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Fabricating Tissue Replacements using a

Hybrid Inkjet-Electrospinner 3D Printer
Amal Duriseti and Uri Nator - University of California Los Angles
Bob Loblaw and Anurag Dikshit - University of Waterloo
Jack Goff - Carnegie Melon
Abstract
In this paper we introduce a technique for producing mechanically superior fibrous scaffolding and controlling
cell differentiation when fabricating tissue. This approach uses a inkjet head working in tandem with an
electrospinning head to deposit a layers of induced pluripotent stem cells interspersed with layers of growth
factor laced PCL. These implants have excellent mechanical properties, and outperform alternate gel based tissue
manufacturing strategies in both stiffness and tensile strength, making them suitable for applications like cartilage
replacement. Cells in these implants have high survival rates both in vitro and after incubation because the initial
cell suspension can be relatively sparse, allowing each cell access to ample resources. The growth factor based
method of controlling cell differentiation has a highly localized effect in the implant, allowing for a high level of
precision when controlling expressed tissue type. This study demonstrated the viability of this method in creating
a complex circulatory system - additonally, these findings indicate that not only can cell differentiation be used to
create complex circulatory structures, but also that this artificial vascular system can improve cell proliferation and
survival rates for other cells in the implant.

I. I NTRODUCTION
In addition to the more obvious applications of
3D printing in mechanical and industrial engineering, researchers in the medical field have explored
the possibility of using 3D printing to fabricate
replacement organs. While the intricacy and inherently variable nature of living tissue lends itself
well to the strengths of 3D printing, the crucial
yet subtle differences in makeup between tissues
of close proximity and the fragility of any solution
of cells used for deposition preclude transplants
of all but the most simplistic of organs 1 . The
two biggest obstacles to any 3D printed organ are
the fragility of the deposit in both solution form
and after addition. Because cells require proximity to others, but paradoxically also require ample
access to resources (oxygen, food, etc...) in their
environment, a solution of bioactive material with
sufficient cellular density cant be sustained for
long prior to deposition. Additionally any cluster of
tissue without intelligent differentiation (i.e. a well
developed circulatory system) will become necrotic
after deposition4.

In this paper we explore a method of creating

mechanically superior tissue implants with a complex circulatory system. Traditionally most methods
of tissue fabrication have attempted to create a
biodegradable matrix similar to that of the bodys
own extracellular matrix (ECM) using ink-jet 3D
printing techniques2 . Cells are embedded in this
matrix in sufficient numbers to replace their housing
as the body breaks it down, ensuring the implant
turns into healthy tissue. This method has the advantage of generating bio-active tissue artificially,
at the cost of a fragile implant. During the time
active cells replace the matrix, a patient must be
careful to limit stress placed on the implant area
to avoid damaging the structure of the matrix.
Additionally, it is unclear as to whether the tissue
matrix produced by the implant seed cells will be
as strong, if the initial 3D printed matrix is nonfibrous. This technique also has the disadvantage
of limited control over what hormones and growth
factors seeded cells see within the matrix. This
makes controlling cell differentiation within tissue
challenging2 . To address these shortcomings, we

attempt to create an initial biodegradable implant

II. M ATERIALS AND M ETHODS
laced with cell differentiation hormones and stem A. Printer Design and Additive Manufacturing Procells with a fibrous structure similar to the ECM. cess
This artificial structure is achieved using a novel
Our hybrid 3D-printing testbed was constructed
combination of electrospinning and ink-jet 3D printby incorporating an electrospinning mechanism onto
ing technologies.
an ink jet printing platform. Below is a diagram
showing the setup:
2
Figure 1: Experimental Apparatus

and additional structural integrity to the implant.

