THE PRELIMINARY EXTRUDING TECHNOLOGY WITH
TWO-MATERIAL SOURCE FOR 3D ASSEMBLY
H.X. Liu1, S.J. Li2, Y.N. Yan2, F. Lin2
1. School of Instrument Science and Opto-electronic Engineering, Beihang University, Beijing 100191
2. Department of Mechanical Engineering, Tsinghua University, Beijing 100084
Correspondence should be addressed to Liu Haixia (e-mail:liuhx02@mails.tsinghua.edu.cn)
Keywords: multi-cell assembly, extruding, hydrogel, nozzle,
rapid prototype.
Abstract
Cell direct controlled assembly technology with one material
source is grown-up increasingly, but can’t satisfy the multi-
cell assembly requirements. On this condition, it is becoming
a needed research program to develop an assembly
technology which can distribute a variety of cells and
biological activity materials to a specifically location
precisely and noninvasively, so that a multi-cell structure
similar to natural tissue structure and function can be
constructed. Here utilizing an in-house single nozzle
assembly technology based on rapid prototyping and
controlled forming process with cell gelatin-based hydrogels,
develop a kernel of multi-cell directly controlled assembly
technology, namely the two-material source controlled
extruding technology. It mainly includes a multi-nozzle
switched on-demand extruding technology and a single
nozzle with a multi-material source delivery extruding
technology, it is possible to directly manipulate cells with
bionic extracellular matrices and arrange them in proper
spatial sites according to predefined digital models to form
living constructs. The experimental formed 3D cell structure
was 3-4 mm high and cultured for 8 weeks, actually, there is
no limitation for the size and height of the 3D organ analogs.
This technology will direct promote the development of
multi-cell controlled assembly technology; consequently
promote the manufacturing engineering of artificial organs
greatly.
1 Introduction
At the end of the 20th century, tissue engineering technology
began using formed scaffolds to indirectly assembly cells.
This technology has resulted in a new way to fabricate tissues
artificially. However, unlike those successfully
commercialized structural tissues (such as skin, bone, and
cartilage), many tissues or organs with vascular systems are
much more complicated, both in structure and physiological
functions, fettered with the indirect fabrication techniques,
unredeemed distribution of primary cells and other necessary
cells, it is very difficult to create a large amount of tissues
with vascular systems [1–3], by now the fabrication of
different types of cells in one single construct has not yet
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been reported widely. An analog of tissue or organ with
multi-cell, which spatial location and proportional component
according to the numerical models, has a characteristic of
non-homogeneous distribution in the composition and
structure [4]. As early as in 2000 years, R. F. Service
published an article in Science, pointed out it needs to have a
manufacturing technology with the ability of distributing
materials accurately and quantificationally to create complex
organ analogs [5]. To overcome deficient vascularization,
many new manufacturing techniques have been proposed to
improve tissue engineering. For example, in Harvard medical
school, a group used micro-electromechanical system to
create capillary network on a silicon plate then cultured cells
on it and layered sheets of cells to make a vascular network
for hepatocyte infusion culture [6–7]. This is a very time-
consuming method with very low efficiency to produce
complex vascular tissue structures. In recent years, rapid
prototyping technique based on discrete/deposit layered
forming principle is applied increasingly in biological
material forming and cell assembly [8]. Based on the
technology, more complex 3D structures can be manufactured,
both the macro structure forming and micro pore constructing
can be coupled to the same manufacturing process, the
structure shape and pore is more controllable, the process is
relatively easy to formation [9-10]. Recently, with the
advantages of rapid prototyping, we have explored
extensively the direct cell assembly and low-temperature
deposition technology [11], developed a cell assembly
technology with two-material source. It is possible to directly
manipulate two type cells with bionic extracellular matrices
and arrange them in proper spatial location according to
predefined digital models to form tissue or organ analog; it is
a basis technology for multi-cell assembly. Therefore there is
a hope to directly create capillary network or organ.
2 Materials and Methods
2.1 Materials
The biomaterials applied to cell assembly should have the
bionic features both in composition and structure, were
chosen according to two aspects, biocompatible and good
formability. Most natural hydrogels are more favourable in
the soft tissue construction [12-13]. Through experiments,
select the part of the natural hydrogels as assembly materials,
such as gelatine (Tianjin Green-Island Company, China),
alginate and chitosan (Sigma-Aldrich, Louis, Mo). Gelatine, a
degradation product of collagen, exhibits reversible
thermosensitive gelation properties and can be irreversibly
cross-linked with glutaraldehyde. In view of this, we use
gelatine-based composite hydrogel system as forming
materials. The concentration and the ratio between gelatine
and chitosan are listed in reference [14]. Experiments show
that the hybrid of gelatine-based hydrogels possess a good
conglutination property, is prone to implement the
discrete/deposit cell assembling process.
2.2 Using the pre-extrusion mechanism
Due to the strong viscosity of selected natural polymer
hydrosol, it is needed an incidental time when the material
were delivered from material cavity to nozzle terminal,
produces the response delay. This response delay could be
reduced as much as possible through optimize the design of
cavity and nozzle, but not completely eliminated. Therefore,
through adopting a control method to compensate for the
response delay in the actual forming process, based on
material characteristics, the response delay time IJ basically
satisfies the model of Equation (1), V1, material cavity
volume; V2, nozzle volume; v, scan rate; d, nozzle diameter.
V1 + V2
= 1

