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:[email protected])

Keywords: multi-cell assembly, extruding, hydrogel, nozzle, been reported widely. An analog of tissue or organ with
rapid prototype. multi-cell, which spatial location and proportional component
according to the numerical models, has a characteristic of
Abstract non-homogeneous distribution in the composition and
structure [4]. As early as in 2000 years, R. F. Service
Cell direct controlled assembly technology with one material published an article in Science, pointed out it needs to have a
source is grown-up increasingly, but can’t satisfy the multi- manufacturing technology with the ability of distributing
cell assembly requirements. On this condition, it is becoming materials accurately and quantificationally to create complex
a needed research program to develop an assembly organ analogs [5]. To overcome deficient vascularization,
technology which can distribute a variety of cells and many new manufacturing techniques have been proposed to
biological activity materials to a specifically location improve tissue engineering. For example, in Harvard medical
precisely and noninvasively, so that a multi-cell structure school, a group used micro-electromechanical system to
similar to natural tissue structure and function can be create capillary network on a silicon plate then cultured cells
constructed. Here utilizing an in-house single nozzle on it and layered sheets of cells to make a vascular network
assembly technology based on rapid prototyping and for hepatocyte infusion culture [6–7]. This is a very time-
controlled forming process with cell gelatin-based hydrogels, consuming method with very low efficiency to produce
develop a kernel of multi-cell directly controlled assembly complex vascular tissue structures. In recent years, rapid
technology, namely the two-material source controlled prototyping technique based on discrete/deposit layered
extruding technology. It mainly includes a multi-nozzle forming principle is applied increasingly in biological
switched on-demand extruding technology and a single material forming and cell assembly [8]. Based on the
nozzle with a multi-material source delivery extruding technology, more complex 3D structures can be manufactured,
technology, it is possible to directly manipulate cells with both the macro structure forming and micro pore constructing
bionic extracellular matrices and arrange them in proper can be coupled to the same manufacturing process, the
spatial sites according to predefined digital models to form structure shape and pore is more controllable, the process is
living constructs. The experimental formed 3D cell structure relatively easy to formation [9-10]. Recently, with the
was 3-4 mm high and cultured for 8 weeks, actually, there is advantages of rapid prototyping, we have explored
no limitation for the size and height of the 3D organ analogs. extensively the direct cell assembly and low-temperature
This technology will direct promote the development of deposition technology [11], developed a cell assembly
multi-cell controlled assembly technology; consequently technology with two-material source. It is possible to directly
promote the manufacturing engineering of artificial organs manipulate two type cells with bionic extracellular matrices
greatly. and arrange them in proper spatial location according to
predefined digital models to form tissue or organ analog; it is
1 Introduction a basis technology for multi-cell assembly. Therefore there is
a hope to directly create capillary network or organ.
At the end of the 20th century, tissue engineering technology
began using formed scaffolds to indirectly assembly cells. 2 Materials and Methods
This technology has resulted in a new way to fabricate tissues
artificially. However, unlike those successfully
commercialized structural tissues (such as skin, bone, and 2.1 Materials
cartilage), many tissues or organs with vascular systems are
much more complicated, both in structure and physiological The biomaterials applied to cell assembly should have the
functions, fettered with the indirect fabrication techniques, bionic features both in composition and structure, were
unredeemed distribution of primary cells and other necessary chosen according to two aspects, biocompatible and good
cells, it is very difficult to create a large amount of tissues formability. Most natural hydrogels are more favourable in
with vascular systems [1–3], by now the fabrication of the soft tissue construction [12-13]. Through experiments,
different types of cells in one single construct has not yet select the part of the natural hydrogels as assembly materials,

