–Pulling Back the Curtain

All of the preceding articles in this series have concentrated on current challenges to 3D bioprinting as well as ones on the horizon that are now coming into focus. By identifying key barriers to the field, we gated the technology into four major stages which are:

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

As we learn to print soft extracellular matrix (ECM) proteins with higher precision (Stage 1 to 2), we will begin to print structures that better recapitulate blood vessels that aid in keeping tissues alive with the assistance of more complicated bioreactors to mature them for implantation (Stage 2 to 3). Stage 4, however, is the furthest level of advancement possible to the point of being science fiction. Much of the difficulty in Stage 3 lies in combining siloed areas of research together into a coherent pipeline. To use a historical example, successfully launching a rocket led to controlling its trajectory which led to returning it to earth intact. These gated steps were each realistic challenges and had all been done in separate lower risk scenarios, but the successful lunar landing in 1969 represented a monumental achievement. Stage 4, however, represents something of jumping to light speed to continue using space exploration as an analogy. In Part 1 of this series, we discussed lifting the veil off the headlines surrounding the field to gain a sober view of its future. While the first three parts of this series focused mainly on technical challenges, this final piece is meant to follow through on dispelling some of the more fantastical claims on the potential future of bioprinting a full organ as frequently seen in headlines (Fig. 1).

Simply put, the challenges of bioprinting fall into two major categories:

• Addressable: a discrete challenge that is not currently solved, but a path to a solution can be hypothesized.
• Fundamental: a core assumption upholding the field that has yet to be answered.

Addressable problems: Referring to space exploration as an archetypal example, an addressable challenge is creating a zero-waste environment where all resources like water and food are replenishable with the aid of solar energy. This challenge is not trivial, but already being tackled in the International Space Station. Some of the large, but addressable, challenges to printing an organ are listed below.

• Patient-specific cell culture: Using patient-derived cells reduces a large risk factor of the immune response, but the challenge here is analogous to the clothing industry. Off-the-rack clothes are affordable but come in limited sizes while bespoke garments fit perfectly at a high labor cost. The difficulty in culturing numerous patient-derived cells is the labor and logistical tracking required. Although one cell type might make up a substantial portion of an organ (cardiomyocytes in the heart), that does not mean the minority components (pacemaker, endothelial, vascular smooth muscle cells, etc.) can be done away with. Obtaining and culturing all these various cell lines in separate media prior to printing will be a nightmare in coordination.
• High–throughput cell culture: A major bottleneck to tissue engineering research is simply the rate at which human cells can be cultured. Expanding culture by increasing surface area scales only as a squared function, while volume scales cubically. The current practice of culturing in more 2D flasks will eventually reach a breaking point of inefficiency when billions of cells are needed per round of an experiment. Fortunately, there are several areas of research trying to grow cells efficiently in 3D such as carrier beads and tissue scaffolds.
• Multi–material printing: If each cell type can be made into its own bioink, then printing with a litany of inks accurately becomes crucial. The difficulty here can become that print time increases drastically with each new bioink added. The good news is that this challenge is mechanical (for syringe-based printers), and more of a matter of implementation than creating from scratch. Many machine systems can move with extreme precision in 3D, they have just yet to be combined on a bioprinter system.
• Regulations: Without getting drawn into a discussion about what size of a role regulatory agencies should play in medicine, it is important to acknowledge that it will be a significant gatekeeper. The time and money required to administer animal and human trials in pharmaceuticals take roughly a decade and a billion dollars for one drug. To attempt to shift this over to a bespoke, patient-specific, cell-based medical device where the recipe changes for literally every patient is a quality control challenge for both manufacturers and regulators.

Fundamental problems: Reverting back to space exploration, a fundamental problem with travel between the stars lies in achieving speeds even 10% of the speed of light. For reference, Voyager (the farthest man-made object from earth) is travelling at 1/18,000^th the speed of light. In the same sense, there are many far-off, fundamental challenges to bioprinting an organ that have yet to be answered, of which some are listed below.

