Osteoarthritis is a leading cause of disability in older adults, currently with no resolutive solution. In-vitro models are powerful tools for studying tissue repair processes and developing potential treatments. Human Mesenchymal Stromal Cells (hMSCs) are often used in these models due to their ability to differentiate into chondrocytes when exposed to active Transforming Growth Factor Beta-1 (TGF-β1). Endogenous TGF-β1 is secreted in an inactive(latent) form and can be activated by mechanical loading (Li et al.2010;Gardner et al.2017). Additionally, 3D printing is an established technique for engineering scaffolds for in-vitro models (Armiento et al.2018). Polyurethane (PU) is a promising biomaterial with its low cell toxicity and excellent load-bearing capacity. This study aims to characterize 3D-printed PU scaffolds and test their performance for the activation of latent TGF-β1 under controlled mechanical loading, using a custom-made bioreactor. Scaffolds (8mm diameter, 4mm height) were punched from thermoplastic PU sheets with varying infill densities (15%,16.88%,19%,25%) and a gyroid infill pattern. Compression tests were performed (Instron 5866, Instron). Scaffolds were cleaned, sterilized, filled with cell-free fibrin, loaded in the bioreactor (20% dynamic compression at 2Hz, shear at 2Hz) for 6h covered with 2mL culture medium containing 50 ng/mL of latent TGF-β1. Active and total TGF-β1 was detected in the medium using ELISA. To evaluate cell metabolic activity, 16.88% scaffolds were seeded with 2.5 million hMSCs in fibrin and cultured for up to 14 days, in differentiation medium with or without exogenous TGF-β1. Quantification was done with Cell Titer Blue assay at different time points. The test was repeated for 3 separate donors. All tests were performed in triplicates. Statistical analysis was performed with GraphPad Prism. The mechanical tests revealed that scaffolds with higher infill densities exhibited increased stiffness, and for each infill percentage, higher compression led to greater stiffness. Active TGF-β1 concentrations were significantly higher in the loaded groups compared to the control groups, indicating that mechanical loading successfully activated latent TGF-β1. The 16.88% group was chosen due to its mechanical properties and it showed to not decrease metabolic activity of hMSCs over time, both in the presence and absence of TGF-β, suggesting low cytotoxicity of the scaffolds. In conclusion, 3D-printed PU scaffolds with tunable mechanical properties were effective in activating latent TGF-β1, establishing them as a promising candidate for in-vitro cartilage models. Ongoing research is focused on evaluating the ability of these scaffolds to promote mechanically induced chondrogenesis in hMSCs.
Mecchi et al. (Mon,) studied this question.