Page 167 - IJB-10-4

International Journal of Bioprinting Printing collagen type IV membrane

2.3. Rheological characterization 2.5. Construction and physical examination of
Rheology experiments were conducted to examine the collagen type IV membranes by molding vs. printing
viscosity of the Col-IV solution under increased shear The Col-IV membranes were constructed using two
rates and to investigate its crosslinking response when methods: molding and 3D printing. The molding method
exposed to UV (365 nm filter). Viscosity was measured was conducted by a simple in-house molding device. In this
using a cone-plate geometry (2˚/40 mm) on 100 μL of setting, a base glass slide with two glass coverslips, placed
Col-IV solution. on opposite ends, created a depth of ~100 μm. Col-IV ink
(50 μL) was added to the base glass slide and then flattened
The crosslinking properties were studied using a
stainless-steel parallel plate geometry (20 mm diameter) by the addition of a rectangular coverslip on top. The bioink
at constant frequency and strain of 1Hz and 1%, was then crosslinked, through the coverslip, using UV for 2
respectively. The Col-IV solution was subjected to light min. After lifting the top coverslip, the crosslinked Col-IV
curing, which started 3 min after the test began and membrane was cut using an 8 mm trephine and washed
continued for an additional 3 min. Subsequently, the off the slide using phosphate-buffered saline (PBS). The
test proceeded for an additional 4 min with the light off. membrane was incubated two to three times (5 min each)
All tests were performed using an AR-G2 Rheometer in the PBS solution on a shaking platform until clear. To
(TA Instruments, USA) at RT and were repeated at least print the Col-IV membrane, we used a two-layered spiral
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three times. printing, and the parameters were selected by the above
printability test: printing speed of 150 mm/min and flow
2.4. Printability evaluation rate of 0.5 mm/min. The line spacing of the print was
The printability (Pr) of the generated Col-IV photo- reduced to 0.5 mm to allow the newly extruded bioink to
crosslinkable solution was conducted as previously fuse, forming a uniform and smooth surface. The outcome
published using an extrusion-based Edu3D printer was 10 mm diameter Col-IV membranes printed on a 35
(TRICEP facility, University of Wollongong, Australia), mm culture dish or a glass coverslip.
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with an external UV light source (Figure S1A). The Col-IV membranes generated by both methods were
Col-IV bioink (pH 6.7–7.4) in solution was loaded examined using an Olympus OSL5000 3D confocal
into a syringe (1 mL) covered with aluminum foil and microscope (Evident, Japan), which generated the height
fitted with a 25 ga tip. The syringe was then inserted profile for thickness and a 3D heatmap for surface
into the printer. UV light was turned on during the roughness measurement. The thickness of the membrane
printing process to crosslink the bioink into the was generated by averaging height profile values from
hydrogel. Standard printing parameters were used as four randomly selected areas (Figure S3). For roughness
baseline values: temperature of 22˚C, printing speed measurements, we used the 3D heatmap build-in function
of 250 mm/min, and a flow rate of 0.8 mm/min. A Root Mean Square (RMS). RMS has been previously used
two-layered lattice (9 × 9 mm) structure was printed. to measure the surface roughness of corneal endothelium
Additional printing parameters tested included printing using averaged RMS values from selected area. Using
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temperatures of 7 and 10˚C, printing speeds of 150 the same approach in this study, six randomly selected
and 350 mm/min, and flow rates of 0.5 and 1 mm/min. areas of 1 mm² were used. The membrane RMS was
Only one printing parameter was changed at a time. calculated by averaging the values of the examined areas.
The influence of printing parameters on the printed The transparencies of printed and molded Col-IV films
structure was evaluated by Pr using the formulation were measured using a ColorQuest XE (HunterLab, USA)
developed by Ouyang et al.: 17 spectrophotometer. Coverslips were used as blanks. Total
light transmittance was generated using the built-in CIE
L 2 L*a*b* system. Tests were repeated at least three times. The
Pr = (I) compression test was performed using an EZ-L Table-Top
16 A
Universal Testing Instrument (Shimadzu, Japan) equipped
with a 2 N load cell (Figure S2).
where L is the internal parameter of the pore and A is
the internal area of the pore. The L and A values of the The tensile test was performed on Col-IV membranes
pores printed were first imaged under a microscope and using Dynamic Mechanical Analyzer 850 (TA instruments,
subsequently measured by ImageJ. A Pr = 1 indicates USA) equipped with a film tension clamp. Descemet’s
optimum Pr, where the printed Col-IV lattice structures membranes were surgically removed from corneas
display regular grids and square holes, while Pr > 1 and (unsuitable for transplantation and provided by the New
Pr < 1 indicate poor Pr, where the structures are irregular South Wales Tissue Bank [NSWTB]; ethics approval no.:
(Figure S1B). HREC 13/1041) and used as a comparison. All procedures

Volume 10 Issue 4 (2024) 159 doi: 10.36922/ijb.3258