• Assuming function will follow form: A core assumption in the field of tissue engineering is that if we use a native tissue as a cheat sheet and organize cells in the same general manner, we will develop an equivalent tissue. A mature tissue is a result of an immensely complex system of developmental biology where form and function complement one another from the microscopic scale upward. Simply placing cells in the same general spatial arrangement and thinking it equivalent to an organ could turn out to be analogous to throwing lumber, tubing, insulation, and wiring together in the shape of a building and thinking it will work just like the neighbor’s house. Current engineered tissues are fortunate if they produce even 10% of native tissue function. The belief that a printed organ will function equivalent to a native organ immediately after being printed assumes a lot about the value (or lack thereof) in allowing tissues to mature over long periods of time.
• Creating a tumor instead of a tissue: Tissue engineering is heavily focused on keeping large numbers of cells alive by trying to vascularize 3D tissues. There is a likelihood that placing such an emphasis on vascularizing a tissue that we will then realize what we have created is more akin to a tumor than healthy tissue. Aside from the microvascular organizational differences serving as a potential canary in the coalmine, the next question becomes, “When this engineered tissue is implanted, will it display contact inhibition?”. Contact inhibition is a regulatory phenomenon in healthy cells in which cells slow their proliferation rate when they begin to contact one another. Tumor cells, on the other hand, continue to grow unregulated, resulting in tumor progression and metastasis. A potential risk to tissue engineering is focusing on maximizing cell growth and angiogenesis to speed up tissue maturation while increasing the risk of unintentionally building tumor-like tissues that grow out of control when implanted.
• Developing better alternatives: No technology exists solely on its own. Alternatives can spring up nearly overnight and decimate established players similar to how smartphones have largely replaced personal digital cameras. While 3D bioprinting is being researched, so are other technologies that could intentionally or unintentionally threaten it. These disruptions can come in the form of either mechanically or bioengineered alternatives. Potential mechanical alternatives could be ventricular assist devices, dialysis machines, and insulin pumps that continue to improve over their current versions as to potentially lower the need for engineered hearts, kidneys, and pancreases, respectively. In terms of bioengineered solutions, the genetic modification of pigs to remove the threat of donor rejection in humans could potentially solve many organ shortage problems by effectively relying on the pig as the bioprinter and incubator. While none of these solutions are guaranteed, the potential still exists. Even if one or several solutions end up a reality, it will come at the cost of bioprinting, but it is undoubtedly an immense gain for society.

The combination of both addressable and fundamental problems runs in opposition to the frequent claim that artificial organs are “5 to 10 years away” just as they allegedly were 5 to 10 years ago. Even if funding to the field continues to increase, I am reminded of the phrase “9 women cannot produce a child in 1 month”, as money is not the only barrier to technological innovation. The persistent stiff-arming of expectations runs the risk of a funding ‘winter’ for bioprinting as has happened in other fields. Notable funding winters occurred in artificial intelligence (AI) which went through two in the 1970s and 1990s as promises about AI potential did not match with the computational power required at the time (Fig. 2).

There is a concerning parallel between the claims touted in media headlines and the current ability to 3D bioprint organs just as AI researchers in the 1970s claimed a Jetsons-style future was only years away. Although 3D bioprinting is a new and endlessly exciting field, I ask that researchers demonstrate the courage to be realistic, lest we risk trapping our expectations in the Land of Oz, where bioprinted “organs” are forever and always 5 years away.

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.

Related Articles:

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

The Yellow Brick Road of 3D Bioprinting (Part 2): Soft Is Hard

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

3D Bioprinting Substrate Stiffness – Often Overlooked, But Always at Work

3D Bioprinting in Space?

Printing the Future: An Introduction to Additive Manufacturing in Space

3D Bioprinting: Chiasm of Art, Design, Science, Technology, Evolution

The post 3D Bioprinting: The Yellow Brick Road (Part 4) appeared first on 3DHeals.