To ensure the stem cells had access to sufficient
resources, but also had sufficient density to populate
the implants artificial matrix, the cell suspension
was made to have a density of 4106 cells ml1 .
The solution feeding into the electrospinner head
used PCL as its artificial polymer. Pluronic F-127
was used as an additive to reduce the viscosity and
surface tension of the solution, allowing for greater
control over the printing process.
Multiple resevoirs fed to the electrospinning head
- the main difference between the solutions they
housed being the type and concentration of growth
factors suspended with the PCL polymer. Because
these additives affected the viscosity and surface
tension of the solution, Pluronic F-127 was used
B. Inkjet and Electrospinner Solution
The solution fed to the ink jet head contained in different quanities for each solution to ensure
a suspension of induced pluripotent stem cells. homogeneity across solutions.
These stem cells were derived from adult fibroblasts
through isolation and introduction of genes: Oct3/4, C. Printing Process
Implant fabrication involved deposition of a laySox2, Klf4, and c-Myc with a retroviral system.
These stem cells were suspended in a gel con- ers of artificial fibrous matrix alternating with desisting of fibrinogen and collagen to add viscosity position of the cell suspension containing the stem
The inkjet printing platform used consisted of an
XYZ step motor driven CNC base with a solenoid
inkjet deposition system. The suspension containing
the non-differentiated stem cells was supplied to
this print head using pressurized air. The printhead
positing and deposition were controlled with custom
software build using Microsofts .NET Framework.
The electrospinning head was mounted in tandem
with the XYZ plotter housing the inkjet deposition
apparatus. The electrospinning mechanism used a
DC voltage supply to draw fibres from an array
of reservoirs containing ECM fibers laced with
different combinations and concentrations of growth
factors and differentiation hormones.

cells. During deposition, the substrate was kept wet

because electrospinning completely dries deposited
fibers, turning them into a dessicant.
Cell differentiation was controlled during the
deposition of the electrospun layer. These implants
were designed with the goal of producing tissue
with a functional circulatory system, so the electrospinning head deposited PCL laced with vascular endothelial growth factor, which promotes
differentiation into blood vessel cells, into a pregenerated 3D model of the desired structure of the
circulatory system. Elsewhere, the PCL matrix was
deposited with fibroblast growth factor to encourage
differentiation into connective tissue.

C. In-Vitro Testing

Because the solution bath designed to mimic body

conditions couldnt control for factors like immune
response and hormone proliferation external to the
implant, the behavior of the implants in vitro was
also examined. Fibroblasts were harvested from
subjects separately and reverted to pluripotent stem
cells. They were then grown in sufficient quantities
to make a cell suspension for the inkjet head of the
printing apparatus. After fabricating the implant, the
prosthetics were inserted back into the subjects who
donated cells for the cell suspension.
The implants were removed from subjects at 2,
4, and 8 weeks, and cell differentiation and survival
right were examined in the same way as for the ones
III. T ESTING
suspended in a solution bath. The hybrid natural
A. Bio-viability and differentiation of Implant
and artificial ECM was examined for any signs of
The survival rate and degree of differentiation immunological response. Additionally, the subjects
of implants was assessed a week after deposition. implant areas were examined for any effects of the
During this week, the implants were allowed to sit growth factors laced into the implant.
in a relatively nutrient rich and well oxygenated
solution designed to mimic internal body condiIV. R ESULTS AND D ISCUSSION
tions. To test for cell survival, the constructs were
rinsed with PBS and examined under a fluoresThis research demonstrated the viability of a
cent microscope. Number of live cells stained with
calcein AM (fluorescing green) was compared to novel approach to both tissue scaffolding fabrication
total number of cells. Cell counts were conducted and cell differentiation control using 3D printing.
on implants with and without PCL deposited with PCL was used because of its ease of manufacture, its
vascular endothelial growth factor, testing whether a biodegradability , and its high mechanical quality.
developed circulatory system can help maintain cell The apparatus used for deposition proved capable of
depositing a consistently fibrous matrix layer, with
proliferation and survival.
To test for differentiation, flourescent molecules minimal beading more typical of gel scaffolding
were bonded with ligands known to associate with techniques. The resulting matrix had a higher tensile
proteins expressed only on endothelial cells. After strength and greater thickness than implants of ala bath in these floursecet ligands, flourescence pat- ternate manufacture, improving chances of implant
terns for the implants were cross referenced with the integration. Cells deposited in this matrix and incu3D model for endothelial growth factor laced PCL bated in a body-like bath demonstrated high survival
rates and were able to proliferate as well as replace
deposition.
the artificial ECM with a natural one. Additionally,
cell counts for the implants with PCL laced with
B. Mechanical Viability
vascular endothelial growth factor were higher for
Mechanical quality of hybrid scaffolds with and all test cases, indicating that some vascular behavior
without cells was compared to that of implants was achieved.
generated without electrospinning using alginate or
The technique of controlling cell differentiation
a collagen/fibrin gel. The thickness of the samples by lacing the artificial ECM with targeted growth
was measured, and the maximum tensile strength factors also proved successful. The matching quality
relative to distance stretched was measured and used of the florescent pattern to deposition XY coordito calculate Youngs modulus and ultimate tensile nates was given by the formula, where X and Y
strength UTS.
are the shortest distances in the X and Y directions

