BioprintingMedical ResearchResearch & Education

Utrecht Medical and EPFL researchers volumetrically bioprint living tissue constructs in seconds

State of the art research in volumetric bioprinting (VBP) method proves highly effective

A team of researchers from the Utrecht Medical Center and the École Polytechnique Fédéral Lausanne (EPFL) demonstrated the bioprinting of large living tissue constructs by processing cell‐friendly hydrogel‐based bioresins with a volumetric, visible light laser‐based printer. Authors of the study include bioprinting pioneers such as Professors Jos Malda and Riccardo Levato from the Utrecht Medical Center, as well as Professor Christophe Moser, from EPFL, who is an expert in photonics. Their research could open up new possibilities in terms of reproducing complex human organ tissue grafts by enabling the additive creation of highly complex geometries without the limitations of a layer-based method. Their research was published in the prestigious Advanced Materials journal.

Biofabrication technologies in general, including stereolithography and extrusion‐based printing, are revolutionizing the creation of complex engineered tissues. Current bioprinting – and in most additive manufacturing processes – relies on the additive layer‐by‐layer deposition and assembly of repetitive building blocks, typically cell‐laden hydrogel fibers or voxels, single cells, or cellular aggregates. The scalability of these additive manufacturing technologies is limited by their printing velocity, as lengthy biofabrication processes impair cell functionality.

The volumetric method described in this new study overcomes such limitations by bioprinting clinically relevant sized, anatomically shaped constructs, in a time frame ranging from seconds to tens of seconds. The scientists developed a custom bioprinter specifically for this project. In the future, a spinoff company, exclusively dedicated to these applications, could evolve and commercialize it.

Fast tissue

An optical‐tomography‐inspired printing approach, based on visible light projection, was developed to generate cell‐laden tissue constructs with high viability from gelatin‐based photoresponsive hydrogels. This method enables the creation of free‐form architectures, difficult to reproduce with conventional printing: these include anatomically correct trabecular bone models with embedded angiogenic sprouts and meniscal grafts.

By printing functional hydrogel‐based ball‐and‐cage fluidic valves, the researchers demonstrated the ability to generate free‐floating structures which are key in complex tissue and organ production. Volumetric bioprinting permits the creation of geometrically complex, centimeter‐scale constructs at an unprecedented printing velocity, thus opening new avenues for upscaling the production of hydrogel‐based constructs and for their application in tissue engineering, regenerative medicine, and soft robotics.

volumetric bioprinting
Overview of the volumetric bioprinting process, showing A) the cell‐laden gelRESIN reservoir connected to a rotating platform, B) a schematic of tomographic projections used to print the human auricle model, and C) a rendering of the resulting printed hydrogel structure. The inset in (C) shows a stereomicrograph of the actual printed hydrogel, stained with alcian blue to facilitate visualization (printing time = 22.7 s; scale bar = 2 mm). Image courtesy of Advanced Materials.

The VBP technology

The technology works by depositing a 3D light dose distribution into a cylindrical container of photopolymer gel to permit its spatially selective crosslink, as shown in the image above. To build up this 3D dose distribution, the resin container is set into the rotation and synchronously irradiated with a sequence of 2D light patterns, computed by a Radon transform, and applying the principles of medical tomographic imaging in reverse, which is the basis for all volumetric 3D printing.

In other words, the light patterns represent projections of the object to fabricate along with multiple rotational angles of the cylindrical volume of photopolymer. These dynamic light patterns are displayed into the build volume by irradiating a DLP modulator with a 405 nm laser source. The polymer solidifies only in selective areas where the accumulation of multiple angular exposures results in an absorbed dose (shown in the video below). The main issue is that, unlike traditional layered photopolymerization, any overcuring results in the part’s deformation so the minimum exposure time to gelate the 3D object within the optical field must be very carefully determined.

In living colors

Living tissues owe their functionality predominantly to their complex architecture. Their complex extracellular composition. Just like any 3D printed part, organs need to be anisotropic (meaning they have the same mechanical properties in all directions), however, this is even more relevant for living organs than for any inanimate part.

