Saarang Panchavati et al

5th Period Capstone

18 November 2016

Computational and Experimental Bioprinting of Efficient 3D Vascular Networks

Introduction
The advent of 3D printing has inspired engineers, doctors, and artists alike to take full
advantage of its ability to rapidly create objects out of inexpensive materials. Its versatility
offers new and exciting prospects - especially in the field of Bioengineering (Kinstlinger et al).
3D bioprinting is increasingly becoming an avenue for the development of tissue and organ
printing(Murphy et al). Imagining a world where organ donors are replaced by inexpensive, rapid
3D printers is no longer an implausible idea. Additive bioprinting functions by depositing
materials in successive layers with precise instructions from a computer, the pattern of each layer
determined by successive masks of the model to be printed. However, the development of organ
printing has been hindered by an inability to 3D print biologically viable vasculature in large,
physiologically relevant synthetic tissues(Miller). While it is feasible to successfully 3D print
biological materials containing cells, without sufficient vasculature, cells towards the center of
the gel which are not exposed to oxygen or media will undergo cell death due to insufficient
nutrient transport(Miller et al).
Recent developments at Miller Lab in Rice University, however, are making this a
reality. Through various 3D printing methods, including Selective Laser Sintering(SLS), and
stereolithography, Miller Lab has successfully printed various vascular architectures that may
prove to be successful in maintaining the viability of cells inside centimeter-sized synthetic

tissues. While complete biomimicry is still a distant goal, several different types of vasculature
models based on various mathematical, and functional figures have been successfully explored.
While each of the various models has its own benefits, none fit all the criteria that was
imperative for an effective vascular network - one that allows maximum space for cells, with the
greatest zone of viability, and with the best fluidic and interstitial flow - that would allow cells
to grow.
We can fabricate many dozens of models, but we have little knowledge on what we
should focus our efforts on. Thus, I created an open source algorithm based on the natural
mechanism of angiogenesis that would provide a functional method to visualize, and fabricate
3D vasculature that would allow the modification of certain parameters to work towards an ideal
vasculature. Furthermore, I aimed to identify the best vasculature by developing a metric that
could narrow researchers search for the most viable vascular network. I believe that such a
metric would go far in helping narrow the focus on vascular networks to a select few, as well as
in developing the most adequate network that will ensure high cell viability, with the maximum
space for cells and minimal fluidic resistance - allowing cells to grow, mature and develop with
all their necessary needs - a step closer to a future of 3D bioprinting.
Why Bioprinting?
3D bioprinting applications can revolutionize organ transplants and patient treatments. In
fact, researchers are already beginning to delve into this by using 3D printed material to work
towards a functional replacement for nervous tissue. [Nervous tissue donations] have significant
limitations...The combination of these limitations often prevents full functional recovery from a
segmental nerve injury. As such, synthetic materials provide an attractive alternative therapy.
Synthetic scaffolds can be engineered to possess the desired dimensions, mechanical properties,

and degradation profiles (Runge). Thus we can take full advantage of 3D printing to fabricate
necessary tissues for nervous function and effectively customize and print unique architectures
per patient necessities. Furthermore, we can start to envision a future where we can 3D print
scaffolds of stem cells, which can then be frozen and removed and allowed differentiate into
whatever organ the patient requires. This will allow us effective and functional translation from
organ donations to organ printing.
The Vascular Problem
Imagine a crowd of people, perhaps at a concert, all packed tightly together. People on
the outside perimeter of the crowd receive the most air and space, and are generally more
comfortable than the people that are within the center who may feel unnaturally hot,
claustrophobic and uncomfortable. In a similar vein, 3D printed tissues with cells feel the same
way, often with the cells in the middle dying. Due to this, 3D printed tissues with cells require
some method to ensure that all cells in the bulk of the gel are able to survive. Thus, in addition to
the presence of functional vasculature, 3D printed gels must also have structural integrity and the
ability to allow for diffusion of nutrients and oxygen. Diffusion in any given gel with vasculature
can be simply tested by using the field of microfluidics(Huang). By running something as simple
as a mixture of water and food coloring through a gel, we can observe how the food coloring
diffuses out into the bulk of the gel and thus visually ascertain how many cells will receive
nutrients and thus survive.
An ideal vascular network has certain qualities that make it the most suitable for cell
viability. Such a network is characterized by 3 main qualities. The ideal vascular network takes
up a minimal amount of space in a gel, leaving the most space for cells. Such a network also has
the maximum fluidic flow and allows for the most complete diffusion across the bulk of the gel

