Multi-material 3D Bioprinting of Complex Constructs for In-vitro Mechanobiology Studies in Pulmonary Fibrosis

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Abstract Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease characterized by progressive tissue stiffening, which modifies fibroblasts behavior and drives to disease progression. Previous mechanobiology studies have demonstrated that mechanical cues affect fibroblasts activation and ECM remodeling in the lung. However, current in-vitro models fail to capture the heterogeneous stiffness found in fibrotic lung tissue. Multi-material 3D bioprinting is a technology that enables the fabrication of physiological relevant constructs with different stiffness regions that can mimic in detail the mechanical heterogeneity of diseased tissue. This study aimed to create complex, heterogeneous constructs with stiffness variations to assess their potential as models for mechanobiology studies, focusing on human lung fibroblast (HLF) activation, morphology and density. We developed two- and three- material 3D bioprinted constructs with defined stiffness regions going from 1 to 35 kPa modulus. Gelatin methacrylate (GelMA) or poly(ethylene glycol) diacrylate (PEGDA) based biomaterial inks at different concentrations were used to control stiffness. To obtain rheological consistency for extrusion bioprinting, Carbopol (CBP) was incorporated to the inks formulations as a rheological modifier. Geometrically complex structures, including meshes and cylinders, were printed to demonstrate the versatility and precision of these inks to create multi-material constructs with regions of defined stiffnesses. We evaluated how material mechanical properties and single- vs multi-material constructs impacted HLF behaviour by culturing them on GelMA-three-material constructs, using α-SMA expression to assess fibroblast-to-myofibroblast transition. In parallel, F-actin and nuclear stains were used to observe changes in cell morphology and density. As expected, HLFs showed stiffness-dependent activation, with increased α-SMA expression in stiffer regions. Multi-material constructs also enhanced regional differences in fibroblast activation compared to the single-material ones. This suggests that fibroblasts in heterogeneous environments may respond more strongly to local stiffness gradients, potentially due to mechanical coupling or signaling across the different regions. These findings align with prior lung mechanobiology studies, reinforcing the role of ECM stiffness in fibroblast activation and disease progression in IPF. By comparing single- and multi-material systems, this study demonstrates the potential of multi-material 3D bioprinting for modeling lung fibrosis, highlighting the unique insights that can be accessed by having a heterogeneous model. This platform could provide a robust tool for disease modeling, drug screening, and therapeutic development for IPF.

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