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

Liesbeth Tytgat,Lana Van Damme,Heidi Ottevaere,Hugo Thienpont,Fabrice Bray,Agnes Dobos,Jasper Van Hoorick,Marica Markovic,Sandra Van Vlierberghe,Peter Dubruel,Aleksandr Ovsianikov

doi:10.1021/acs.biomac.0c00386

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.

Highlights

Animal-derived gelatin, especially the methacrylamide-modified derivative (Gel-MA), can be considered the gold standard within the field of tissue engineering and biofabrication as reflected by numerous articles published to date.[1−5] the use of animal-derived materials has raised increasing concerns due to the occurrence of batch-to-batch variations resulting in issues with reproducibility as well as the possibility for disease transmission toward humans.[6−9] researchers have recently shifted their attention toward recombinant proteins as an attractive alternative
The primary amines of RCPhC1 were modified with either norbornene (RCPhC1-NB), or thiol (RCPhC1SH), or methacrylamide (RCPhC1-MA) functionalities using 5-norbornene-2-carboxylic acid, N-acetyl-homocysteine thiolactone, or methacrylic anhydride, respectively, to subsequently enable thiol-ene photoclick or chain-growth polymerization in the presence of a suitable photoinitiator
1H-NMR spectroscopy was applied to determine the degree of substitution (DS) of RCPhC1-NB and RCPhC1-MA by comparing the characteristic peaks of norbornene and methacrylamide with the reference peak corresponding to the hydrogen atoms of the chemically inert Leu, Ile, and Val amino acids at 1.0 ppm (Figure 4).[16]

Summary

Introduction

Animal-derived gelatin, especially the methacrylamide-modified derivative (Gel-MA), can be considered the gold standard within the field of tissue engineering and biofabrication as reflected by numerous articles published to date.[1−5] the use of animal-derived materials has raised increasing concerns due to the occurrence of batch-to-batch variations resulting in issues with reproducibility as well as the possibility for disease transmission toward humans.[6−9] researchers have recently shifted their attention toward recombinant proteins as an attractive alternative As these recombinant materials are produced in a controlled laboratory process, the drawbacks associated with animal-derived materials can be eliminated.[6,7] In literature, both transgenic crops and eukaryotic organisms are described to produce heterotrimeric human recombinant type I collagen, which can be used for tissue engineering purposes.[6,10] recombinant self-assembling peptides can be used as a biomaterial.[11] In the present study, a recombinant protein based on human collagen type I (RCPhC1) was modified to allow the light-based processing of the polymer. We explored the RCPhC1 processing via HD bioprinting using two-photon polymerization (2PP), while the effect of crosslinking chemistry on the 2PP processing capabilities was evaluated. 2PP is a HD 3D-printing technique that enables the production of scaffolds with a spatial resolution in the submicrometer range due to the nonlinear nature of the 2PP process, thereby often using near-infrared femtosecond pulsed

Methods

Results

Conclusion