such that all cells get intended nutrients. And while complete biomimicry is still a distant goal,
several different types of vasculature models based on various mathematical, and functional
figures have been successfully explored. While each of the various models has its own benefits,
none fit all the criteria that was imperative for an effective vascular network one that allows
maximum space for cells, with the greatest zone of viability, and with the best fluidic and
interstitial flow that would allow cells to grow.
Intussusception Algorithm
We can fabricate many dozens of models, but we have little knowledge on what we should focus
our efforts on. Thus, I created an open source algorithm based on a natural mechanism of
angiogenesis that would provide a functional method to visualize, and fabricate 3D vasculature
that would allow the modification of certain parameters to work towards an ideal vasculature.
Intussusception is the natural process by which one blood vessel splits into two and provides the
perfect biological template by which we can develop branching structures. I developed code that
could generate individual aspects of such vasculature with high precision and customizability.
This code was then written using python and implemented as a free add-on to a free, open-source
3D modeling software(Blender) that could be used to visualize complex 3D architectures. I
create fractal vasculature by starting with a single tube and sequentially, dichotomously dividing
all available tubes with each fractal generation. This provides the generational quality that was
imperative for our model. In addition, the regularity of the branching as well as its easily
mathematically definable parameters of the process allow it to be easily algorithmically defined.

Figure 1 demonstrates how the algorithm functions.

Fig 1: 3 step process by which the algorithm defines a model. 1) Creating vertices of branching points. 2)
Connecting the vertices 3) Skinning and curving the edges

Vascular Resistance
Measuring fluidic resistance in vascular networks is nontrivial and requires the use of
expensive modeling software. Instead, we can use electrical resistance as an analog for fluidic
resistance. By assuming that vasculature is similar in structure to a circuit in parallel, we can
easily measure electrical resistance and relate it to fluidic resistance. This was accomplished
through the use of conductive ink purchased from Bare Conductive. Per Bare Conductives
suggestion, a 1:4 dilution of ink to water was created and perfused through the vasculature. The
resistance was then measured by putting the leads of a multimeter in the inlet and outlet of the
model. Because the channel diameters were so small (800 m) it was necessary to attach
minimal resistance wires to the ends of the leads to ensure accurate readings. Intussusception

models were perfect for measuring resistance because of its uniformity and its patent similarity
to a parallel circuit. We can easily develop a theoretical equation for all generations of
Intussusception which is useful because we can compare both theoretical and actual resistance
measurements, creating a more accurate and precise metric for efficiency.
Using resistance values for each generation model along with their respective surface
area to volume ratios, we can easily calculate the Vascular Efficiency of each generation of
Intussusception. The efficiency was found by dividing the ratio between the volume of the
vasculature to the volume of the gel by the architectures respective resistance.
Vascular Efficiency Metric
So far in the field of bioprinting, we are fully aware of what we can print. However, we
are not certain of what we should print. This development of a functional vascular efficiency
metric will help define the direction that we go in choosing what types of architectures to print
and what architectures will be most beneficial for cells in 3D printed tissues. With the addition of
an electricity aspect, the work presented here can allow 3D bioprinting to be defined in new,
exciting ways. A vascular efficiency metric is essential to identify the most beneficial vascular
networks. Such a metric could distinguish between complex, interpenetrating architectures
shown in Fig. 2, and simpler branching networks rendered in Fig 3.

Fig 2. - Hilbert Curve

Fig.3 - Intussusception

At first glance, these two architectures are extraordinarily different, and both of them interact in
vastly different ways with the gel and the cells that surround them. The use of a metric could

help determine which network would most benefit the cells around them. Which network allows
for the most amount of cells? Which metric allows the greatest amount of cells to stay alive? The
metric that I developed is based on the idea that fluidic resistance can be used as an analog for
electrical resistance. This means that we can view fluidic resistance through the lens of electricity
- far easier to measure, and with relatively basic technology. By perfusing the vascular networks
with electrically conductive ink I can easily measure the resistance values across a given
network. So for Fig. 1, imagine that all the red lines are resistors in parallel, and I touch the leads
of a multimeter to the inlet and outlet of the vascular architecture. The method to measure
and discover this metric is fairly trivial, and thus can be easily and inexpensively be implemented
by researchers. The metric can be formally defined by the following equation:

where V is the volume and is resistance.