Capturing such a shape–function relationship within engineered biomaterials holds great potential for the creation of new cell‐instructive implants and in general for all regenerative medicine based lab-grown cells.

Irregular and anisotropic architectures are fundamental, for example, in the load‐bearing function of cancellous bone as the trabecular framework aligns along the main direction of stress, in the shock‐absorber function of menisci, where geometry and zonal architecture distribute applied loads, or in the contractile function of cardiac and skeletal muscle, as cell alignment provides directionality for force generation.

In this regard, volumetric printing technologies introduce a paradigm shift, as they enable the creation of entire objects at once, rather than through the sequential addition of basic building blocks.

Volumetric bioprinting
Main volumetric printing process parameters and printed construct features. A) Graphical representation of the light penetration pattern through the entire build volume in the presence of a photoinitiator. B) Fabrication time for the human auricle model scaled 1× (0.15 cm3), 2× (1.23 cm3), and 3× (4.14 cm3) using different bioprinting techniques: volumetric printing, extrusion‐based (bio)printing, and digital light processing. C) Close‐up images of the surface features of the auricle fabricated through (i) volumetric printing, (ii) extrusion‐based printing, and (iii) digital light processing (scale bars = 500 µm). D) Volumetric printing of a fluidic ball‐cage valve with free‐floating elements from a (i) computer‐generated 3D model of the valve. (ii) Top view of the printed valve (scale bar = 2 mm), (iii) close‐up of the cage segment that permits flow (scale bar = 1 mm), and video stills showing unidirectional flow functionality with an (iv) open and a (v) closed valve (black arrow represents the flow direction). Image courtesy of Advanced Materials.

In this study, VBP showed a promising volume accuracy, with printed human auricle models, when comparing the printed constructs acquired via microcomputed tomography (µCT) and the original STL files. Remarkably, and in contrast to other additive bioprinting approaches, printing time is not bound to the dimensions of the construct.

In extrusion‐based bioprinting, the printing time increases cubically with the scaling factor, quickly reaching an unfavorable magnitude when cell‐laden centimeter‐scale objects are required without greatly compromising on resolution.

Instead, in DLP processes (but not in conventional SLA), the printing time increases linearly as a function of the height of the construct, independently on the area to be printed in each individual layer. DLP processes can be accelerated by reducing the lifting time of each printed layer, as shown by the recent development of the continuous liquid interface printing (the high-speed, planar technology successfully commercialized by Carbon).

However, even with planer approaches, the overall fabrication time would be one order of magnitude higher than that achieved with volumetric bioprinting. Conversely, in volumetric bioprinting, the printing time can be consistently found in the range of tens of seconds, regardless of the volume of the construct, as long as the same irradiation intensity is supplied to the photopolymer. In order to keep a constant printing time for a build volume that is twice as large, it is sufficient to use a four times more powerful laser output. Furthermore, volumetric bioprinting results in seemingly artifact‐free surface features, without the surface roughness that characterizes all 3D printing processes.

Free floating

Finally, the researchers demonstrated the possibility, unique to volumetric bioprinting, to print free‐floating parts without the need for sacrificial support materials or two‐photon polymerization approaches. This feature is considered paramount to generate systems able to reversibly modify their shape post-printing, and similar free‐moving parts could be included also in structures printed with stimuli‐responsive materials (often used in 4D printing – and it doesn’t get any more 4D than living organs).

For instance, to facilitate shape changes, hydrogels can be combined with nanoparticles. On the other hand, the freedom of design provided by volumetric bioprinting approaches permits the production of such actuators through the direct fabrication of movable or articulating parts. To confirm this, the researchers also printed a fluidic valve inspired by the ball‐and‐cage cardiac valve prosthesis (shown in the figure above, Di–iii)

Such a model, unlike other valve designs such as the anatomically inspired bi‐ and tri‐leaflets, cannot be directly fabricated by extrusion or DLP/DMD technologies in the absence of sacrificial supports. When connected to a fluidic system, the valve could function correctly, enabling unidirectional flow within the circuit. This feature can have potential applications in hydrogel‐based microfluidics or in hydrodynamic‐actuated soft robots. To achieve this type of complex constructs, the thermoreversible gelation of gelRESIN is particularly advantageous, however the results were so successful to suggest that, given the rapidity of the VBP process, homogenous cell suspension could be achieved even when processing alternative bioresins that lack the thermal gelation behavior of gelatin.

