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Concentric bioprinting of alginate-based tubular constructs using multi-nozzle extrusion-based technique

lethal to the patient in the long run [19] . Furthermore, of xanthan gum (XG) from Xanthomonas campestris
the degradation residues of these materials could po- (Sigma-Aldrich) was dissolved into the alginate solu-
tentially cause substantial damage to the tissue around tion. Thereafter, 0.022 g/mL of calcium chloride
it [20] . (CaCl 2) (Sigma-Aldrich) was added dropwise to the
In light of such issues, researchers have begun to mixture. Additionally, 500 mmol/L of CaCl 2 was pre-
develop hydrogel tubular structures for TE purposes. pared as an additional cross-linking solution.
These tubular structures are usually cast using a
mold [21, 22] , centrifuged in tubes [23, 24] or co-axially 2.2 Hydrogel Characterisation
extruded [25, 26] . Although the strength of such gels are Rheology of the hydrogel was characterized using a
much weaker than polymers in terms of tensile Discovery Hybrid Rheometer 2 (TA Instruments) us-
strength and ductility, the use of some of these gels ing a 40 mm parallel plate geometry with a measure-
improves cell compatibility and have been shown to ment gap of 0.5 mm and Peltier plate thermal control.
reduce autoimmune responses [27] . After loading, the samples were conditioned by sub-
–1
Recently, bioprinting has emerged as a potential jecting to 30 s pre-shear at 500 s followed by one
method for fabricating cell-encapsulated hydrogel minute equilibrium before measurements were taken.
tubular construct [28, 29] . This method of fabrication Shear-dependent viscosity was evaluated using a
–1
increases the flexibility and versatility in the printing stepped ramp of shear rate from 1–1000 s and the
°
process, allowing various forms and sizes of tubular process was done at 25 C. In this method, the hydro-
structures to be manufactured with design-driven re- gel was used as it is with the exception of the addition
peatability [30] . of excess CaCl 2. Measurements were taken at 10
Tubular structures fabricated using bioprinting have points per decade.
been demonstrated with multiple materials in several
reports. Examples of some of these materials used in 2.3 Printing Process
printing include hyaluronan [31] , alginate [32] and gela- Tubular structure design and process was input into
tin-derived products [33] . They are usually built hori- the bioprinter using BioCad (RegenHu). First, a cir-
zontally as the weight from the structure itself would cular structure with radius of 6 mm was defined in the
not allow for sufficient structural integrity of the base system as the extrusion route for the hydrogel. Next, a
if it was to be built vertically. However, such method secondary loop of radius of 4 mm which is concentric
of producing tubular structure would cause irregulari- with the hydrogel path was made for the dispensing
ties in the diameter if the diameter is too large. Fabri- route for CaCl 2. The hydrogel was placed into a time-
cating a tubular structure in the horizontal configura- based extruder while CaCl 2 was placed in a micro-
tion also restricts the potential in constructing tubular valved controlled dispenser. The printing process was
branches on a 3D scale. done at room temperature. The printing process is
The main objective of this study is to investigate shown in Figure 1.
the feasibility of fabricating vertical tubular structure The pressure of the bioprinter was set at 1.5 bar for
using a multi-nozzle extrusion-based technique. This the hydrogel and 0.5 bar for CaCl 2. Tubular constructs
method was derived based on concurrent deposition of were printed using RegenHu’s Biofactory. Hydrogel
cross-linking agent into the concentric tubular wall was printed through a 0.25 mm syringe needle
during each layer of deposition. Alginate was selected (Needle DD-135N-N4) while the CaCl 2 solution was
as the model material to demonstrate the feasibility of dispensed through a 0.3 mm needle tip. The path
this versatile and simple method. This method could speed of the hydrogel was 500 mm/min while the path
be extended for different hydrogels and their cross- speed of the CaCl 2 was 100 mm/min. CaCl 2 solution’s
linking agents. path speed was much lower than the hydrogel to allow
it to sufficiently fill up the tube during dispensing. The
2. Materials and Methods layer thickness of the hydrogel was set at 0.2 mm. To
allow sufficient time for the layers to fuse before the
2.1 Hydrogel gel cross-linked, the printer was programmed to dis-
Sodium alginate powder (Sigma-Aldrich) was dissol- pense CaCl 2 only after 3 layers have been built. Sub-
ved at 0.06 g/mL in phosphate-buffered saline solution sequent layers were added in the vertical axis after
under constant stirring. Next, 0.01 g/mL to 0.03 g/mL gelling interaction was achieved in the first 3 layers,

50 International Journal of Bioprinting (2015)–Volume 1, Issue 1