vd 2
Set the specific value of the material cavity volume V1 and
nozzle volume V2 in the modeling software, the response
delay time IJ will be calculated approximately according to
Equation (1). Before the nozzles arrive at a certain assembly
position, the material cavity corresponding to the assembly
position was pre-extrusion for a time IJ, when the nozzle
arrives at the position, continue the extrusion action, the pre-
extrusion material just reach the nozzle terminal, and then
realize the extrusion and deposition.
2.3 The implementations of non-homogeneous distribution
When construct the non-homogeneous distribution structure
with multi-material source, different material cavity is need to
be switched on demand and were expected to complete its
own material extrusion separately at the specific delamination.
During this control process, the different material cavities are
fixed together and immovable each other, their switch and
extrusion are achieved by the drive unit. When some material
cavity obtains an action command, the drive unit of this
material cavity extrudes the material, forming platform carries
out plane movement simultaneously.
From the spray forming point of view used the heterogeneous
continuous distribution with multi-material cavity, there are
two extrusion implementations, one is every cavity
corresponding to a nozzle, forming a nozzle unit, multiple
nozzle units combined to form a multi-nozzle composite
structure, the other is all the material cavity corresponds to
one nozzle, namely the multi-input and single-output nozzle
structure. These two structures each have its advantages and
disadvantages, and its control way is in different. In
comparison, the former control method is simple, do not take
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into account the residual materials of nozzle cavity during the
extrusion processing; the latter does not exist the position
alignment problems when switch nozzles, forming efficiency
is higher. Because of the more than three material cavities has
the same control method with the double cavities; here
introduce the non-homogeneous distribution of the double
cavities as a representative.
For the multi-nozzle switched structure, the implement
of non-homogeneous distribution process is shown in Figure
1. The different materials cavity stores the different material,
there is an absolute displacement ǻx between the each two
material cavities, if the location of the material cavity A1 is
P1 (x0, y0, z0), A2 is P2 (x0+ǻx, y0, z0), when forming at
the specific delamination, the forming platform move the
absolute displacement ǻx for switch the material cavity, and
the nozzles group adjust in the Z direction timely.
For the single nozzle with a multi-material source
structure, the implement of non-homogeneous distribution
process is shown in Figure 2. The different materials cavity
stores the different material, there is an absolute displacement
ǻx between the each two material cavities, if the location of
the material cavity A1 is P1 (x0, y0, z0), A2 is P2 (x0+ǻx, y0,
z0), when forming at the specific delamination, the forming
platform does not need to move the absolute displacement ǻx
for switch the material cavity, just need to adjust the single
nozzle in the Z direction timely. However, it needs to use the
pre-extrusion method reasonably for forming control, needs
the material cavity A2 start extrusion action before the
materials A1 cavity ending the extrusion, the time difference
is decided by the specific material delivery path and the
nozzle chamber.
 
h 2 h 1
Z
O/Y X

Figure 1 Non-homogeneous distribution process of the multi-
nozzle switched device
 