from a deposition cycle to an observed flourescence percent, indicating that the fluorescence patterns
generated after ligand based isolation of differenpoint.
tiated cardiovascular and connective tissue matched
Xavg + Yavg
statistically significantly with the 3D model used
M atching quality =
largest sample dimension to guide deposition of PCL. Below is a display of
The matching quality for samples was 0.032, which the computer model used to guide PCL deposition
corresonds to a percent difference of less than 5 compared to observed flourescence patterns:
2
Figure 2: 3D Model to Guide Endothelial Growth Factor Laced PCL

Figure 3: Observed Flourescence Patterns at 2 weeks(left), 4 weeks(middle), and 8 weeks(right)

In vitro testing proved sucessful from an implant

survivability and differentiation standpoint. The host
had no immunological response to the implant or
the PCL matrix. Additionally, the hosts natural
hormone balances didnt affect cell differentiation
within the implant. However, the effects of the
implant on surrounding tissue is cause for some
concern. High levels of both fibroblast and endothelial growth factor are linked to diseases most organ
systems. Additionally, high levels of these hormones
may be carcinogenic. That said, this study did not
track effects over a long enough period of time to
provide useful data on the subject.

V. C ONCLUSION AND F UTURE D IRECTION

This research demonstrated the viability of a hybrid electrospinner-inkjet deposition system in producing implants with high mechanical quality and
good cell differentiation. Not only was it possible to
control differentiation to produce complex vascular
structures, but cell proliferation rates indicated that
this differentiation improved implant bioavailability.
Despite these promising results, this technology
is currently very limited. The method of growth
factor lacing demonstrated in this study limits differentiation processes to single hormone cultivation

cycles. As a result this method cant differentiate a

homogeneous stem cell solution into all the tissue
types needed for organs like a kidney or a liver. A
3D printing technology with this capability could
still use the growth factor laced PCL matrix, but
it would have to use a more complicated inkjet
head capable of depositing many different types
of already partially differentiated cells, and could
possibly have to be organ specific. Future research
could explore this idea.
The electrospinning process proved capabale of
selectively differentiating cells, but it also gave the
implant excellent tensile strength and stiffness. As a
result, implants generated using this method could
have high stress applications like cartilage or connective tissue replacement. Although not supported
in this study, it is possible that greater control
over the structure of the PCL matrix could guide
the artificial ECM replacement to produce highly
ordered tissue with mechanical quality equal to or
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greater than natural tissue.

Despite the many advantages of this tissue fabrication process, the health risks of inserting high
concentrations of growth factor into host were not
addressed in this study. Abnormally high levels of
any type of growth factor are almost universally
bad and are generally linked to health complications in other organ systems. Additionally, any
growth factor, while necessary for development and
maintenance, is inherently carcinogenic because it
encourages cell growth and reproduction. None of
the subjects of this study exhibited any abnormal
growth around the implant area or seemed noticeably unhealthy. However, because of the short
timeframe in this study, this insufficient to rule
out health complications. Before this technology
becomes available to the public, further research
must be conducted to determine this the safety of
this method of cell differentiation.