Fully Defined 3D Hybrid System for Bone Tissue Engineering: Integration of MeHA–RGD/PCL–TCP Scaffolds With Human Stem Cells via 3D-Printed Vacuum-Assisted Cell Loading Device

Despite ongoing efforts, the regeneration of critical-sized bone defects remains a significant challenge for clinicians due to the absence of a standard clinically compliant bone tissue engineering protocol. These challenges are mostly attributed to the inadequacies of current methods, characterized by their high variability and the reliance on animal-derived components, such as fetal bovine serum (FBS) in cell culture. To address these shortcomings, our approach diverges from conventional practices by prioritizing consistency and reproducibility, and the complete elimination of animal derivatives throughout the entire process. We have developed a novel method that utilizes a peptide-functionalized photocrosslinkable methacrylated hyaluronic acid (MeHA–RGD) hydrogel as a cell sealant for loading human adipose-derived stem cells (hASCs) into a 3D porous polycaprolactone–tricalcium phosphate (PCL–TCP) scaffold. Additionally, we created a 3D-printed vacuum-assisted cell loading device to facilitate this process and ensure efficiency and consistency during cell loading. Our findings indicate that the MeHA–RGD hydrogel supports both stem cell viability and osteogenic differentiation, demonstrating outcomes comparable to those achieved with fibrin glue, a conventional cell sealant widely used in BTE from autologous or xenogeneic sources, even under serum- and xeno-free conditions. In the pursuit of clinical translation, it is vital that biomaterials exhibit low variability, are easily accessible, readily available, and completely free of animal derivatives. To our knowledge, this is the first study to employ a 3D-printed vacuum-assisted cell loading device system within a fully defined hybrid 3D system under complete serum- and xeno-free conditions. These findings unravel and encourage alternative approaches in addressing the existing challenges in BTE, thereby facilitating and accelerating clinical translation in the future.