h 2 h 1
Z
O/Y X

Figure 2 Non-homogeneous distribution process of a single
nozzle with a multi-material source device
2.4 Two-nozzle switched device
The extruded sol-state materials have a high viscosity, due to
the discrete accuracy is not very high, considering the discrete
accuracy, material characteristics, cell damage rate, the
characteristics of the nozzles, here using the volume-driven
method as the extrusion technology, one nozzle unit
integrates the stepper drive extrusion unit (including the
stepping motor and the screw slide), disposable medical
material cavity and stainless steel needle, as shown in Figure
3. Installed and fixed several nozzle units in together to
achieve multi-nozzle composite device. Figure 4 is a double-
nozzle device. For the multi-nozzle composite device, cell
assembly is primarily achieved through switching the nozzle
unit; the different nozzle unit switched need to be alignment
according to delamination position, therefore, the multi-
nozzle composite device is intended for using as in the
construction of the simple and clear hierarchical tissues.
Figure 3 Multi-nozzle switched unit sketch
Figure 4 Two-nozzle switched device, scale bar is10mm
2.5 Single nozzle and multi-material source device
The single nozzle and multi-material source device has the
same drive units and material cavities as the multi-nozzle
composite device, different cell-hydrogel stored in a different
material cavity, there is only one specific nozzle as a common
export, during the forming process, after the forming material
was selected according to the forming location, the
corresponding drive unit acts and extrudes materials from
cavity to the common nozzle. Figure 5 is a model of the
single nozzle and two-material source device. For this type
nozzle, it doesn’t need to align after switching the drive unit,
so the forming accuracy and efficiency is higher, moreover, it
can more easily achieve continuous forming for mixing
proportion of the different material adopting the natural two-
phase flow. Although the uniformity of mixing is not
controllable, the shear force during the mixing extrusion is
smaller; the resulting hybrid materials are also more stable, so
it is provide a special facility for some non-homogeneous
distribution.
Figure 5 Single nozzle and two-material source device, scale
bar is10mm
3 Results
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3.1 Forming example of two-nozzle switched device
Multi-cell assembly aims for the construction of large soft
tissue and internal organs in vitro, typical characteristics of
the tissue or organ is rich of vascular providing the nutrition
for the surrounding cells. Commonly, the larger vessels have
two layers, the inner layer contains the endothelial cells, the
outer layer contains the smooth muscle cells. Here using the
two-nozzle switched device to form a structure simulated the
lager vessel. Material cavity N1 corresponding to the nozzle
A1 stored the material 12G1.6AG (it is on behalf of this
solution containing 12% (w/w) gelatine, 1.6% (w/w) of
sodium alginate, with the solvent PBS.); material cavity N2
corresponding to the nozzle A2 stored material 8G2.4AG
supplemented with 10mg/mL phenolphthalein. The assembly
result is as shown in Figure 6; the outer structure was
achieved by the nozzle A1, the inner structure was achieved
by the nozzle A2. It is shown that the designed two-nozzle
composites device has a good forming applicability in multi-
cell controlled assembly technology. It is suggested to use this
multi-nozzle switched device to form the simple tissues,
particularly those with gradient layers tissues, such as
cartilage-bone composite, bladder, cornea, big blood vessels,
etc. This nozzles switched device can satisfy the forming
requirements through using simple control method.
3.2 Forming example of the single nozzle and multi-
material source device
Here using the single nozzle and double-material source
device to form a structure simulated the nerve fibres. Material
cavity N1 stored the Schwann cells plus the material
12G1.6AG; material cavity N2 stored neurons plus the
material 8G2.4AG supplemented with 10mg/mL
phenolphthalein. The assembly result is as shown in Figure 7.
It is shown that the designed single nozzle and double-
material source device has a good applicability in multi-cell
controlled assembly technology. It is suggested to use this
single nozzle and multi-material source device to form the
complicated tissues, particularly those with intricate gradient
tissues, such as heart, liver, vascular network, etc. Based on
this nozzle structure, the high forming precision and
complicated shape can easily be achieved under the direct
CAD model driving.
Figure 6 Assembly process using double-nozzle switched
device, the scale bar is 10mm
Figure 7 Assembly process using single nozzle and double-
material source device, the scale bar is 10mm
4 Discussions
Now, the two materials controlled assembly was
experimentally realized and the more materials units
controlled assembly is under development. In the
development process, on the one hand, it is need to optimize
the structure of the single nozzle and multi-material source
device; on the other hand, explore the precise quantitative
control ways through extruding experiments and theory
analysis, build the structure CAD model corresponding
exactly to the digital model information, play a guiding and
predictability for the preparation of expensive materials
(biological materials, biological factors and cellular, etc.).
The specific implementation process is as follows: Through
reversing the signs of the cell assembly structure model
information, extract the important parameters of the forming
process, such as the volume concentration, microfilament
diameter, the channel size of the blood vessels, storey height,
stack rate between layer and layer, extrusion speed and other
parameters. After input all the identified information, the
computer will output corresponding information at a friendly
user interface, such as the prepared material volume, the cells
and biological factors amount, estimated forming time, etc,
while using the corresponding icon to display a different cell
source material cavity, all these will help the researchers to
prepare and load materials correctly. Finally, the forming
equipment can complete the assembly process automatically
without human intervention. It is an artificial intelligence
terminal of the multi-cell assembly technology.
4 Results
The proposed multi-source extruding technology and test
systems can satisfy the discrete/deposits forming operation of
a variety of viscous gelatine-based materials with cells;
through adopting the pre-extrusion as a compensation control
method, built the double-nozzle switched device and the
single nozzle with double-material source device, realized the
continuous distribution and assembly with non-homogeneous
materials for the most tissue analogy forming.
Based on the forming system above designed, to build the
large soft tissue and internal organs, the test results show that
the forming system has a well-controlled ability to achieve
the multi-cell heterogeneity distribution structure. This
technology will be the core extruding method for multi-cell
controlled assembly and be widely used in many fields of the
artificial organs construct.
Furthermore, the combination of clinical imaging data with
CAD-based freeform fabrication techniques may offer the
capability to form constructs that are customized to the shape
of the defect or injury. Finally, such fabrication technology
may provide a means for large-scale production of multiple
identical organic constructs for use in drug discovery or
fundamental scientific studies.
Acknowledgements
This work was supported by the National Natural Science
Foundations of China (50905009).
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