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1. Stiffness-matched extracellular matrices comprising collagen or fibrin differentially affect cell-mediated remodeling and peri-cellular stiffness

   https://doi.org/10.21203/rs.3.rs-1348112/v1  

   Jagiełło, A. ; Castillo, U. ; Botvinick, E. ( February 2022 , Research square)

   Cells are known to continuously remodel their local extracellular matrix (ECM) and in a reciprocal way, they can also respond to mechanical and biochemical properties of their fibrous environment. In this study, we measured how stiffness around dermal fibroblasts (DFs) and human fibrosarcoma HT1080 cells differs with concentration of rat tail type 1 collagen (T1C) and type of ECM. Peri-cellular stiffness was probed in four directions using multi-axes optical tweezers active microrheology (AMR). First, we found that neither cell type significantly altered local stiffness landscape at different concentrations of T1C. Next, rat tail T1C, bovine skin T1C and fibrin cell-free hydrogels were polymerized at concentrations formulated to match median stiffness value. Each of these hydrogels exhibited distinct fiber architecture. Stiffness landscape and fibronectin secretion, but not nuclear/cytoplasmic YAP ratio differed with ECM type. Further, cell response to Y27632 or BB94 treatments, inhibiting cell contractility and activity of matrix metalloproteinases, respectively, was also dependent on ECM type. Given differential effect of tested ECMs on peri-cellular stiffness landscape, treatment effect and cell properties, this study underscores the need for peri-cellular and not bulk stiffness measurements in studies on cellular mechanotransduction. 

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2. Dermal fibroblasts and human breast cancer cells differentially stiffen their local matrix

   Jagiello, A ; Lim, M. ; Botvinick, E ( July 2021 , EUROPEAN BIOPHYSICS JOURNAL WITH BIOPHYSICS LETTERS)

   Bulk ECM stiffness measurements are often used in research on cell mechanobiology. However, past studies by our group have shown that peri-cellular stiffness can span few orders of magnitude and diverges from the bulk properties. Us- ing optical tweezers active microrheology (AMR) we can de- scribe stiffness landscape around individual cells. In this study, we show how different cell lines cultured in 1.0 and 1.5 mg/ml type 1 collagen (T1C) create disparate patterns of peri-cellular stiffness. Dermal fibroblasts (DFs) increase peri-cellular stiffness, when embedded in 1.0 mg/ml T1C hy- drogels, but do not alter stiffness in 1.5 mg/ml T1C hydro- gels. In contrast, invasive human breast cancer MDA-MB- 231 cells (MDAs) do not significantly change the stiffness of T1C hydrogels, as compared to cell-free controls. Results indicate that while MDAs adapt to the bulk ECM stiffness, DFs regulate local stiffness to levels they intrinsically “fa- vor”. Further, cells were also subjected to treatments that were previously shown to regulate their migration, prolifera- tion and contractility. Following each treatment, cells estab- lished dissimilar stiffness patterns. Stiffness magnitude and extent of anisotropy varied with the cell line, T1C concen- tration and treatment. In summary, we demonstrate that AMR can reveal otherwise masked mechanical properties of the local ECM, which are known to affect cell behavior. 

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3. Cells utilize strain hardening and crosslinking to establish their extracellular niche in fibrous tissue

   Jagiello, A. ; Lim, M. ; Botvinick, E. ( March 2021 , APS March Meeting 2021)

