The first steps toward the creation of a bioink for endochondral bone regeneration

Abstract

The treatment of critical-sized bone defects poses significant challenges as these defects often require medical intervention. The gold standard treatment, autologous bone grafts, has drawbacks like donor site morbidity and limited tissue availability. In response, the field of bone tissue engineering (BTE) emerged, aiming to develop biological substitutes or scaffolds utilizing biomaterials, cells, and growth factors to regenerate bone. However, challenges such as insufficient vascularization still hinder clinical translation. Nonetheless, endochondral bone regeneration (EBR) is a BTE approach that holds promise. It involves mimicking a soft callus to serve as template for bone regeneration.

This thesis focuses on advancing the EBR research of our group by developing a bioink containing chondrogenic micro pellets. A microwell system was selected to generate micro pellets, which were cultured across four mesenchymal stem cell donors. Seven different cell densities (1500, 3000, 6000, 12000, 24000, 36000, and 50,000 cells per micro pellet) and a control (250,000 cells per macro pellet) were analyzed for size and chondrogenic differentiation with a safranin-O, toluidine blue, and collagen II staining. In continuation, micro pellets were combined with Gelatin Methacryloyl (GelMA) to create a bioink, which was cast and volumetrically printed to demonstrate functionality by fabricating an incus bone shape.

The microwell system of the Inge Zuhorn group was chosen for further optimization instead of microwell stamps. Micro pellets under 6000 cells displayed limited to no GAG and collagen II deposition. Above 6000 cells, micro pellets demonstrated unwanted aggregation but exhibited complete GAG and collagen II deposition. Due to these issues, a second bioink, composed of crushed macro pellets and GelMA, was formulated. The macro pellets were effectively crushed and integrated into bioink and used to volumetrically print an incus bone.

In conclusion, a microwell system was selected, the initial steps to optimize micro pellet culture were taken, and a successful bioink for EBR was formulated and printed. Optimizing and standardizing chondrogenic micro pellet culture, bioink formulation, and printing through further experimentation is recommended for future applications.