Integrating 3D Bioprinting and Pluripotent Stem Cell Technologies for Articular Cartilage Tissue Engineering

Abstract

Osteoarthritis is a major cause of disability characterised by articular cartilage degeneration and pathological changes in the joints. Tissue engineering strategies address osteoarthritis through the combination of cells, biomaterials, and biochemical factors. Human pluripotent stem cells (hPSCs) are a promising cell source for articular cartilage tissue engineering due to their unique ability to differentiate into chondroprogenitors through the developmental route. However, protocols for hPSC chondrogenesis are limited due to the extensive use of growth factors, high complexity and variability of current methods, and lack of translation into 3D hydrogel culture systems. Moreover, combining hPSC chondrogenesis with technologies such as 3D bioprinting remains challenging, further limiting the ability to replicate the structural, functional, and mechanical organisation of native cartilage tissue. This project aimed to develop an articular cartilage model through the integration of hPSC and 3D bioprinting technologies. First, strategies for human embryonic stem cell (hESC) chondrogenesis were evaluated and a cost-efficient protocol was validated using transcriptomic and proteomic analyses. By controlling WNT and retinoic acid receptor signalling, it was possible to gain selective control over the expression of lateral plate mesoderm (HAND1, HAND2), limb bud (PRRX1, TBX5), and chondrogenic (SOX9, SOX5, COL2A1, ACAN) markers during differentiation. By eliminating the need for growth factor supplementation, it was also possible to significantly reduce the cost of differentiation. Then, alginate-based polymeric inks were developed and their rheological properties were evaluated. All inks displayed shear-thinning properties but had low levels of viscosity, which can be detrimental to retain shape fidelity after printing. Using oscillatory rheology, it was possible to determine that after gelation, 2% (w/v) alginate had the highest shear moduli, followed by 1% (w/v) alginate / 0.25% (w/v) collagen, and 1% (w/v) alginate hydrogels. To improve the shape fidelity of printed constructs without affecting cell viability, a suspended layer additive manufacturing (SLAM) strategy was adopted. Alginate-based inks could be processed using microvalve jetting and pressure-assisted extrusion. However, only pressure-assisted extrusion enabled bioprinting of cell aggregates with high cell viability. Then, hESC chondrogenic differentiation protocols were translated into 3D cell-laden hydrogel culture systems. Culture of hESC chondroprogenitors as cell aggregates (1000 cells) in hydrogels resulted in high levels of cell viability and chondrogenic gene expression. Finally, hESC chondroprogenitor cell aggregates were bioprinted using SLAM. This led to high levels of cell viability up to 14 days in 3D culture and the ability to generate constructs with different shapes, including patient-specific cartilage models produced by reverse engineering of human joint tissues. These findings suggest that the combination of hPSCs with 3D bioprinting can offer new opportunities for biofabrication of personalised articular cartilage tissue implants.

Date of Award	31 Dec 2022
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Michael Buckley (Supervisor), Susan Kimber (Supervisor) & Marco Domingos (Supervisor)