2154
such as gelatine (Tianjin Green-Island Company, China), into account the residual materials of nozzle cavity during the
alginate and chitosan (Sigma-Aldrich, Louis, Mo). Gelatine, a extrusion processing; the latter does not exist the position
degradation product of collagen, exhibits reversible alignment problems when switch nozzles, forming efficiency
thermosensitive gelation properties and can be irreversibly is higher. Because of the more than three material cavities has
cross-linked with glutaraldehyde. In view of this, we use the same control method with the double cavities; here
gelatine-based composite hydrogel system as forming introduce the non-homogeneous distribution of the double
materials. The concentration and the ratio between gelatine cavities as a representative.
and chitosan are listed in reference [14]. Experiments show For the multi-nozzle switched structure, the implement
that the hybrid of gelatine-based hydrogels possess a good of non-homogeneous distribution process is shown in Figure
conglutination property, is prone to implement the 1. The different materials cavity stores the different material,
discrete/deposit cell assembling process. there is an absolute displacement ǻx between the each two
material cavities, if the location of the material cavity A1 is
2.2 Using the pre-extrusion mechanism P1 (x0, y0, z0), A2 is P2 (x0+ǻx, y0, z0), when forming at
the specific delamination, the forming platform move the
Due to the strong viscosity of selected natural polymer absolute displacement ǻx for switch the material cavity, and
hydrosol, it is needed an incidental time when the material the nozzles group adjust in the Z direction timely.
were delivered from material cavity to nozzle terminal, For the single nozzle with a multi-material source
produces the response delay. This response delay could be structure, the implement of non-homogeneous distribution
reduced as much as possible through optimize the design of process is shown in Figure 2. The different materials cavity
cavity and nozzle, but not completely eliminated. Therefore, stores the different material, there is an absolute displacement
through adopting a control method to compensate for the ǻx between the each two material cavities, if the location of
response delay in the actual forming process, based on the material cavity A1 is P1 (x0, y0, z0), A2 is P2 (x0+ǻx, y0,
material characteristics, the response delay time IJ basically z0), when forming at the specific delamination, the forming
satisfies the model of Equation (1), V1, material cavity platform does not need to move the absolute displacement ǻx
volume; V2, nozzle volume; v, scan rate; d, nozzle diameter. for switch the material cavity, just need to adjust the single
V1 + V2 nozzle in the Z direction timely. However, it needs to use the
= 1

vd 2 pre-extrusion method reasonably for forming control, needs

Set the specific value of the material cavity volume V1 and the material cavity A2 start extrusion action before the
nozzle volume V2 in the modeling software, the response materials A1 cavity ending the extrusion, the time difference
delay time IJ will be calculated approximately according to is decided by the specific material delivery path and the
Equation (1). Before the nozzles arrive at a certain assembly nozzle chamber.
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- h 2 h 1
extrusion material just reach the nozzle terminal, and then
realize the extrusion and deposition.

2.3 The implementations of non-homogeneous distribution Z

O/Y X

When construct the non-homogeneous distribution structure
with multi-material source, different material cavity is need to Figure 1 Non-homogeneous distribution process of the multi-
be switched on demand and were expected to complete its nozzle switched device
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
h 2 h 1
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. Z
From the spray forming point of view used the heterogeneous O/Y X

continuous distribution with multi-material cavity, there are
two extrusion implementations, one is every cavity Figure 2 Non-homogeneous distribution process of a single
corresponding to a nozzle, forming a nozzle unit, multiple nozzle with a multi-material source device
nozzle units combined to form a multi-nozzle composite
structure, the other is all the material cavity corresponds to 2.4 Two-nozzle switched device
one nozzle, namely the multi-input and single-output nozzle
The extruded sol-state materials have a high viscosity, due to
structure. These two structures each have its advantages and
the discrete accuracy is not very high, considering the discrete
disadvantages, and its control way is in different. In
accuracy, material characteristics, cell damage rate, the
comparison, the former control method is simple, do not take
characteristics of the nozzles, here using the volume-driven