–Tissue Maturation

The previous articles in this series highlighted the current challenges to 3D bioprinting tissues and organs. The foremost challenge is that the basic building blocks of living tissues are soft and therefore difficult to manufacture precisely. This results in resolution limits that prevent us from bioprinting the capillaries that keep cells alive and tissues function in the body. By identifying key barriers to the field, we gated 3D bioprinting into four major stages which are:

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

Since we said previously that we are transitioning from Stage 1 to 2, talking about Stages 3 and 4 become hypothetical and philosophical at various points. Stage 3 is unique in that it is just beyond the grasp of current research capabilities, however, various elements of Stage 3 have been accomplished independently. Transitioning to Stage 3 is then a matter of concatenating separate areas of complex research together. The barriers to Stage 3 are not defined by the bioprinted tissues themselves, but bioreactors.

A bioreactor is currently defined as, “an apparatus in which a biological reaction or process is carried out, especially on an industrial scale”. This current definition is centered around producing insulin or penicillin in industrial fermentation tanks. Future bioreactors, like ones discussed here, will be far more complicated than bulk tanks. They will conduct a symphony of fluid flow, proteins, and nutrients through the tissue to act as a synthetic womb. Although our bioprinting resolution is not high enough to directly print all tissue microstructure (refer to Part 2 of the series), it is a shortsighted assumption that we must assemble every aspect of the tissue ourselves. Powerful stem cells are genetically programmed to eventually assemble into our major organs. Thanks to developmental biology everyone began life as a single cell which eventually differentiated into all the various cell types in the body with an astonishing success rate. This unmatched success means the womb is the most effective bioreactor in nature, making it the gold standard for engineered bioreactors to be compared to. With this appreciation in mind, the question then becomes, “How can we utilize the power of cell-based developmental biology to fill in the detail that is currently beyond our capability to 3D bioprint?”. To leverage biology, bioreactors must be designed to balance cellular self-assembly with an engineering control. Leaning heavily towards the former increases the risk of unintentional and uncontrolled tumor development while the latter could stymie efficient maturation. Put simply, the full biological complexity of gestation is beyond our level of comprehension, much less our ability to replicate it artificially. This means bioreactors can only be a simplified version of the body, with the new question being, “What must a bioreactor do?”.

The first answer is obvious – keep cells alive. Until now, most engineered tissues have been very small and thin to allow cells to acquire nutrients through diffusion without the need for dedicated blood vessels. The first challenge to printing large (>1cm³) tissues is delivering nutrients to the cells in the center of the construct to prevent the formation of a necrotic core. The second role of a bioreactor system is to promote the function of the specific tissue. It is not simply enough for liver tissue to contain viable liver cells. Liver tissue that cannot produce bile does not have much therapeutic potential. It is creating a tissue that produces a higher level of function close to what is seen in the body that is an even greater challenge than keeping the cells alive. Higher-level tissue function is a concerted effort between a plethora of cell types and often organ systems. Even today, the best dialysis machine is no match for a kidney which hopefully leads you to appreciate its function from an engineering standpoint as well as the fortune that yours (probably) works. That being said, a bioreactor does not have to do everything. The most appropriate role of bioreactors is to act as a bridge between immature tissues post-print (Fig. 1A) and a minimum viable tissue (Fig. 1B) suitable for implantation where it will finish maturing (Fig. 1C).

