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Osteosarcoma is a primary bone tumor characterized by a dismal prognosis, especially in the case of recurrent disease or metastases. Therefore, tools to understand in-depth osteosarcoma progression and ultimately develop new therapeutics are urgently required. 3D in vitro models can provide an optimal option, as they are highly reproducible, yet sufficiently complex, thus reliable alternatives to 2D in vitro and in vivo models. Here, we describe 3D in vitro osteosarcoma models prepared by printing polyurethane (PU) by fused deposition modeling, further enriched with human mesenchymal stromal cell (hMSC)-secreted biomolecules. We printed scaffolds with different morphologies by changing their design (i.e., the distance between printed filaments and printed patterns) to obtain different pore geometry, size, and distribution. The printed PU scaffolds were stable during in vitro cultures, showed adequate porosity (55–67%) and tunable mechanical properties (Young’s modulus ranging in 0.5–4.0 MPa), and resulted in cytocompatible. We developed the in vitro model by seeding SAOS-2 cells on the optimal PU scaffold (i.e., 0.7 mm inter-filament distance, 60° pattern), by testing different pre-conditioning factors: none, undifferentiated hMSC-secreted, and osteo-differentiated hMSC-secreted extracellular matrix (ECM), which were obtained by cell lysis before SAOS-2 seeding. Scaffolds pre-cultured with osteo-differentiated hMSCs, subsequently lysed, and seeded with SAOS-2 cells showed optimal colonization, thus disclosing a suitable biomimetic microenvironment for osteosarcoma cells, which can be useful both in tumor biology study and, possibly, treatment.