High-Resolution 3D Bioprinting of Photo-Cross-linkable Recombinant Collagen to Serve Tissue Engineering Applications

Abstract

Various biopolymers, including gelatin, have already been applied to serve a plethora of tissue engineering purposes. However, substantial concerns have arisen related to the safety and the reproducibility of these materials due to their animal origin and the risk associated with pathogen transmission as well as batch-to-batch variations. Therefore, researchers have been focusing their attention toward recombinant materials that can be produced in a laboratory with full reproducibility and can be designed according to specific needs (e.g., by introducing additional RGD sequences). In the present study, a recombinant protein based on collagen type I (RCPhC1) was functionalized with photo-cross-linkable methacrylamide (RCPhC1-MA), norbornene (RCPhC1-NB), or thiol (RCPhC1-SH) functionalities to enable high-resolution 3D printing via two-photon polymerization (2PP). The results indicated a clear difference in 2PP processing capabilities between the chain-growth-polymerized RCPhC1-MA and the step-growth-polymerized RCPhC1-NB/SH. More specifically, reduced swelling-related deformations resulting in a superior CAD-CAM mimicry were obtained for the RCPhC1-NB/SH hydrogels. In addition, RCPhC1-NB/SH allowed the processing of the material in the presence of adipose tissue–derived stem cells that survived the encapsulation process and also were able to proliferate when embedded in the printed structures. As a consequence, it is the first time that successful HD bioprinting with cell encapsulation is reported for recombinant hydrogel bioinks. Therefore, these results can be a stepping stone toward various tissue engineering applications.

3D Bioprinting of In Vitro Models Using Hydrogel-Based Bioinks

“…The use of hyaluronic acid as a bioink requires chemical modifications and/or mixing with other polymer materials, resulting in solidification of the fabricated 3D structure. Typical chemical modifications of hyaluronic acid for use as bioinks are methacrylation and thiolation to solidify the printed polymer solution [ 85 , 86 ]. In addition, hyaluronic acid has been mixed with other polymer materials (e.g., gelatin and β-cyclodextrin) to overcome the limitations of a single hyaluronic acid application [ 86 , 87 ].…”

Multiphoton Laser Fabrication of Hybrid Photo-Activable Biomaterials

“…Very recently, a thorough analysis of the possibility offered by a recombinant collagen type I protein (RCPhC1), endowed also with RGD sequences for cell adhesion, has been presented by the Wien group [ 129 ]. In this case, the protein was functionalized with photo-cross-linkable methacrylamide (RCPhC1-MA), norbornene (RCPhC1-NB), or thiol (RCPhC1-SH).…”

“…The cubes were printed using different concentrations and laser powers. Reprinted with permission from [ 129 ]. ( D ): Schematic showing two-photon cross-linking of HCC–hydrogel into skeletal muscle across epimysium (left).…”

Controlled Cell Alignment Using Two‐Photon Direct Laser Writing‐Patterned Hydrogels in 2D and 3D

“…We selected a poly(ethylene glycol) diacrylate (PEGDA)based photoresist as the cell-repellent photoresist and a methacrylated recombinant peptide (RCP)-based photoresist as the cell-adhesive photoresist due to the RGD motifs enriching the RCP. [24,25] DLW was performed in distilled water or 0.1X phosphate-buffered saline (PBS, pH 7.4) with the water-soluble photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP). [26] We first evaluated the mechanical properties of two-photon-polymerized PEGDA or RCP hydrogels as a function of laser scan speed.…”