Transfer of assembled collagen fibrils to flexible substrates for mechanically tunable contact guidance cues

Contact guidance or bidirectional migration along aligned fibers modulates many physiological and pathological processes such as wound healing and cancer invasion. Aligned 2D collagen fibrils epitaxially grown on mica substrates replicate many features of contact guidance seen in aligned 3D collagen fiber networks. However, these 2D collagen self-assembled substrates are difficult to image through, do not have known or tunable mechanical properties and cells degrade and mechanically detach collagen fibrils from the surface, leading to an inability to assess contact guidance over long times. Here, we describe the transfer of aligned collagen fibrils from mica substrates to three different functionalized target substrates: glass, polydimethylsiloxane (PDMS) and polyacrylamide (PA). Aligned collagen fibrils can be efficiently transferred to all three substrates. This transfer resulted in substrates that were to varying degrees resistant to cell-mediated collagen fibril deformation that resulted in detachment of the collagen fibril field, allowing for contact guidance to be observed over longer time periods. On these transferred substrates, cell speed is lowest on softer contact guidance cues for both MDA-MB-231 and MTLn3 cells. Intermediate stiffness resulted in the fastest migration. MTLn3 cell directionality was low on soft contact guidance cues, whereas MDA-MB-231 cell directionality marginally increased. It appears that the stiffness of the contact guidance cue regulates contact guidance differently between cell types. The development of this collagen fibril transfer method allows for the attachment of aligned collagen fibrils on substrates, particularly flexible substrates, that do not normally promote aligned collagen fibril growth, increasing the utility of this collagen self-assembly system for the fundamental examination of mechanical regulation of contact guidance

Degradation and Remodeling of Epitaxially Grown Collagen Fibrils

Introduction: The extracellular matrix (ECM) in the tumor microenvironment contains high densities of collagen that are highly aligned, resulting in directional migration called contact guidance that facilitates efficient migration out of the tumor. Cancer cells can remodel the ECM through traction force controlled by myosin contractility or proteolytic activity controlled by matrix metalloproteinase (MMP) activity, leading to either enhanced or diminished contact guidance. Methods: Recently, we have leveraged the ability of mica to epitaxially grow aligned collagen fibrils in order to assess contact guidance. In this article, we probe the mechanisms of remodeling of aligned collagen fibrils on mica by breast cancer cells. Results: We show that cells that contact guide with high fidelity (MDA-MB-231 cells) exert more force on the underlying collagen fibrils than do cells that contact guide with low fidelity (MTLn3 cells). These high traction cells (MDA-MB-231 cells) remodel collagen fibrils over hours, pulling so hard that the collagen fibrils detach from the surface, effectively delaminating the entire contact guidance cue. Myosin or MMP inhibition decreases this effect. Interestingly, blocking MMP appears to increase the alignment of cells on these substrates, potentially allowing the alignment through myosin contractility to be uninhibited. Finally, amplification or dampening of contact guidance with respect to a particular collagen fibril organization is seen under different conditions. Conclusions: Both myosin II contractility and MMP activity allow MDA-MB-231 cells to remodel and eventually destroy epitaxially grown aligned collagen fibrils

The Effect of Stiffness on Cancer Cell Contact Guidance

Statistics have shown approximately 20% of deaths every year in the US are as a result of cancer. Most of these deaths aren’t caused by the primary tumor, but the migration of cancer cells and their subsequent tumor growth. It has been found that many cells migrate directional using different cues. The directional cues have been identified as electrical gradients, stiffness gradients, chemical gradients, or fiber alignment. Cell migration along aligned fibers is called contact guidance. While these cues have been identified to cause directional migration, there has not been an in-depth study on how cells are responding to these cues. Our purpose was to characterize the effect of matrix stiffness on contact guidance migration. Collagen fibers were used as the contact guidance platform, since collagen makes up the fibrous region of the extracellular matrix. Mica has been found by our lab to be a good platform to grow aligned collagen fibers due to its ionic surface chemistry and ordered structure. However, mica is stiff and not tunable. In order to characterize cell alignment, a transplantation method was also developed to transfer fibers to polymer surfaces with tunable stiffness like polyacrylamide and polydimethylsiloxane. Alignment and elongation of the cells were characterized through fixing and staining the cells. Long time-lapse migration was examined and speed and persistence of the cells along the fiber cue was calculated. From preliminary results, we could confirm the effectiveness of our transfer method from mica to functionalized polymer surfaces. In addition, stiffer substrates seem to lead to better elongation and alignment with the matrix. This leads us to believe that stiffer extracellular matrices could be worse for cancer cell metastasis.</p