2155
method as the extrusion technology, one nozzle unit 3.1 Forming example of two-nozzle switched device
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 Multi-cell assembly aims for the construction of large soft
3. Installed and fixed several nozzle units in together to tissue and internal organs in vitro, typical characteristics of
achieve multi-nozzle composite device. Figure 4 is a double- the tissue or organ is rich of vascular providing the nutrition
nozzle device. For the multi-nozzle composite device, cell for the surrounding cells. Commonly, the larger vessels have
assembly is primarily achieved through switching the nozzle two layers, the inner layer contains the endothelial cells, the
unit; the different nozzle unit switched need to be alignment outer layer contains the smooth muscle cells. Here using the
according to delamination position, therefore, the multi- two-nozzle switched device to form a structure simulated the
nozzle composite device is intended for using as in the lager vessel. Material cavity N1 corresponding to the nozzle
construction of the simple and clear hierarchical tissues. 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
Figure 3 Multi-nozzle switched unit sketch cartilage-bone composite, bladder, cornea, big blood vessels,
Figure 4 Two-nozzle switched device, scale bar is10mm etc. This nozzles switched device can satisfy the forming
requirements through using simple control method.
2.5 Single nozzle and multi-material source device
3.2 Forming example of the single nozzle and multi-
The single nozzle and multi-material source device has the material source device
same drive units and material cavities as the multi-nozzle
composite device, different cell-hydrogel stored in a different Here using the single nozzle and double-material source
material cavity, there is only one specific nozzle as a common device to form a structure simulated the nerve fibres. Material
export, during the forming process, after the forming material cavity N1 stored the Schwann cells plus the material
was selected according to the forming location, the 12G1.6AG; material cavity N2 stored neurons plus the
corresponding drive unit acts and extrudes materials from material 8G2.4AG supplemented with 10mg/mL
cavity to the common nozzle. Figure 5 is a model of the phenolphthalein. The assembly result is as shown in Figure 7.
single nozzle and two-material source device. For this type It is shown that the designed single nozzle and double-
nozzle, it doesn’t need to align after switching the drive unit, material source device has a good applicability in multi-cell
so the forming accuracy and efficiency is higher, moreover, it controlled assembly technology. It is suggested to use this
can more easily achieve continuous forming for mixing single nozzle and multi-material source device to form the
proportion of the different material adopting the natural two- complicated tissues, particularly those with intricate gradient
phase flow. Although the uniformity of mixing is not tissues, such as heart, liver, vascular network, etc. Based on
controllable, the shear force during the mixing extrusion is this nozzle structure, the high forming precision and
smaller; the resulting hybrid materials are also more stable, so complicated shape can easily be achieved under the direct
it is provide a special facility for some non-homogeneous CAD model driving.
distribution.

Figure 6 Assembly process using double-nozzle switched

Figure 5 Single nozzle and two-material source device, scale device, the scale bar is 10mm
bar is10mm Figure 7 Assembly process using single nozzle and double-
material source device, the scale bar is 10mm
3 Results

