Cell size is known to mediate the cell’s ability to sense external chemical signals by modulating ligand-receptor interactions (57, 58). The inflammatory cytokine TNFα binds to MSCs and subsequently upregulates genes that modulate the innate immune system, including the chemokine CCL2 (59), which is an important determinant of immunosuppression by therapeutic MSCs (60, 61). Thus, we tested the effect of matrix post spacing, stiffness and heterogeneity on CCL2 expression in MSCs upon TNFα activation. In mechanically homogeneous scaffolds, MSCs in scaffolds with far spacing show 2-fold higher CCL2 upregulation by TNFα than MSCs in scaffolds with close spacing or on 2D substrates—
regardless of stiffness (Fig. 14A). In contrast, MSCs in mixed scaffolds with close spacing show 2-fold higher TNFα-induced CCL2 upregulation than MSCs in soft or stiff-only scaffolds. The basal level of CCL2 remains unchanged across different scaffolds (Fig. 14C). As a result, TNFα-induced CCL2 expression follows a step function with average cell volume (Fig. 14B, i) and with fold change in cell volume by myosin-II inhibition (Fig. 14B, ii). Y27632 does not impact CCL2 expression in mixed scaffolds with close spacing (Fig. 14D). Thus, combining soft and stiff matrix signals maximizes inflammatory activation of MSCs under confinement.
38
Figure 14. TNFα-mediated upregulation of CCL2 is maximized under confinement when MSCs are presented with both soft and stiff matrix signals. (A) CCL2 expression in MSCs on plastic (“P”), 2D PEG gels and microscale post scaffolds with varied stiffness (Stiff: “St”, Soft:
“So”) and spacing as quantified by qPCR after 72h TNFα stimulation. Transcriptional level of CCL2 was was normalized to GAPDH in each sample. Error bars represent the SEM of the mean values across n ≥ 3 independent experiments with a single donor. *p < 0.05, ordinary one-way ANOVA followed by Tukey multiple comparison testing between conditions. (B) Dose response of TNFα-activated CCL2 expression as a step function (cooperativity ∞) of (i) average cell volume, and (ii) fold change in volume due to myosin-II inhibition. (A) The basal CCL2 expression in MSCs on 2D PEG gels and micropost scaffolds as quantified by qPCR after 72h culture without TNFα stimulation. CCL2 expression in each sample was normalized to GAPDH expression. Error bars represent the SEM between biological replicates; n ≥ 3 experiments with a single donor for each experimental condition. (B) CCL2 8 expression in MSCs on mixed
scaffolds in the presence or absence of blebbistatin as quantified by qPCR. Error bars represent the SEM between biological replicates; n = 4 experiments with a single donor. *p < 0.05, ordinary one-way ANOVA followed by Tukey multiple comparison testing between conditions.
* *
* *
39 2.4 Discussion
The freeform stereolithography approach enables precise control of microscale
mechanical properties by curing one matrix post at a time to fabricate a hydrogel scaffold. Our approach is distinct from previous studies with other micropatterned substrates and bulk engineered hydrogels in that cells can be spatially confined and receive distinct mechanical signals simultaneously. Fabricating arrays of posts that serve as distinct matrix signal units has enabled us to derive a quantitative relationship between cell-matrix interactions and cellular phenotypes. By leveraging this approach, we reveal diverse changes in cell size as a function of matrix mechanics, spatial confinement, mechanical heterogeneity, and myosin-II contractile forces—and their implications in communication with external soluble signals in the context of inflammatory activation (Fig. 15).
40
Figure 15. Cell size and inflammatory activation as a function of spatial confinement, matrix mechanics, mechanical heterogeneity, and myosin-II contractility. (A) Schematic illustration of the results. (B) Representative confocal images after 3D reconstruction. Dark red:
stiff post, light red: soft post, green: human MSC labeled with CellMask plasma membrane stain.