There are at least two critical features that must develop to produce this minimum viable tissue: (i) a nutrient delivery network and (ii) the minimization of the immune response. It is important to note that the minimum functional requirements for tissues are organ-specific such as burst pressure for valves, a contractile force for muscle, and protein secretome for GI organs. To transplant larger tissues a blood inlet and outlet system should be present as constructs cannot rely on bulk passive diffusion like 2D cell culture can. This interaction with blood, therefore, means interaction with the immune system, creating immediate and long-term threats. The immediate threat to the engineered tissue is clotting on foreign surfaces. The endothelial cells that line the inside of blood vessels can be thought of as a Teflon barrier that prevents blood platelets and proteins from attaching to healthy blood vessels. A crucial role in bioreactor maturation is the production of a coherent endothelium that acts as a barrier to the body’s natural clotting cascade. Much exciting work has been done on smaller scale models to endothelialize tissue-engineered constructs 1,2, as the path to implantation is predicated on the presence of endothelium. The long-term immunological threat is rejection whereby the body recognizes the cells or materials in the tissue as foreign and either wall off or attacks the implanted tissue. To avoid this potential threat, it is preferable to use the patient’s own cells, however obtaining, growing, differentiating, and printing numerous patient-specific cell lines adds an additional layer of technical and logistical complexity. It is not very feasible to expect a bioreactor to be as coordinated as the womb. When it comes to a bioreactor, the only resources available to developing the tissue are what is put into it. There is no full immune, endocrine, or any organ system already present to help mature the tissue. Instead, we provide exogenous chemical cocktails to act as nascent in vitro substitutes for these systems such as antibiotics and growth factors to stand in for the immune and endocrine systems, respectively.

Bioreactor development is a burgeoning field that has already produced very promising results that hint at the potential to eventually transplant bioreactor-matured tissues. Notably, Syedain et al. placed a fibrinogen gel of Ovine and human dermal fibroblasts in a tubular mold inside a bioreactor³. After pulsing the molds for five weeks, the cells produced a cylindrical tube of the extracellular matrix that could then be sewn onto a traditional heart valve frame. The engineered heart valves were successfully transplanted into sheep for the entire six months of the study with impressive function and minimal immune response. Other studies have also shown the ability for bioprinted vessel networks to maintain cell viability for weeks, however, these tissues have yet to be transplanted into a subject after bioreactor maturation 4,5. The current tissue development pipeline relies on passing the baton of a nascent bioprinted tissue to a bioreactor system. Improving the quality and resolution of a bioprinted tissue will ease the workload on the bioreactor system, meaning bioprinting research will remain a critical endeavor in the future. Although implanting a bioreactor-matured functional tissue that was also bioprinted has yet to be demonstrated, both projects have been published separately. Joining the two halves together remains sizable, but a surmountable task. The first demonstration of a functional tissue production pipeline would provide substantial de-risking of tissue-engineered therapies to large outside investment, making it likely that bioreactor technology will become a linchpin in ushering in the next generation of regenerative medicine.

References:

1.        Miller, J. S. et al. Rapid casting of patterned vascular networks for perfusable engineered three-dimensional tissues. Nat. Mater. 11, 768–774 (2012).

2.        Nguyen, D. H. T. et al. Biomimetic model to reconstitute angiogenic sprouting morphogenesis in vitro. Proc. Natl. Acad. Sci. U. S. A. 110, 6712–6717 (2013).

3.        Syedain, Z. et al. 6-Month Aortic Valve Implantation of an Off-the-Shelf Tissue- engineered Valve in Sheep. 612–626 (2017) doi:10.1016/j.biomaterials.2015.09.016.6-Month.

4.        Kolesky, D. B., Homan, K. A., Skylar-Scott, M. A. & Lewis, J. A. Three-dimensional bioprinting of thick vascularized tissues. Proc. Natl. Acad. Sci. U. S. A. 113, 3179–3184 (2016).

5.        Lee, A. et al. 3D bioprinting of collagen to rebuild components of the human heart. Science (80-. ). 365, 482–487 (2019).

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.

Related Articles:

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

The Yellow Brick Road of 3D Bioprinting (Part 2): Soft Is Hard

3D Bioprinting Substrate Stiffness – Often Overlooked, But Always at Work

3D Bioprinting in Space?

Printing the Future: An Introduction to Additive Manufacturing in Space

The post 3D Bioprinting: The Yellow Brick Road (Part 3) appeared first on 3DHeals.

