Quantifying Cell-Derived Changes in Collagen Synthesis, Alignment, and Mechanics in a 3D Connective Tissue Model

Area of Science:

• Biomaterials Science
• Tissue Engineering
• Cell Biology

Background:

• Extracellular matrix (ECM) dysregulation is central to diseases like fibrosis and cancer.
• Existing in vitro models struggle to replicate the complex, hierarchical structure of native collagen-rich tissues.
• This limitation hinders accurate recapitulation of disease phenotypes in experimental settings.

Purpose of the Study:

• To develop a novel, scaffold-free 3D in vitro model for engineering fibrous connective tissues.
• To enable direct quantification of cell-derived ECM synthesis, organization, and mechanics.
• To investigate the impact of biomolecular perturbations on engineered tissue properties.

Main Methods:

• Primary human fibroblasts were cultured in custom 3D non-adhesive agarose molds to promote tissue morphogenesis.
• A multi-modal characterization approach including histology, multiphoton second-harmonic generation, and electron microscopy was used.
• The model allowed for the assessment of ECM synthesis, collagen alignment, and mechanical properties.

Main Results:

• Engineered 3D ring-shaped tissue constructs with native-like tensile and histological properties were successfully fabricated.
• Structural changes in collagen synthesis and alignment were correlated with functional differences in tissue mechanics and collagen content.
• The scaffold-free nature enabled direct quantification of cell-driven matrix changes in response to various biomolecular factors.

Conclusions:

• The developed 3D agarose mold system provides a robust platform for studying cell-matrix interactions in fibrous connective tissues.
• This model facilitates the direct assessment of how factors like nutrient composition and specific compounds influence ECM production and tissue mechanics.
• It offers a valuable tool for understanding disease mechanisms and for screening potential therapeutic interventions in fibrosis and cancer research.