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Macrophages are phagocytic innate immune cells capable of phenotypical switching in response to the local microenvironment. Studies often use either primary macrophages or immortalized cell lines for hypothesis testing, therapeutic assessment, and biomaterial evaluation without carefully considering the potential effects of cell source and tissue of origin, which strongly influence macrophage response. Surprisingly, limited information is available about how, under similar stimuli, immortalized cell lines and primary cells respond in both phenotypical and functional changes. To address this need, in this work, we cultured immortalized macrophage cell lines derived from different origins (i.e.,blood, lung, peritoneal) to understand and compare macrophage phenotypical responses, including polarization and plasticity, morphological changes, and phagocytic functionalities, as well as compared primary macrophages extracted from peritoneal and bone marrow to their immortalized cell line counterparts. We found significant differences in baseline expression of different markers (e.g., CD86, MHCII, CD206, and EGR2) amongst different cell lines, which further influence both polarization and repolarization of the cells, in addition to their phagocytic functionality. Additionally, we observed that, while RAW 264.7 cells behave similarly to the primary bone marrow-derived macrophages, there are noticeable phenotypical and functional differences in cell line (IC-21) and primary peritoneal macrophages, highlighting tissue-specific differences in macrophage response amongst cell lines and primary cells. Moving to three-dimensional (3D) culture in well-defined biomaterials, blood-derived primary and cell line macrophages were encapsulated within hydrogel-based synthetic extracellular matrices and their polarization profiles and cell morphologies were compared. Macrophages exhibited less pronounced polarization during 3D culture in these compliant, soft materials compared to two-dimensional (2D) culture on rigid, tissue culture plastic plates. Overall, our findings highlight origin-specific differences in macrophage response, and therefore, careful considerations must be made to identify the appropriate cell source for the application of interest. 

Collagen-like peptide heterotrimers are computationally designed to create percolated networks as a function of solvent quality and multifunctional materials of interest to the biomaterials community. 

Assembling peptides allow the creation of structurally complex materials, where amino acid selection influences resulting properties. We present a synergistic approach of experiments and simulations for examining the influence of natural and non-natural amino acid substitutions via incorporation of charged residues and a reactive handle on the thermal stability and assembly of multifunctional collagen mimetic peptides (CMPs). Experimentally, we observed inclusion of charged residues significantly decreased the melting temperature of CMP triple helices with further destabilization upon inclusion of the reactive handle. Atomistic simulations of a single CMP triple helix in explicit water showed increased residue-level and helical structural fluctuations caused by the inclusion of the reactive handle; however, these atomistic simulations cannot be used to predict changes in CMP melting transition. Coarse-grained (CG) simulations of CMPs at experimentally relevant solution conditions, showed, qualitatively, the same trends as experiments in CMP melting transition temperature with CMP design. These simulations show that when charged residues are included electrostatic repulsions significantly destabilize the CMP triple helix and that an additional inclusion of a reactive handle does not significantly change the melting transition. Based on findings from both experiments and simulations, the sequence design was refined for increased CMP triple helix thermal stability, and the reactive handle was utilized for the incorporation of the assembled CMPs within covalently crosslinked hydrogels. Overall, a unique approach was established for predicting stability of CMP triple helices for various sequences prior to synthesis, providing molecular insights for sequence design towards the creation of bulk nanostructured soft biomaterials.