3D Bioprinting: The Yellow Brick Road of (Part 1)

–Bioink

Three-dimensional (3D) bioprinting has exploded in popularity in the recent decade. No longer confined entirely to science fiction novels, headlines emerge frequently touting its near-unlimited potential. It is a field dreaming of providing us with an unlimited supply of beating hearts at the push of a button, revolutionizing human longevity. These grandiose claims make it especially difficult to tell when to apply the old adage “if it sounds too good to be true, it probably is”.

This leads to the title of this series. The aim is for the reader, be it a graduate student or simply one with a penchant for science fiction, to garner a sober view of the trajectory of this field and dispel some fantasies, taking us from the yellow brick road and placing us, along with our expectations, on the red brick road. To do this we must establish where the field has come from, where it is most likely going, and the reasons why certain barriers exist. In the process, we will break bioprinting into four major stages. These stages are defined by technological barriers, or rather the overcoming of them, that allows the field to progress to the next, much like moving from the printing press to the first-word processor. These stages are loosely inspired by the Kardashev scale which seeks to categorize a civilization based on energy consumption. Instead, we categorize a civilization based on its ability to 3D bioprint functional living tissue as follows:

Stage 1: Lacking the ability to print using cells and/or proteins with high fidelity.
Stage 2: Printing cells and/or proteins at high fidelity, but no significant tissue function upon print completion.
Stage 3: Combining an immature 3D printed tissue with a coordinated maturation system, resulting in a functional tissue for use or study.
Stage 4: Producing a tissue or organ that is functional immediately upon print completion.

The term “fidelity” in Stages 1 and 2 is used loosely as it is highly debatable. It refers to being able to 3D print at length scales critical for living tissue – from micrometer to centimeters, not the single-molecule or kilometer-scale – with minimal deformation. While researchers are also attempting to create tissues and organs for humans through processes such as decellularization and genetic modification of pigs, this series focuses on the ability and likelihood for these to be achieved through 3D bioprinting.

To understand 3D bioprinting and why it is currently transitioning from Stage 1 to 2, one should understand where it came from. Fortunately, the field is only about ten years old, making its history brief; however, so is its list of accomplishments. In the late 2000s, critical patents from the largest and oldest 3D printer companies like 3D Systems and Stratasys began to expire on printing methods like Fused Deposition Modeling (FDM), Digital Light Projection (DLP) and Stereolithography (SLA). The resulting wave of expiring patents produced an overnight surge in nascent hobbyist 3D printer companies like MakerBot and Ultimaker that brought an open-source printing movement to the forefront, popularizing thermoplastic 3D printing. Meanwhile, established key players such as Stratasys, Arcam, and General Electric, to name a few, continued to push the field of 3D printing forward toward the automobile and aerospace industries. It didn’t take long for biomedical researchers to try to adapt these new tools in their research with the potential benefits being obvious. The human body and its tissues have immensely intricate 3D architecture and along comes a technology capable of creating complex 3D shapes. Therefore, creating a functional, beating heart should be as simple as pressing ‘print’ in just five to ten years, some claimed. So where are the hearts?

To understand why 3D bioprinting is so difficult, one must understand the materials that make up the body. Largely speaking, one can divide the body into two types of building blocks – cells and extracellular matrix (ECM). When you break down the words, the extracellular matrix is just another way of saying all the stuff (matrix) that is outside of your cells (extracellular). A simple way to think about a piece of tissue is a brick wall in which the cells are the bricks and the ECM is the mortar holding it all together. The cells (bricks) push, pull, chew up, and spit out ECM (mortar) constantly. While the list of ECM proteins that make up the body is extremely long and the cells of the organs are as numerous as they are diverse, the crucial thing to note is that both these building blocks make structures that are extremely soft. While bone is on the same order of stiffness as aluminum or titanium, the rest of your organs are around one million times softer, with your brain tissue being the softest of them all-around ten times softer than fat. This makes building tissues with precision an engineering nightmare.