–Soft Is Hard

In the first part of this article series it was posited that although 3D bioprinting is approximately ten years old, it is already an area of major scientific hype despite its translation to the clinic remaining highly limited. The reason for this lack of translation was suggested to be technological barriers that must first be overcome before 3D bioprinting can begin executing on its lofty claims. By defining these barriers, we gated 3D bioprinting into four major stages, where overcoming one stage’s barrier allows the field to proceed to the next. To reiterate, the four stages are:

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

Part 1 of this series focused on Stage 1 bioprinting, where the field lacks the ability to precisely print the biomaterials we truly wish to use. To briefly summarize, these biomaterials, unlike the plastics and metals used in 3D printing, are too soft to physically support their weight in the air, resulting in significant print deformation and a loss of fidelity. To minimize this print deformation, researchers compromise on bioink formulations with a litany of additives to make materials more rigid at the cost of biological relevance, such as exposing methacrylate inks to UV light. This exposure might not cause the immediate decimation of all cells, but the risk of long-term DNA damage is now elevated while also producing covalent bonds that make cell-based matrix remodeling more difficult. Fortunately, recent advancements have made significant headway to overcoming these barriers and have resulted in a significant increase in bioprinting resolution and have initiated the transition from Stage 1 to Stage 2.

The two major methods of 3D bioprinting are extrusion-based and light-based printing, with each technique possessing unique advantages and disadvantages. It is possible to argue that some techniques are hybrids that first extrude a UV light-sensitive ink, but these techniques do not provide the astounding resolution possible with pure light-based techniques. Instead, they struggle with each technique’s disadvantage by suffering from the lower resolution of extrusion-based printing as well as the chemical limitations of light-based printing. Of the recent advancements, the most potentially impactful in extrusion-based and light-based bioprinting are Freeform Reversible Embedding of Suspended Hydrogels (FRESH)1,2, and Stereolithography Apparatus for Tissue Engineering (SLATE)³, respectively.

FRESH is an extrusion-based printing technique in which bioinks are extruded into a support bath instead of in open air(1,2). This support bath cushions the fluid ink as it is injected inside the bath by the printer, preventing it from collapsing (Fig. 1A). The bath has an important property in that it has yield stress – at rest, it behaves like a solid, but when enough force is exerted upon it, it starts to flow like a fluid. Common household products like mayonnaise, ketchup, and hair gel all have yield stresses. They do not flow unless you shake or squeeze their containers, overcoming their yield stress in the process. This yield stress means the FRESH support bath flows out of the way of the needle and ink that is being injected but returns to behaving like a solid shortly thereafter. This recovery prevents the ink from deforming under its own weight as is common in open-air printing techniques. Not only does the bath physically support the ink, but it also allows for chemical interactions between the bath and the ink. For example, fibrin is a key protein that comprises a major component of blood clots. For fibrin to be formed, its predecessor fibrinogen must be cleaved enzymatically by thrombin as part of the clotting cascade. Instead of mixing fibrinogen and thrombin together at once, fibrinogen can instead be injected into a bath containing trace amounts of thrombin to allow it to be 3D printed without deformation. The bath is made from gelatin which melts at body temperature, meaning upon print completion, the container is warmed, causing the support to melt away, gently releasing the print. This means that FRESH expands the list of chemistries that can be 3D printed. Inks that are sensitive to pH (collagens), ions (alginates), or enzymes (fibrinogen) can be printed into baths containing their respective pH buffer, ion, or enzyme that cause them to gel. By printing acidic collagen into a pH neutral bath, the pH-sensitive collagen gels, allowing researchers to bioprint early-stage heart valves, blood vessel networks, and more (2). Moreover, highly cell-dense constructs can be printed with FRESH whether the cells are in a compact ink (2) or bath (4) phase on the order of hundreds of millions of cells per milliliter.

