Abstract
Osteosarcoma, among all bone sarcomas, remains a challenge despite the unwavering efforts of medical professionals and scientists. To address this, the scientific community is actively pursuing the development of three-dimensional (3D) in vitro models to faithfully replicate the heterogeneity of osteosarcoma, thereby facilitating the reliable preclinical screening of potential therapies. In this study, we present the latest advancements in engineering an in vitro 3D osteosarcoma model comprising enriched Cancer Stem Cells (CSCs) and a hybrid hydroxyapatite-based scaffold (MgHA/CoII). The improvement of the model occurred through two primary steps: (1) serial passaging of sarcospheres as the CSCs enrichment system and (2) the optimization of the structural configuration of the niche in the scaffold. Two injection-mediated approaches of sarcosphere seeding were designed and extensively characterized in vitro and in vivo Chorioallantoic Membrane (CAM) models to explore their biological properties and tumorigenic potential. The combination of the selected enriched-CSCs and custom-made seeding into the scaffold resulted in the development of 3D osteosarcoma models exhibiting tumor-like features in vitro and tumorigenic properties in vivo. The outcomes of this study offer prospects for future endeavors involving more complex systems capable of replicating specific malignant tumor behaviors (metastatic process and drug resistance), pushing the discovery of new therapeutic strategies for clinical applications.