Data collection and studies using this metric has demonstrated the necessity for a balance
between the volume and resistance. For my work specifically, I used an architecture based on the
biological phenomenon of angiogenesis - Intussusception(Fig 1.) - a perfect mimic to resistors in
parallel (Fig. 4).

Fig. 3 - Intussusception and resistors in parallel

This architecture is generational, meaning an increase in generations will lead to an increase in

branches and thus a decrease in resistance (resistance is inversely proportional to the number of

resistors in parallel); demonstrating that a higher generational model is favorable. However, upon
closer inspection, an increase in generations will also lead to an increase in the ratio between
vascular and gel volume - an unfavorable aspect. It is apparent that this metric shows that there
needs to be a balance between number of branches and how big or small a network is in a gel
-implying some sort of threshold of generations, after which the efficiency will regress. Solutions
to this vascular limit are the continuing focus of my research.
Solutions and Future Applications
As I search for different types of vascular networks that could meet my definition of
efficiency, I can take inspiration from the 200 year old problem concerning spacefilling and
space optimization was first proposed by Lord Kelvin in 1887. One of the solutions to this
problem is the Weaire - Phelan model which I aimed to imitate in a design for a vascular

network. The model looks like this :

If you can imagine the edges as the

network of vasculature, then you will get a general idea for how this would look. The Weaire Phelan model, its obvious that fulfills the volume ratio requirement, however, getting liquids to
flow through this complex vasculature is sure to be challenging, the angles of the connecting
edges are sure to be non-conducive to fluidic flow and thus will result in high resistance, low
flow rates and even lower efficiency. I have worked to incorporate this into the algorithm I
created (Intussusception), where it will automatically change to become more spacefilling and

thus have a lower vascular to gel volume ratio. The following image is a spacefilling model

from my algorithm.

The reason that the branches are spread apart

is because of this spacefilling modification - making it more efficient as it allows for more space
for cells and more efficient diffusion(diffusion from one given branch does not intersect with the
diffusion for another given branch.) You can see that it has a low surface area to volume ratio,
and a high diffusion coverage. But is it the best possible model?
The use of a metric to choose adequate vascular networks has huge implications and
future applications of bioprinting with an adequate vascular network are vast and can lead to
massive developments and innovations in the field of medicine. For example, efficient networks
can be used to begin bioprinting on a chip(Homan et al). Bioprinting on a chip allows for
functional bioprinting; a chip can be used as both a vascular attachment and a heart monitor. A
chip can also be used to monitor growth and administer medication as needed, all within the
body and completely biologically sound(Rizzo et al). The better a network functions, the more
possibilities lie with In vitro and in vivo bioprinting wherein structures can be made to grow
along with an organism itself(Syedain et al). Imagine a baby born with microcephaly - functional
brain and skin tissue can be grafted to the babys head and made to grow with the baby as it ages
and matures. Finally, the fact that this metric employs electricity points to a further applications
of conductive ink studies(Shin et al) and uses of a combination of biotechnology and electrical

engineering, perhaps even going as far to make bio-batteries. Not necessarily in the matrix
sense, but that we can grow and construct complex batteries through this combination of
conductivity, simple cell types, and 3D printing.
Conclusion
My capstone work delved into the characterization of 3-dimensional branching networks.
Through exploring these 3D architectures, I can more closely examine their effects on cells that
are growing throughout the bulk of the gel, as well as begin to examine 3-dimensional
architectures for circuits. Furthermore, the exploration of 3-dimensional architectures will allow
to branch out into even more complicated models that can be used to further improve the
viability of cells in functional 3D printed tissues. Additionally, we can begin to explore
interpenetrating networks such as the Hilbert Curve, and the Torus Knot shown in Figure 5.

Fig 5: Hilbert Curve(left), Torus Knot(Right)

Biologically speaking interpenetrating networks can allow for the perfusion of multiple
materials, oxygen and media for example, that will improve cell viability. From the electrical
perspective, such interpenetrating networks that can be made conductive through the use of
conductive ink start to resemble complex 3-dimensional capacitors that may be used to develop
more efficient and powerful batteries and circuits.
So far in the field of bioprinting, we are fully aware of what we can print. However, we
are not certain of what we should print. This development of a functional vascular efficiency
metric will help define the direction that we go in choosing what types of architectures to print

and what architectures will be most beneficial for cells in 3D printed tissues. With the addition of
an electricity aspect, the work presented here can allow 3D bioprinting to be defined in new,
exciting ways - moving one step closer to a world where organ donor lists are replaced by print
queues.

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