The development of commercial collagen inks for extrusion-based bioprinting has increased the amount of research on pure collagen bioprinting, i.e., collagen inks not mixed with gelatin, alginate, or other more common biomaterial inks. New printing techniques have also improved the resolution achievable with pure collagen bioprinting. However, the resultant collagen constructs still appear too weak to replicate dense collagenous tissues, such as the cornea. This work aims to demonstrate the first reported case of bioprinted recombinant collagen films with suitable optical and mechanical properties for corneal tissue engineering. The printing technology used, aerosol jet® printing (AJP), is a high-resolution printing method normally used to deposit conductive inks for electronic printing. In this work, AJP was employed to deposit recombinant human collagen type III (RHCIII) in overlapping continuous lines of 60 µm to form thin layers. Layers were repeated up to 764 times to result in a construct that was considered a few hundred microns thick when swollen. Samples were subsequently neutralised and crosslinked using EDC:NHS crosslinking. Nanoindentation and absorbance measurements were conducted, and the results show that the AJP-deposited RHCIII samples possess suitable mechanical and optical properties for corneal tissue engineering: an average effective elastic modulus of 506 ± 173 kPa and transparency ≥87% at all visible wavelengths. Circular dichroism showed that there was some loss of helicity of the collagen due to aerosolisation. SDS-PAGE and pepsin digestion were used to show that while some collagen is degraded due to aerosolisation, it remains an inaccessible substrate for pepsin cleavage.

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Collagen has grown increasingly present in bioprinting, however collagen bioprinting has mostly been limited to the extrusion printing of collagen type I to form weak collagen hydrogels. While these weak collagen hydrogels have their applications, synthetic polymers are often required to reinforce gel-laden constructs that aim to replicate dense collagenous tissues found in vivo. In this study, aerosol jet printing (AJP) was used to print and process collagen type I and II into dense constructs with a greater capacity to replicate the dense collagenous ECM found in connective tissues. Collagen type I and II was isolated from animal tissues to form solutions for printing. Collagen type I and II constructs were printed with 576 layers and measured to have average effective elastic moduli of 241.3 ± 94.3 and 196.6 ± 86.0 kPa (±SD), respectively, without any chemical modification. Collagen type II solutions were measured to be less viscous than type I and both collagen type I and II exhibited a drop in viscosity due to AJP. Circular dichroism and SDS-PAGE showed collagen type I to be more vulnerable to structural changes due to the stresses of the aerosol formation step of aerosol jet printing while the collagen type II triple helix was largely unaffected. SEM illustrated that distinct layers remained in the aerosol jet print constructs. The results show that aerosol jet printing should be considered an effective way to process collagen type I and II into stiff dense constructs with suitable mechanical properties for the replication of dense collagenous connective tissues.

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