Simple SummaryDevelopment of new therapeutics to treat osteosarcoma is fundamental to decreasing its current health impact. 3D in vitro models are gaining tremendous momentum as, compared to traditional 2D in vitro models and in vivo models, can speed up new treatment discovery and provide clarification of the pathology development, by ultimately offering a reproducible and biomimetic tool. However, engineering a 3D osteosarcoma in vitro model is challenging, since the reliability of the models strictly depends on their ability to correctly mimic the physical, mechanical, and biological properties of the pathological tissue to be replicated. Here, we designed 3D printed polyurethane scaffolds enriched by in vitro pre-generated bone extracellular matrix, synthesized by osteo-differentiated human mesenchymal stromal cells, to replicate in vitro an osteosarcoma model, which can be potentially used to study tumor progression and to assess new treatments.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.
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