Mechanical maturation of human dermal fibroblast-laden microporous annealed particle scaffolds during long-term in vitro culture.

The use of biomaterials to model diseases and tissues in vitro has updated our understandings of cellular processes through the inclusion of a 3D microenvironment that better mimics physiological conditions. A major limitation that remains is the ability to probe these cultures on longer timescales while maintaining a simple, reproducible 3D-structure. Microporous annealed particle (MAP) scaffolds demonstrate potential in circumventing some drawbacks of traditional bulk hydrogels, including lowered nutrient diffusivity and early inhibition of cell-to-cell interactions, that may limit efficacy over long timescales. Previous research explored impacts of degradation and cell-adhesion ligands on MAP's ability to promote creation of cell-mediated extracellular matrix (ECM) within its porous network over an 8-day period. This manuscript expands on this work by culturing a high density of human dermal fibroblasts in poly(ethylene glycol) (PEG) MAP scaffolds over 63 days. We evaluated mechanical maturation and viability over time between enzymatically insensitive (PEG-PEG) and sensitive (PEG-peptide) formulations of MAP. Mechanical modulus significantly increased in PEG-peptide scaffolds at 28 days, but viability was sustained for both conditions up to 63 days. Bulk RNAseq was performed on both conditions at equivalent timepoints, with many ECM-related genes highlighted as significant to the overall model and largest differences in gene profiles between conditions occurring at 28 days, corroborating mechanical data. Overall, this study demonstrates MAP's use for long-term in vitro cultures with sustained viability and begins to optimize scaffold formulation to promote material-tissue hybridization. Future work will further analyze mechanisms for the differential behaviors, including decoupling degradability from material composition. STATEMENT OF SIGNIFICANCE: Three-dimensional in vitro cultures better mimic the physiological environment compared to traditional 2D cultures, but extending their lifetime remains challenging. Microporous annealed particle (MAP) scaffolds address limitations of bulk hydrogels by providing a porous microenvironment that enables immediate cell migration and proliferation without extensive polymer breakdown. This study demonstrates sustained viability and mechanical maturation of human dermal fibroblasts in MAP scaffolds for over 63 days - significantly longer than previous reports. We demonstrate that protease-sensitive peptide crosslinkers, compared to protease-insensitive alternatives, lead to enhanced ECM deposition and mechanical maturation, supported by transcriptomic evidence. These findings establish MAP scaffolds as a platform for long-term in vitro modeling of tissue development and potential engineering of tissues for transplantation.