2156
4 Discussions References
Now, the two materials controlled assembly was [1] H. X. Liu, Y. N. Yan, X. H. Wang, et al. Construct
experimentally realized and the more materials units hepatic analog by cell-matrix controlled assembly
controlled assembly is under development. In the technology, Chinese Sci. Bull., 51(15), pp. 1830-1835,
development process, on the one hand, it is need to optimize (2006).
the structure of the single nozzle and multi-material source [2] X. H. Wang, Y. N. Yan, R. J. Zhang. Rapid prototyping
device; on the other hand, explore the precise quantitative as a tool for manufacturing bioartificial livers, Trends
control ways through extruding experiments and theory Biotechnol., 25(11), pp. 505-513, (2007).
analysis, build the structure CAD model corresponding [3] W. Xu, X. H. Wang, Y. N. Yan, et al. A Polyurethane-
exactly to the digital model information, play a guiding and gelatin hybrid construct for manufacturing implantable
predictability for the preparation of expensive materials bioartificial livers, J. Bioact. Compat. Polym., 23(5), pp.
(biological materials, biological factors and cellular, etc.). 409-422, (2008).
The specific implementation process is as follows: Through [4] I. Nishimura, R. L. Garrell, M. Hedrick, et al. Precursor
reversing the signs of the cell assembly structure model tissue analogs as a tissue engineering strategy, Tissue
information, extract the important parameters of the forming Eng., 9(11), pp. 77-89, (2003).
process, such as the volume concentration, microfilament [5] R. F. Service. Tissue engineering builds new bone,
diameter, the channel size of the blood vessels, storey height, Science, 289(5478), pp. 1498-1500, (2000).
stack rate between layer and layer, extrusion speed and other [6] J. I. Borenstein, C. J. Bettinger, D. J. Carter, et al.
parameters. After input all the identified information, the Nanotopographic tools for vascular tissue engineering,
computer will output corresponding information at a friendly In Vitro Cell Dev.An., 41, pp. 10, (2005).
user interface, such as the prepared material volume, the cells [7] S. Kaihara, J. Borenstein, R. Koka, et al. Silicon
and biological factors amount, estimated forming time, etc, micromachining to tissue engineer branched vascular
while using the corresponding icon to display a different cell channels for liver fabrication, Tissue Eng., 6(2), pp. 105-
source material cavity, all these will help the researchers to 117, (2000).
prepare and load materials correctly. Finally, the forming [8] F. Lin, Y. N. Yan, R. J. Zhang, et al. From rapid
equipment can complete the assembly process automatically prototyping to bio-manufacturing, Tissue Eng., 12(4), pp.
without human intervention. It is an artificial intelligence 1023-1028, (2006).
terminal of the multi-cell assembly technology. [9] S. J. Li, Y. N. Yan, Z. Xiong, et al. Gradient
hydrogel construct based on an improved cell
4 Results assembling system, J. Bioact. Compat. Polym.,
24(1Suppl.), pp. 84-99, (2009).
The proposed multi-source extruding technology and test [10] Y. N. Yan, S. J. Li, R. J. Zhang, et al. Rapid
systems can satisfy the discrete/deposits forming operation of prototyping and manufacturing technology:
a variety of viscous gelatine-based materials with cells; principle, representative technics, applications and
through adopting the pre-extrusion as a compensation control developmental trends, Tsinghua Sci. and Tec.,
method, built the double-nozzle switched device and the 62(7), pp. 15-16, (2008).
single nozzle with double-material source device, realized the [11] L. Liu, Z. Xiong, Y. N. Yan, et al. Multi-nozzle low-
continuous distribution and assembly with non-homogeneous temperature deposition system for construction of
materials for the most tissue analogy forming. gradient tissue engineering scaffolds, J. of Biomed. Mat.
Based on the forming system above designed, to build the Res. Part B, 88B (1), pp. 254-263, (2009).
large soft tissue and internal organs, the test results show that [12] L. L. Hench, J. M. Polak. Third-generation biomedical
the forming system has a well-controlled ability to achieve materials. Science, 295(5557), pp. 1014-1016, (2002).
the multi-cell heterogeneity distribution structure. This [13] J. Cheng, F. Lin, H. X. Liu, et al. Rheological properties
technology will be the core extruding method for multi-cell of cell-hydrogel composites extruding through small
controlled assembly and be widely used in many fields of the diameter tips, J. of Manu. Sci. and Eng., 130, pp. 1-5,
artificial organs construct. (2008).
Furthermore, the combination of clinical imaging data with [14] H. X. Liu, S. J. Li, Y. N. YAN. Cell direct assembly
CAD-based freeform fabrication techniques may offer the technology adopting hybrid of gelatin-based hydrogels,
capability to form constructs that are customized to the shape Adv. Mater. Res., 189-193, pp. 2986-2992, (2011).
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).

2157