Speaker
Description
Physiologically relevant in vitro models of connective tissues are critical for advancing tissue engineering and disease modeling. However, replicating the hierarchical organization and extracellular matrix (ECM) richness of native stromal environments remains a significant challenge. Traditional scaffold-based approaches often lack the resolution and biological complexity to support proper tissue organization and maturation. In this study, we introduce a versatile extrusion-guided bioprinting strategy for the spatial organization and fusion of biofabricated connective microtissue precursors (µTPs) into structured, ECM-rich constructs. This bottom-up approach is compatible with stromal microtissues derived from different human sources, including pulmonary and breast fibroblasts, and enables the fabrication of scaffold-free tissues through controlled fusion post-printing.
Connective µTPs were generated by culturing primary human fibroblasts onto porous gelatin-based microcarriers under dynamic conditions in spinner flasks. After 18–20 days, the resulting microtissues reached sizes up to ~500 µm for lung-derived fibroblasts (L-µTPs) and ~800 µm for breast-derived fibroblasts (B-µTPs), with early ECM deposition evident in both cases. The µTPs were suspended in a 30% w/v thermoresponsive Pluronic® F127 hydrogel (P30) to create a printable bioink. This formulation demonstrated shear-thinning behavior, homogenous µTP distribution, and temperature-responsive gelation, ensuring filament stability and high print fidelity.
Bioprinting was performed using an extrusion-based system into a 40% w/v Pluronic® F127 support bath (P40), which provided mechanical support and promoted fusion of adjacent µTPs. The P40 bath exhibited self-healing and thixotropic properties, allowing the fabrication of stable, high-resolution constructs with minimal deformation. Multiple geometries were printed to assess fusion kinetics and structural remodeling. Post-printing, constructs were cultured dynamically for up to 28 days to support tissue maturation and ECM remodeling. Tissue fusion, compaction, and matrix organization were monitored via confocal microscopy, live/dead staining, second harmonic generation imaging, and immunofluorescence analysis for fibronectin and collagen.
Both L-µTP and B-µTP constructs exhibited rapid fusion behavior within the first week post-printing. Quantitative imaging confirmed progressive compaction and continuous tissue formation by day 8. Matrix deposition intensified over time, with increasing alignment along the print direction. Importantly, live/dead analysis revealed high cell viability (>90%) throughout the culture period. Structural remodeling was confirmed by second harmonic generation and mechanical stiffening of the constructs, consistent with functional ECM development. Fusion efficiency was closely linked to initial µTP proximity and pattern resolution, demonstrating the importance of extrusion control in directing construct architecture.
This work establishes a modular, scaffold-free strategy to fabricate engineered stromal tissues from biologically active microtissues. The platform is adaptable across tissue types and enables precise control over microtissue organization and fusion, key features for building complex connective tissue models. The method’s flexibility, printability, and biological performance support its potential use in both fundamental studies and translational applications, such as organ-on-chip systems and fibrotic disease modeling. By demonstrating this approach with lung and breast fibroblast-derived microtissues, we highlight its robustness and extendability for connective tissue engineering beyond a single anatomical context.
Work performed within BREATH project (CUP E53D23016840001) – Next Generation EU within the PNRR, Mission 4, Component 2, Investment 1.1, PRIN PNRR 2022 program (D.D. 1409 14/09/2022 MUR).
42705205679