Studies with 3D nanoporous hydrogels show that matrix degradation or stress relaxation plays a dominant role in mediating cell volume expansion (48, 49). In contrast, our approach controls degree of spatial confinement without material degradation or stress relaxation—thereby preserving the material’s intrinsic mechanical properties. Using this approach, we show that in microporous environments, stiffness of elastic posts determines the sensitivity of cell size to spatial confinement. While it is known that cells on soft 2D or in 3D nanoporous hydrogels
41
generate low contractile forces (42), we show that myosin-II contractility is essential for cells to interact with posts and bend them in soft scaffolds. This observation is consistent with a previous study that used electrospinning to show that cells recruit more matrix fibers when they are softer to increase spreading (9). At the molecular level, our results further suggest that ROCK, an upstream regulator of myosin-II, may tune myosin-II activity to deform posts, likely via phosphorylation of myosin-II light chain (62), while myosin-II activity itself is essential to maintain the number of interacting posts when cell size increases. The results raise a possibility that different regulators of myosin-II could play distinct roles in adhesion to and deformation of matrix posts in soft scaffolds.
We show a surprising nonlinear effect of combining soft and stiff matrix signals on cooperatively increasing cell size: cell volume (V) ~ interacting post number (n)1.3. This leads to a fundamental question of how cells meet metabolic demands as they increase in size in different environments (63). Coincidentally, the scaling exponent 1.3 is close to the inverse of 0.75—the scaling exponent of Kleiber’s law: metabolic rate (B) ~ mass (M)0.75 (64). Combining our equation with Kleiber’s law leads to: B ~ n.(M/V)0.75 ~ n.(intracellular mass density)0.75 for mixed scaffolds. On the other hand, B ~ n0.75.(intracellular mass density)0.75 for soft or stiff-only scaffolds, since V ~ n1.0. This analysis suggests increased metabolic demands, such as ATP production, when cells undergo hyperallometric growth to generate sufficient force to bend stiff posts in mixed scaffolds. This mode of cell size regulation appears to be designed to distribute myosin-II contractility to bend stiff posts while keeping soft posts bent, most likely by
polarization of myosin-II contractility towards stiff posts as observed previously in durotaxis (44). In contrast to soft posts where myosin-II inhibition decreases either post bending or the number of interacting posts by MSCs, ROCK inhibition decreases stiff post bending but
42
increases the number of interacting posts while maintaining cell size. This is reminiscent of a recent study showing that matrix stiffness increases cell area independently of myosin-II but by force generation from actin polymerization (65). Thus, cells may still be able to utilize actin polymerization or other means of force generation, such as membrane associated myosin-I (66) when they interact with stiff posts in mixed scaffolds to increase cell size in the absence of myosin-II contractility.
When cells are spatially confined, they undergo changes in plasma membrane surface area and chromatin modification (67), both of which can influence the ability of cells to respond to external soluble signals via ligand-receptor interactions. In the context of inflammation, previous studies show that fibroblasts cultured on circular micropatterned 2D substrates exhibit low myosin contractility and transient response to TNFα (68), while macrophages under spatial confinement exhibit reduced actin polymerization and activation of macrophages by
lipopolysaccharide (LPS) (50). Our results not only support these findings, but also suggest that combining soft and stiff matrix signals helps cells maximize inflammatory activation under spatial confinement. Thus, one functional role of the synergy between soft and stiff matrix signals is to switch on maximal inflammatory activation without a need to remodel the matrix.
Since MSCs have been tested clinically because of their ability to modulate immune cells (69), our results reveal the potential utility of engineered scaffolds with alternating soft and stiff matrix signals in maximizing immunomodulatory functions of MSCs while retaining them within scaffolds. The results also suggest that mechanical heterogeneity, as observed in the bone marrow microenvironment (52), is likely a key attribute to optimize inflammatory activation of marrow-resident MSCs that can impact monocyte emigration in response to remote infections (59).