   Bulk measurements of ECM stiffness are commonly used in mechanobiology. However, peri-cellular stiffness can be quite heterogenous and divergent from the bulk properties. Here, we use optical tweezers active microrheology (AMR) to quantify how two different cell lines embedded in 1.0 and 1.5 mg/ml type 1 collagen (T1C) establish dissimilar patterns of peri-cellular stiffness. We found that dermal fibroblasts (DFs) increase local stiffness of 1.0 mg/ml T1C hydrogels, but surprisingly do not alter stiffness of 1.5 mg/ml T1C hydrogels. In contrast, MDA-MB-231 cells (MDAs) predominantly do not stiffen T1C hydrogels, as compared to cell-free controls. Results suggest that MDAs adapt to the bulk ECM stiffness, while DFs regulate local stiffness to levels they intrinsically “prefer”. Further, cells were subjected to treatments, that were previously shown to alter migration, proliferation and contractility of DFs and MDAs. Following treatment, both cell lines established different levels of stiffness magnitude and anisotropy, which were dependent on the cell line, T1C concentration and treatment. In summary, our findings demonstrate that AMR reveals otherwise masked mechanical properties such as spatial gradients and anisotropy, which are known to affect cell behavior at the macro-scale. 

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4. Effects of Cell-Adhesive Ligand Presentation on Pentapeptide Supramolecular Assembly and Gelation: Simulations and Experiments

   https://doi.org/10.1159/000534280  

   Thede, Andrew T. ; Tang, James D. ; Cocker, Clare E. ; Harold, Liza J. ; Amelung, Connor D. ; Kittel, Anna R. ; Taylor, Phillip A. ; Lampe, Kyle J. ( September 2023 , Cells Tissues Organs)

   The extracellular matrix (ECM) is a complex, hierarchical material containing structural and bioactive components. This complexity makes decoupling the effects of biomechanical properties and cell-matrix interactions difficult, especially when studying cellular processes in a 3D environment. Matrix mechanics and cell adhesion are both known regulators of specific cellular processes such as stem cell proliferation and differentiation. However, more information is required about how such variables impact various neural lineages that could, upon transplantation, therapeutically improve neural function after a central nervous system injury or disease. Rapidly Assembling Pentapeptides for Injectable Delivery (RAPID) hydrogels are one biomaterial approach to meet these goals, consisting of a family of peptide sequences that assemble into physical hydrogels in physiological media. In this study, we studied our previously reported supramolecularly-assembling RAPID hydrogels functionalized with the ECM-derived cell-adhesive peptide ligands RGD, IKVAV, and YIGSR. Using molecular dynamics simulations and experimental rheology, we demonstrated that these integrin-binding ligands at physiological concentrations (3–12 mm) did not impact the assembly of the KYFIL peptide system. In simulations, molecular measures of assembly such as hydrogen bonding and pi-pi interactions appeared unaffected by cell-adhesion sequence or concentration. Visualizations of clustering and analysis of solvent-accessible surface area indicated that the integrin-binding domains remained exposed. KYFIL or AYFIL hydrogels containing 3 mm of integrin-binding domains resulted in mechanical properties consistent with their non-functionalized equivalents. This strategy of doping RAPID gels with cell-adhesion sequences allows for the precise tuning of peptide ligand concentration, independent of the rheological properties. The controllability of the RAPID hydrogel system provides an opportunity to investigate the effect of integrin-binding interactions on encapsulated neural cells to discern how hydrogel microenvironment impacts growth, maturation, or differentiation.

    

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5. Using light to establish and measure stiffness gradients in three-dimensional engineered tissues

   https://doi.org/10.1117/12.2595590  

   Jagiello, A ; Hu, Q ; Castillo, U ( August 2021 , spie: Optical Trapping and Optical Micromanipulation XVIII)

   Studies of cell-extracellular matrix (ECM) interactions within fibrous systems such as collagen or fibrin are challenging, particularly if peri-cellular stiffness cannot be monitored. Here we present our light-based method for non-invasive patterning of molecular crosslinking combined with multi-axes optical tweezers active microrheology to map ECM stiffness landscapes. This method allows us to generate prescribed stiffness gradients and associated anisotropies, which model stiffness of the natural peri-cellular ECM. Patterned crosslinking induces strain hardening and measured stiffness gradients are in agreement with predicted strain fields. Migratory cells respond to these gradients as assessed by change in F-actin distribution and morphological properties. 

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