Three-dimensional cell culture systems are relevant in vitro models for studying cellular behavior. In this regard, this present study investigates the interaction between human osteoblast-like cells and 3D-printed scaffolds mimicking physiological and osteoporotic bone structures under simulated microgravity conditions. The objective is to assess the effects of scaffold architecture and dynamic culture conditions on cell adhesion, proliferation, and metabolic activity, with implications for osteoporosis research. Polylactic acid scaffolds with physiological (P) and osteoporotic-like (O) trabecular architectures were 3D-printed by means of fused deposition modeling technology. Morphometric characterization was performed using micro-computed tomography. Human osteoblast-like SAOS-2 and U2OS cells were cultured on the scaffolds under static and dynamic simulated microgravity conditions using a rotary cell culture system (RCCS). Scaffold biocompatibility, cell viability, adhesion, and metabolic activity were evaluated through Bromodeoxyuridine incorporation assays, a water-soluble tetrazolium salt assay, and an enzyme-linked immunosorbent assay of tumor necrosis factor-α secretion. Both scaffold models supported osteoblast-like cell adhesion and growth, with an approximately threefold increase in colonization observed on the high-porosity O scaffolds under dynamic conditions. The dynamic environment facilitated increased surface interaction, amplifying the effects of scaffold architecture on cell behavior. Overall, sustained cell growth and metabolic activity, together with the absence of detectable inflammatory responses, confirmed the biocompatibility of the system. Scaffold microstructure and dynamic culture conditions significantly influence osteoblast-like cell behavior. The combination of 3D-printed scaffolds and a RCCS bioreactor provides a promising platform for studying bone remodeling in osteoporosis and microgravity-induced bone loss. These findings may contribute to the development of advanced in vitro models for biomedical research and potential countermeasures for bone degeneration.

Background/Objectives: Three-dimensional cell culture systems are relevant in vitro models for studying cellular behavior. In this regard, the present study investigates the interaction between human osteoblast-like cells and 3D-printed scaffolds mimicking physiological and osteoporotic bone structures under simulated microgravity conditions. The objective is to assess the effects of scaffold architecture and dynamic culture conditions on cell adhesion, proliferation, and meta-bolic activity, with implications for both osteoporosis research and space medicine. Methods: Poly (lactic acid) (PLA) scaffolds with physiological (P) and osteoporotic-like (O) tra-becular architectures were 3D printed by means of the used deposition modeling (FDM) tech-nology. Morphometric characterization was performed using micro-computed tomography (µCT). Human osteoblast-like SAOS-2 and U2OS cells were cultured on the scaffolds under static and dynamic simulated microgravity conditions using a rotary cell culture system (RCCS). Cell viability, adhesion, and metabolic activity were evaluated through BrdU, WST-1, and ELISA assays, with TNF-α secretion assessed to determine biocompatibility. Results: Both scaffold models supported osteoblast-like cell adhesion and growth, with enhanced colonization observed on the high-porosity O scaffolds under dynamic conditions. The dynamic environment facilitated increased surface interaction, amplifying the effects of scaffold archi-tecture on cell behavior. No inflammatory response was detected, confirming scaffold biocom-patibility.