SLATE is a light-based printing technique in which yellow food coloring (tartrazine) acts as a  photo absorber while Lithium phenyl-2,4,6-trimethyl-benzoyl phosphinate (LAP) acts as a photoinitiator for a photo-sensitive polymer such as (poly(ethylene glycol) diacrylate [PEGDA])(3) (Fig. 1B). Long names aside, a critical aspect of light-based printing is that one needs a chemical that will start a polymer polymerizing (a photoinitiator) when exposed to UV light while also making sure that only the thickness you want to print solidifies, hence the use of a photo absorber to prevent stray light from scattering into the unpolymerized resin. Previous techniques used photo absorbers and photoinitiators that were more cytotoxic or had other compromises such as printing slower or less precisely. The optimizations in SLATE allow for quick printing at high resolution with less toxicity. With these improvements, researchers printed a model of a lung alveolus in which an air sac was surrounded by a complex 3D channel network to mimic the way capillaries surround alveoli to facilitate gas exchange.

This is not to say that either FRESH or SLATE is perfect. As economist Thomas Sowell put it, “There are no solutions, only trade-offs.”. The pros and cons to FRESH and SLATE are listed (Fig. 1 C and D). It is important to note that much of the pros and cons of these bioprinting techniques are now the same as the pros and cons of their regular plastic 3D printing counterpart techniques (FDM and SLA). This indicates that biological researchers have discovered the modifications they needed to make to traditional FDM and SLA techniques to translate 3D printing to 3D bioprinting.

Although bioprinting cells and proteins at high fidelity are starting to become possible, there is still a large disparity at the microscopic structural level between bioprinted and native tissues. Figure 2 seeks to compare notable bioprinted analogs of tissues to the actual microstructure of native tissue. The most crucial detail in this figure is to pay close attention to the scale bars when juxtaposing the native tissue and bioprinted microstructure. The level of microstructural detail of bioprinted tissues still falls well short of matching the microarchitecture of native tissue. The glomerulus of a kidney is still far more complex than a channel with kidney cells5 (Fig. 2 A to D). A dense capillary network wrapped around alveolar sacs is still orders of magnitude smaller than its SLATE-printed counterpart (Fig. 2 E to H). Cardiac muscle tissue contains muscle fibers, capillaries, and electrically conductive Purkinje fibers all organized in a dense fashion and it is still far smaller than what is printed with FRESH (Fig. 2 I to L). This lack of true microstructure is now the large barrier that prevents functional tissue from being 3D bioprinted. Now that we can 3D bioprint with high fidelity, the next challenge becomes printing functional tissue, which in turn means replicating tissue-specific microarchitecture. To expect 3D bioprinting to produce a functional, mature tissue immediately upon print completion is a rather unrealistic expectation. Every human is given 9 months of gestation to develop these organs that continue to mature for years after birth. Why should we expect engineered tissue to outperform developmental biology billions of years in the making? The next most reasonable step is to, therefore, combine an immature bioprinted tissue with a maturation system that seeks to encourage cells to assemble into the microarchitecture seen in native tissues, promoting function. Although a vast chasm of research must be crossed to get to Stage 3, that is where the translational potential explodes, and where 3D bioprinting must start to follow through on its promises.

References:

1.        Hinton, T. J. et al. Three-dimensional printing of complex biological structures by freeform reversible embedding of suspended hydrogels. Sci. Adv. 1, (2015).

2.        Lee, A. et al. 3D bioprinting of collagen to rebuild components of the human heart. Science (80-. ). 365, 482–487 (2019).

3.        Grigoryan, B. et al. Multivascular networks and functional intravascular topologies within biocompatible hydrogels. Science (80-. ). 364, 458–464 (2019).

4.        Skylar-Scott, M. A. et al. Biomanufacturing of organ-specific tissues with high cellular density and embedded vascular channels. Sci. Adv. 5, (2019).

5.        Homan, K. A. et al. Bioprinting of 3D Convoluted Renal Proximal Tubules on Perfusable Chips. Sci. Rep. 6, 1–13 (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.

Related Articles:

The Yellow Brick Road of 3D Bioprinting

3D Bioprinting Substrate Stiffness – Often Overlooked, But Always at Work

3D Bioprinting in Space?

Printing the Future: An Introduction to Additive Manufacturing in Space

The post 3D Bioprinting: Yellow Brick Road (Part 2) appeared first on 3DHeals.

–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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