago. Dynamic photo-patterning of a light source allows instantaneous projection of a complex 2D pattern (x-y plane) onto a reservoir of bio-ink, thereby solidifying the liquid bio-ink on that plane. As the focal plane of the light source moves along the axis perpendicular to the 2D plane (z axis), 3D objects are crafted in a layer-by-layer manner.

A long-standing concern for product fidelity in stereolithography is the resolution along the z axis, since excess light that scatters in the pool of bio-ink may lead to unwanted polymerisation and smear the desired layer thickness.

Last year, scientists from the University of Washington found synthetic and natural additives that can potently absorb scattered light, yet are biocompatible and easy to wash away from the printed product1. With these, the team successfully generated entangling 3D structures, from mathematical space-filling curves to intertwined vasculature, with high fidelity.

To take one step further, they reconstructed an even more complex structure – lung alveolus, the morphology of which has been mathematically modelled as tessellations of 14-face polyhedra2. Within the hydrogel containing the printed alveolus model, they periodically ventilated the air sac structure in the centre. This oxygenated the red blood cells being perfused through the vasculature wrapping around the air sac at a distance of 300 µm (approximately three times the diameter of a hair).

A printout of the heart

Extrusion-based printing, on the other hand, has a relatively simple design, where bio-ink and cells are printed voxel-by-voxel in 3D space (as with ‘pixel’ for 2D) from the printer nozzle. Scientists have been attempting to improve its performance in printing live cells and soft biostructures such as extracellular matrix. This is largely limited by the lack of suitable support for these soft materials during printing, which results in poor resolution and fidelity.

To address this, Adam Feinberg and his team developed a technique named FRESH (freeform reversible embedding of suspended hydrogel). In their latest report in 20193, the second generation of FRESH printing was able to use unmodified collagen as the bio-ink with a resolution at 20 µm. This is a 10 times improvement compared to its predecessor, and remains superior to other extrusion-based approaches to date.

The team gradually scaled up their creation of cell-laden structures, starting with porous collagen scaffolds that exhibited micro-vascularisation after being implanted in mice, eventually to contracting heart ventricles containing interconnected and striated stem cell-derived cardiomyocytes. Finally, they printed a neonatal scale human heart completely out of collagen, which, according to microscale CT imaging, recapitulated all anatomical structures. 

Printing from inside

Organ-scale bioprinting definitely brings hope for whole organ transplant. But is there any possibility to avoid invasive procedures, if all we want is to repair a tiny fraction of a damaged organ?

A team based in China explored this possibility in a study published earlier this month4. Compared to conventional light sources for photo-polymerisation including ultraviolet and blue light, near-infrared light has a far better tissue penetrance, and hence should be more capable to initiate printing in deeper layers of the body without exposing any wounds.

To test this hypothesis, the team first injected bio-ink under the skin of a mouse. Then, using the structural coordinates of a human ear generated by computer, they instructed their printing device to project near-infrared light accordingly at the skin where the bio-ink (containing live chondrocytes, or cartilage cells) resided underneath. This successfully printed an ear-like structure subcutaneously, which retained a clear form after one month with observable cartilage formation.

A. Non-invasive bioprinting under the skin using near infra-red (NIR) light.
B. Subcutaneously bioprinted ear-like construct in a mouse. Scale bar = 5 mm
Chen et al., Sci Adv, 20204 (CC BY-NC 4.0)

Future of bioengineering

3D bioprinting has demonstrated great potential in organ-scale therapeutics, and even in guiding spinal cord regeneration5. But there is still a long way to go before it can meet clinical standards. And there are numerous challenges ahead, including ethical regulations and practical technical issues, such as the demand for billions of cells for merely one printed organ. Nevertheless, on-demand organ printing for medical purposes seems to be no longer impossible.

References:
1. Science. 2019;364(6439):458-464
2. J Biomech. 1980;13(10):865-73
3. Science. 2019;365(6452):482-487
4. Sci Adv. 2020;6(23):eaba7406
5. Nat Med. 2019;25(2):263-269

Feature photo from pixabay.com

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