When it comes to 3D printing, there is a simple, obvious reason why plastics and metals are the go-to materials – they are strong enough to support their own weight during the printing process. This leads to the first major barrier in 3D bioprinting. The materials that we would like to print with, are not the materials that we can print with; they are simply too soft. To make biological inks, or “bioinks”, more rigid, a litany of additives such as alginate, gelatin, and agarose are used to thicken and mechanically stabilize native ECM proteins (if they’re even used at all) to support their own weight. Despite these compromises, these bioinks still deform under their own weight when printing onto a build platform (Fig. 1 A and B). To make materials solidify even faster and harder, many researchers chemically modify their inks via a process called methacrylation. Methacrylation simply means the ink has been modified to be sensitive to UV light so that it solidifies quickly upon exposure. Such is the demand for rigid, stable building materials that researchers are willing to subject living cells to UV light designed to sterilize surfaces. Light-sensitive inks (like gelatin methacrylate (GelMA) and Poly(ethylene glycol) diacrylate (PEGDA)) are mandatory when trying to print with light-based techniques, but even FDM printers add in UV-sensitive inks to further increase the ink’s rigidity. The result is a field caught in a zeitgeist of trying to make bioinks rigid enough to support their own weight to be compatible with the 3D printing techniques handed to it.

Even after these modifications, bioinks are still only loosely capable of supporting their weight, meaning they sag and deform far more than their plastic and metal counterparts. As a result, most bioinks cannot be printed more than a few centimeters tall as print deformation becomes too severe. To mask the limitations inherent to the inks, researchers bioprint geometries that are surreptitiously safe. The most popular are structures that have vertical walls with limited or gently overhanging structures, lest they collapse from their instability. Hence the popularity of printing vertical logpile structures (Fig. 1C and D)¹, “ears” and “noses” – they are inherently more structurally stable geometries. However, to imply we are truly bioprinting ears and noses is still an exaggeration. Just because the overall shape of an object is in the form of an ear, for example, does not make it equivalent to an actual ear. To start, the tissues in the body are fed by a complex system of blood vessels branching from the aorta to the capillaries less than a fraction of the width of a hair, just as a tree branches from a single trunk. The bioprinting techniques widely being used today do not have the resolution capable of printing these fine capillaries using ECM or even the still-unstable bioinks, meaning the cells inside the prints slowly die and that the noses and ears being printed today have more in common with a jello mold than it does an actual ear.

The difficulty of bioprinting is multifactorial to say the least. Despite material choice being a major component, there are others to be discussed later that loom over the field regardless. Whereas plastic printers deal with stable, predictable polymers, bioprinting requires one to build with a soft, dynamic, living material. No other construction material needs food and dies if it strays too far from body temperature in a matter of hours. Despite the relative stagnation of the field, news headlines still emerge touting the printing of a heart, or similar organ, furthering the chasm between public expectation and scientific reality. Nonetheless, new technologies are emerging that are beginning to address limits to FDM or light-based techniques. These new methods seek to elevate us from Stage 1 to Stage 2 by addressing resolution and material choice limitations to allow us to start building the blood vessels and capillaries upon which all living tissues are dependent.

In the next article in this series, we will take a deeper dive into technologies that have allowed us to take that leap towards Stage 2, where we believe the field of bioprinting currently resides. At the same time, we hope readers will develop an understanding for the obstacles that currently pushes back on our ability to proceed to Stage 3 where bioprinting functional tissue models is the norm.

References:

1. Jia, W. et al. Direct 3D bioprinting of perfusable vascular constructs using a blend bioink. Biomaterials 106, 58–68 (2016).

About the Author:

Andrew Hudson

Chief Operations Officer, Fluidform

Andrew is a co-founder of FluidForm and leads the development, manufacturing and scale-up efforts of LifeSupport™. His research focuses on developing the next generation of techniques for vascularizing 3D-bioprinted tissues to improve the clinical translational potential of tissue engineered therapies.

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