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International Journal of Bioprinting Redefined collagen inks in cartilage printing

candidate for creating scaffolds, most studies focus on from 100 to 8%, controlled by the M221 instruction in the
mechanical properties, degradation rates, and biological machine code (.gcode file) (Figure 6). Utilizing the 22GA
performance rather than printability. This gap in the 420 µm conical nozzle, the finest strand diameter achieved
literature underscores the need for more rigorous without repeated interruptions was approximately 440 µm
assessments of printability, such as extrusion parameters, (Figure 6).
viscosity measurements, and post-printing stability,
to fully realize the potential of pure collagen bioinks The 2% collagen ink exhibits exceptional shape fidelity
in 3D bioprinting applications. Following preliminary during 3D printing, primarily due to its optimal viscosity
printability tests and to avoid extensive cell death due and effective water retention capacity. Notably, a 10-layer
to a too-high density, the 2% collagen ink was selected construct maintains its height with minimal change within
for the bioprinting experiments reported below. Thus, the first 5 min and only a slight decrease of up to 10% after
here, we describe the comprehensive characterization of 20 min at ambient conditions (20°C, 45% relative humidity
its printability. [RH]) (Figure S4a, Supporting Information). This contrasts
with previous studies where a 50% reduction in height was
The good extrudability of the 2% collagen ink was observed within 10 min, but in this study, the ink preserves
ascertained using a pneumatic extrusion bioprinter (BioX) structural integrity for up to 90 min, akin to hydroxyethyl
to facilitate comparison with the prevalent literature, cellulose – sodium alginate (HEC-SA) ink in water-vapor-
which predominantly utilizes pneumatic systems. The saturated environments. Thus, humidity control could
50
ink was successfully extruded through a 420 µm conical further enhance the integrity of the collagen strands.
nozzle (22GA) at a pressure as low as 10 kPa, yielding
continuous strands. The subsequent printability tests Additionally, the ink’s precision is demonstrated by
were conducted on a mechanical extrusion bioprinter forming pores as narrow as around 80 µm wide (Figure S4b,
(FiBotBio), which offers a distinct advantage in terms Supporting Information). This capability, along with the
of direct control over the flow rate. Unlike pneumatic test results from printing a strand over various gap spans
systems where the flow rate is influenced by different (Figure 7), where collagen strands maintain structural
variables (applied pressure, nozzle geometry, bioink integrity across gaps of 1 to 16 mm with minimal sagging
viscosity under shear, and piston friction) and must be except at the widest span, underscores the robustness of its
determined experimentally, mechanical systems directly formulation. The ink’s higher viscosity curtails the initial
control the plunger’s displacement rate. This feature allows spread upon deposition, effectively preserving height
for precise management of the flow rate, independent and form, while its ability to retain moisture prevents
of the bioink’s rheological properties and other factors, deformation or collapse. Taken together, these properties
enhancing reproducibility and accuracy in bioprinting are crucial for achieving high resolution and maintaining
applications. As a result, the width of the extruded strand the structural fidelity of bioprinted tissues, illustrating
consistently decreases as the flow percentage is reduced how the ink adapts to different stresses and maintains

Figure 6. Printing 2% collagen ink with decreasing flow rates as defined by an extrusion factor. (a) A photograph of the resulting strands; The extrusion
factor (EF) was 1: 100%; 2: 60%; 3: 36%; 4: 22%; 5: 13%; 6: 8%. (b) Average width of the continuous non-interrupted strands with EF 1: 100%; 2: 60%; 3:
36%; 4: 22%. Error bars represent the 95% confidence interval for the mean from five replicates.

Volume 10 Issue 6 (2024) 505 doi: 10.36922/ijb.4566