Volumetric bioprinting
A) Volumetric bioprinted, ACPC‐laden gelRESIN disc‐shaped constructs were cultured for 7 d and exhibited (i) high cell viability (>85%), (ii) increased metabolic activity over time, and (iii) comparable cell survival compared to hydrogel casting and other commonly used bioprinting techniques: extrusion‐based bioprinting and digital light processing. B) A complex trabecular bone model was fabricated to examine the development of salient characteristics post‐printing. (i) The printed bone exhibited a porosity that extended throughout the extended 3D construct (scale bar = 2 mm), (ii) as shown through μCT imaging. C) The O‐MSC‐laden (pink) bone construct was cultured for 7 d (scale bar = 1 mm). D) Constructs were then seeded with ECFCs (green) and P‐MSCs (yellow) to induce capillary formation. (i) ECFCs were shown to interconnect after three additional days of culture, filling the pores of the printed construct (scale bar = 500 µm), (ii) and were also shown to begin invading the bone compartment of the construct (scale bar = 250 µm). (iii) Vessel interconnectivity and length were also measured quantitatively and compared to a control 2D culture condition. Image courtesy of Advanced Materials.

Multimaterial volumetric bioprinting 

The actual paper  goes much more in-depth with additional samples and we invite you to read the full document here. The bottom line is that the volumetric bioprinting process is showing great promise (as we had predicted a few months ago) and with its adoption in the field of biofabrication, several future developments can be expected. There is virtually no limitation on the use of different photopolymers, and several photoresponsive natural or synthetic hydrogels could be optimized for this process (including but not limited to materials based on hyaluronan, PEG, alginate, or decellularized ECM) or even stimuli‐responsive biomaterials for remote stimulation of the construct or controlled patterning of bioactive molecules.

Next steps pertaining to the technology should introduce the potential for printing multiple materials within the same process, as this will be important to further mimic the heterogeneous composition of living tissues. For example, multimaterial volumetric printing could be used to address the zonal architecture of certain tissues (i.e., cartilage, menisci), create cell and material gradients, replicate biological interfaces, introduce vascularization in a single step, or even coprint mechanically strong polymers to reinforce the cell‐laden bioprinted hydrogels.

The rapid speed of volumetric bioprinting is an important benefit for the production of tissues and disease models. The generation of large constructs with arbitrary shape can aid patient‐specific regenerative medicine, in light of potential translation of clinically relevant grafts. At the same time, drug discovery and testing typically require testing of a large number of molecule combinations on identical models, which can easily be produced on a large scale with the proposed method, also reducing costs related to personnel and machine time necessary per constructs. The researchers go on to indicate that this capability can complement and even reduce animal testing in the intermediate phases of drug development, leading to lower development costs and fewer ethical issues. Complemented by these perspectives for future developments, our results and the volumetric bioprinting technology proposed herein pave the way for the next generation of large and functional biofabricated grafts, with a wide array of envisioned applications for tissue regeneration, in vitro tissue and disease models, and soft robotics.

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Davide Sher

Since 2002, I have built up extensive experience as a technology journalist, market analyst and consultant for the additive manufacturing industry. Born in Milan, Italy, I spent 12 years in the United States, where I received my Bachelor of Arts undergraduate degree. As a journalist covering the tech industry - especially the videogame industry - for over 10 years, I began covering the AM industry specifically in 2013, as blogger. In 2016 I co-founded London-based 3D Printing Business Media Ltd. (now 3dpbm) which operates in marketing, editorial, and market analysis & consultancy services for the additive manufacturing industry. 3dpbm publishes 3D Printing Business Directory, the largest global directory of companies related to 3DP, and leading news and insights websites 3D Printing Media Network and Replicatore. I am also a Senior Analyst for leading US-based firm SmarTech Analysis focusing on the additive manufacturing industry and relative vertical markets.

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