Bioprinting of human dermal microtissues precursors as building blocks for endogenous in vitro connective tissue manufacturing.

The advent of 3D bioprinting technologies in tissue engineering has unlocked the potential to fabricate in vitro tissue models, overcoming the constraints associated with the shape limitations of preformed scaffolds. However, achieving an accurate mimicry of complex tissue microenvironments, encompassing cellular and biochemical components, and orchestrating their supramolecular assembly to form hierarchical structures while maintaining control over tissue formation, is crucial for gaining deeper insights into tissue repair and regeneration. Building upon our expertise in developing competent three-dimensional tissue equivalents (e.g., skin, gut, cervix), we established a two-step bottom-up approach involving the dynamic assembly of microtissue precursors (μTPs) to generate macroscopic functional tissue composed of cell-secreted extracellular matrix (ECM). To enhance precision and scalability, we integrated extrusion-based bioprinting technology into our established paradigm, to automate, control and guide the coherent assembly of μTPs into predefined shapes. Compared to cell-aggregated bioink, our μTPs represent a functional unit where cells are embedded in their specific ECM. μTPs were derived from human dermal fibroblasts (HDF) dynamically seeded onto gelatin-based microbeads. After 9-days, μTPs were suspended (50%v/v) in Pluronic-F127 (30%w/v) (µTP:P30), and the obtained formulation was loaded as bioink into the syringe of the Dr.INVIVO-4D6 extrusion based bioprinter. µTP:P30 bioink showed shear-thinning behavior and temperature-dependent viscosity (gel at T>30°C), ensuring µTPs homogenous dispersion within the gel and optimal printability. The bioprinting involved extruding several geometries (line, circle, and square) into Pluronic-F127 (40%w/v) (P40) support bath, leveraging its shear-recovery property. P40 effectively held the bioink throughout and after the bioprinting procedure, until µTPs fused into a continuous connective tissue. µTPs fusion dynamics was studied over 8-days of culture, while the resulting endogenous construct underwent 28-days culture. Histological, immunofluorescence analysis, and Second Harmonic Generation (SHG) reconstruction revealed an endogenous collagen and fibronectin increased production within the bioprinted construct, closely resembling the native connective tissue composition.