Bio-inks for 3D printing of Cartilage Implants: Tailoring gelMA and polyHPMA-lac-PEG hydrogels for the fabrication of spatially organized constructs

Abstract

A promising approach to treat cartilage defects is the implantation of stratified cell-laden hydrogel implants that mimic native cartilage. To fabricate such constructs, three-dimensional (3D) bioprinting techniques are promising, as they allow accurate deposition of (cell-laden) biomaterials, the so-called bio-inks, as well as biological cues and reinforcement structures. Several hydrogels have been suggested as bio-inks, including hydrogels based on gelatin-methacryloyl (gelMA) with gellan gum or triblock copolymers of polyethylene glycol (PEG) and partially methacrylated poly(N-(2-hydroxypropyl)methacrylamide mono/dilactate (polyHPMA-lac). However, tobioprintsuccessful constructs with a high resolution, the bio-ink properties are crucial. Therefore, the main aim was to investigate the application of gelMA and polyHPMA-lac-PEG based hydrogels, as bio-ink platforms for the 3D bioprinting of cell-laden organized cartilage implants. Here, the optimal cartilage bio-ink properties are based on the ability to print the material with high shape-fidelity and the ability of the material to support chondrogenesis. To accomplish this, the rheological properties governing the printability and cell encapsulation of gelMA/gellan hydrogels were investigated. Secondly, the possibility to improve the bio-ink properties of gelMA and polyHPMA-lac-PEG hydrogels, by the incorporation of additives or reinforcement was explored. More specifically, the bio-ink and construct properties after addition of gellan gum or methacrylated hyaluronic acid (HAMA) in gelMA hydrogels, and HAMA, methacrylated chondroitin sulfate (CSMA), or poly-ε-caprolactone (PCL) reinforcement in polyHPMA-lac-PEG hydrogels were investigated. Zone-specific matrix production was explored for chondrocytes, articular cartilage progenitor cells (ACPCs), and multipotent mesenchymal stromal cells (MSCs) in gelMA/gellan(/HAMA) hydrogels. Finally, the optimal spatial positioning of chondrocytes in hydrogel constructs for cartilage repair was investigated using an ex vivo osteochondral plug model. The feasibility of bioprinting and cell encapsulation with gelMA/gellan was governed by the yield stress. The printability and construct stiffness after crosslinking was enhanced for gelMA hydrogels by the incorporation of gellan gum and/or HAMA.These characteristics were improved for polyHPMA-lac-PEG hydrogels with the incorporation of HAMA or to a lesser extent CSMA. Co-printing of a hydrogel with PCL provided porous constructs with Young’s moduli comparable to those of native cartilage. The chondrogenic potential of embedded chondrocytes could only be improved with specific concentrations of HAMA. For the generation of stratified implants, ACPCs were found to be the most suitable cell type to produce superficial zone-like matrix. Additionally, the highest amount of cartilage-like tissue was produced by MSCs, which are therefore interesting for the fabrication of middle/deep zone cartilage. A homogeneous spatial cell distribution within hydrogel constructs was beneficial for defect filling with hyaline-like cartilage, while a dense cell layer at the bottom of the defect improved construct integration in full thickness cartilage defects in the plug model. Altogether, the work in this thesis resulted in two optimized cartilage bio-inks: gelMA/gellan/HAMA and polyHPMA-lac-PEG/HAMA hydrogels. Although several steps towards the bioprinting of organized cartilage implants have been made, some challenges still need to be overcome, such as finding the optimal combination of factors to stimulate zone-specific cartilage production by embedded cells. However, the results of this thesis encourage further development of organized cartilage implants using gelMA/gellan/HAMA and polyHPMA-lac-PEG/HAMA bio-inks.