Mechanical Maturation of Human Dermal Fibroblast-Laden Microporous Annealed Particle Scaffolds During Long-Term in Vitro Culture

Abstract

The use of biomaterials to model diseases and tissues in in vitro cultures has expanded and updated our understandings of many cellular processes through the inclusion of a 3D microenvironment that better mimics in vivo conditions. A major limitation that remains, though, is the ability to probe and monitor these cultures on a longer timescale while still maintaining a simple, reproducible 3D-structure. Porous scaffolds, particularly microporous annealed particles (MAP) scaffolds, demonstrate potential in circumventing some drawbacks of traditional bulk hydrogels, including lowered diffusivity of nutrients and early inhibition of cell-to-cell interactions, that may limit efficacy over long timescales. Previous research explored the impact of degradation and cell-adhesion ligands on MAP’s ability to promote the creation of a cell-mediated extracellular matrix (ECM) by human mesenchymal stem cells (hMSCs) within its porous network over an 8-day period. This manuscript expands on this work by culturing a high cellular density of human dermal fibroblasts (hDFs) in poly(ethylene glycol) (PEG) MAP scaffolds over 63 days. Comparing two iterations of MAP, including an enzymatically insensitive formulation (PEG-PEG) and an enzymatically degradable formulation (PEG-peptide) mediated by inclusion of a matrix metalloprotease (MMP) sensitive peptide linker, we evaluated mechanical maturation and viability over time. We note a significant increase in the modulus of PEG-peptide at 28 days, no difference in the modulus of PEG-PEG, and sustained viability for both conditions up to 63 days. To further probe for mechanistic differences, bulk RNAseq was performed at 4, 28, and 63 days. Importantly, many ECM-related genes were highlighted as highly significant to the overall model, such as fibronectin and collagen 1 and 12. Furthermore, the gene profile of PEG-peptide scaffolds diverged from that of PEG-PEG scaffolds at 28 days, indicated through PCA analysis and likely attributed to many of those ECM-related genes. The genomic results both corroborate differences found in mechanical maturation of these systems and delineate a much longer timescale for observed cellular behavior changes to these material types. Overall, this study demonstrates the ability to use MAP for long-term in vitro cultures with sustained viability and begins to question optimization of formulation for these scaffolds, such as the inclusion of peptide motifs and linkers, that may better promote material-tissue hybridization. Future work will further analyze the mechanism for the different mechanical and genomic profiles, including decoupling degradability from material composition (PEG vs. peptide).

Keywords: Microporous annealed particle (MAP) scaffold, Extracellular Matrix, Enzymatic degradation, Mechanical maturation, Long-term 3D culture, Human dermal fibroblasts, Transcriptomics, Tissue Engineering