43 2.5 Conclusion
Together, this study presents a freeform stereolithography approach that can be applied to systematically control degree of spatial confinement and mechanics of matrix signals at the microscale. Future efforts in printer design may be directed toward fabrication of structures at higher spatial and structural resolution, and investigating functionalization with other
biomolecules. Additionally, adapting the printing platform to directly print scaffolds in multi-well plates would scale up fabrication efficiency and increase the throughput of downstream applications—including tissue patterning, drug screening and other parallel experiments.
The findings with MSCs emphasize mechanical cooperativity between soft and stiff matrix signals as a key mediator of cell size and inflammatory activation under confinement, and offer insight into how biomaterials can be designed to tune immunomodulatory functions of MSCs. Future efforts seek to investigate the directionality of cell contractile forces in response to different stiffness patterns to regulate volume. We also aim to define the myosin-II independent mechanisms involved in MSC interaction with heterogeneous mechanical input, and how they impact downstream cell functions. This study opens new avenues of investigation into how distinct matrix biophysical properties work together to influence other aspects of
mechanobiology, such as durotaxis under confinement and mechanically directed stem cell differentiation.
44
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48 VITA
NAME Daniel M. Devine
EDUCATION
Master of Science in Bioengineering, University of Illinois at Chicago, expected graduation Dec. 2019
Bachelor of Science in Biology – Cellular & Molecular, University of Wisconsin – La Crosse, May 2015
EXPERIENCE & SKILLS
Research Assistant, University of Illinois at Chicago, Mar. 2018 – Sep.
2019.
Designed platform for printing hydrogel micropost arrays while
controlling mechanical properties of individual posts. Used scaffolds to study the independent effects of post stiffness and spacing on human stromal cell size regulation, contractility and inflammatory activation Skills: UV stereolithography 3D printing – design, construction, use
hydrogel reaction chemistry and preparation human stromal cell culture
confocal microscopy and live cell 3D imaging
3D image reconstruction and quantitative image analysis
Research Associate, University of Wisconsin – Madison, Sep. 2015 – Jun. 2017
Studied reprogramming of cell fate; measured changes in epigenetic regulators at transcriptional and translational levels. Derived knockout mouse stem cell lines using cloning, CRISPR/Cas9, siRNA, and shRNA based methods. Studied role of histone demethylase Kdm3b in
reprogramming somatic murine cells into iPSCs. Managed mouse colony and lab logistics.
Skills: mammalian cell isolation, culture and reprogramming Cell line derivations, mutagenesis and cloning
CRISPR/Cas9 genome modifications siRNA, shRNA and lentiviruses Flow Cytometry/FACS
Immunoprecipitation (IP) and pull-down assays Western blots, RT-qPCR, immunofluorescence Mouse colony management and breeding
49
Undergraduate Researcher, University of Wisconsin – La Crosse, Jun.
2014 – Sep. 2015
Studied structural perturbations in the protein calmodulin (CaM) during oxidative stress. Performed long timescale molecular dynamic simulations and analyzed changes in chemical bonds during structural collapse events and conformational change.
Skills: Nanoscale molecular dynamics simulations Batch scripting (python/shell/tcl)
Structural modeling and biophysical analysis
Circular Dichroism (CD), Isothermal Titration Calorimetry (ITC) PUBLICATIONS
Lenzini S, Devine D & Shin J-W (2019) Leveraging Biomaterial Mechanics to Improve Pluripotent Stem Cell Applications for Tissue Engineering. Frontiers in Bioengineering and Biotechnology 7(260). doi:
10.3389/fbioe.2019.00260
Devine D, Wong SW, Vijayakumar V & Shin JW (2019) Soft and stiff matrix signals cooperate to maximize cell size and inflammatory activation under spatial confinement. Manuscript submitted Sep. 2019.
PRESENTATIONS
Devine, Daniel M. and Jae-Won Shin. Three-dimensional scaffold spacing directs changes in cell morphology.
Talk, Functional & Regenerative Medicine Workshop. UIC, Oct. 3, 2018.
Devine, Daniel M., Newman, Peter L. and Jae-Won Shin. Ligand
Devine, Daniel M., Newman, Peter L. and Jae-Won Shin. Ligand