The Effect of Stiffness on Cancer Cell Contact Guidance

Statistics have shown approximately 20% of deaths every year in the US are as a result of cancer. Most of these deaths aren’t caused by the primary tumor, but the migration of cancer cells and their subsequent tumor growth. It has been found that many cells migrate directional using different cues. The directional cues have been identified as electrical gradients, stiffness gradients, chemical gradients, or fiber alignment. Cell migration along aligned fibers is called contact guidance. While these cues have been identified to cause directional migration, there has not been an in-depth study on how cells are responding to these cues. Our purpose was to characterize the effect of matrix stiffness on contact guidance migration. Collagen fibers were used as the contact guidance platform, since collagen makes up the fibrous region of the extracellular matrix. Mica has been found by our lab to be a good platform to grow aligned collagen fibers due to its ionic surface chemistry and ordered structure. However, mica is stiff and not tunable. In order to characterize cell alignment, a transplantation method was also developed to transfer fibers to polymer surfaces with tunable stiffness like polyacrylamide and polydimethylsiloxane. Alignment and elongation of the cells were characterized through fixing and staining the cells. Long time-lapse migration was examined and speed and persistence of the cells along the fiber cue was calculated. From preliminary results, we could confirm the effectiveness of our transfer method from mica to functionalized polymer surfaces. In addition, stiffer substrates seem to lead to better elongation and alignment with the matrix. This leads us to believe that stiffer extracellular matrices could be worse for cancer cell metastasis

Transfer of assembled collagen fibrils to flexible substrates for mechanically tunable contact guidance cues

Contact guidance or bidirectional migration along aligned fibers modulates many physiological and pathological processes such as wound healing and cancer invasion. Aligned 2D collagen fibrils epitaxially grown on mica substrates replicate many features of contact guidance seen in aligned 3D collagen fiber networks. However, these 2D collagen self-assembled substrates are difficult to image through, do not have known or tunable mechanical properties and cells degrade and mechanically detach collagen fibrils from the surface, leading to an inability to assess contact guidance over long times. Here, we describe the transfer of aligned collagen fibrils from mica substrates to three different functionalized target substrates: glass, polydimethylsiloxane (PDMS) and polyacrylamide (PA). Aligned collagen fibrils can be efficiently transferred to all three substrates. This transfer resulted in substrates that were to varying degrees resistant to cell-mediated collagen fibril deformation that resulted in detachment of the collagen fibril field, allowing for contact guidance to be observed over longer time periods. On these transferred substrates, cell speed is lowest on softer contact guidance cues for both MDA-MB-231 and MTLn3 cells. Intermediate stiffness resulted in the fastest migration. MTLn3 cell directionality was low on soft contact guidance cues, whereas MDA-MB-231 cell directionality marginally increased. It appears that the stiffness of the contact guidance cue regulates contact guidance differently between cell types. The development of this collagen fibril transfer method allows for the attachment of aligned collagen fibrils on substrates, particularly flexible substrates, that do not normally promote aligned collagen fibril growth, increasing the utility of this collagen self-assembly system for the fundamental examination of mechanical regulation of contact guidance.This is a pre-copyedited, author-produced version of an article accepted for publication in Integrative Biology following peer review. The version of record Wang, Juan, Joseph Koelbl, Anuraag Boddupalli, Zhiqi Yao, Kaitlin M. Bratlie, and Ian C. Schneider. "Transfer of assembled collagen fibrils to flexible substrates for mechanically tunable contact guidance cues." Integrative Biology 10, no. 11 (2018): 705-718 is available online at DOI: 10.1039/c8ib00127h. Posted with permission.</p

Degradation and Remodeling of Epitaxially Grown Collagen Fibrils

Introduction: The extracellular matrix (ECM) in the tumor microenvironment contains high densities of collagen that are highly aligned, resulting in directional migration called contact guidance that facilitates efficient migration out of the tumor. Cancer cells can remodel the ECM through traction force controlled by myosin contractility or proteolytic activity controlled by matrix metalloproteinase (MMP) activity, leading to either enhanced or diminished contact guidance. Methods: Recently, we have leveraged the ability of mica to epitaxially grow aligned collagen fibrils in order to assess contact guidance. In this article, we probe the mechanisms of remodeling of aligned collagen fibrils on mica by breast cancer cells. Results: We show that cells that contact guide with high fidelity (MDA-MB-231 cells) exert more force on the underlying collagen fibrils than do cells that contact guide with low fidelity (MTLn3 cells). These high traction cells (MDA-MB-231 cells) remodel collagen fibrils over hours, pulling so hard that the collagen fibrils detach from the surface, effectively delaminating the entire contact guidance cue. Myosin or MMP inhibition decreases this effect. Interestingly, blocking MMP appears to increase the alignment of cells on these substrates, potentially allowing the alignment through myosin contractility to be uninhibited. Finally, amplification or dampening of contact guidance with respect to a particular collagen fibril organization is seen under different conditions. Conclusions: Both myosin II contractility and MMP activity allow MDA-MB-231 cells to remodel and eventually destroy epitaxially grown aligned collagen fibrils.This is a manuscript of an article published as Wang, Juan, Anuraag Boddupalli, Joseph Koelbl, Dong Hyun Nam, Xin Ge, Kaitlin M. Bratlie, and Ian C. Schneider. "Degradation and Remodeling of Epitaxially Grown Collagen Fibrils." Cellular and Molecular Bioengineering 12, no. 1 (2019). The final publication is available at Springer via DOI: 10.1007/s12195-018-0547-6. Posted with permission.</p

Association of Intraventricular Fibrinolysis With Clinical Outcomes in Intracerebral Hemorrhage: An Individual Participant Data Meta-Analysis

Background: In patients with intracerebral hemorrhage (ICH), the presence of intraventricular hemorrhage constitutes a promising therapeutic target. Intraventricular fibrinolysis (IVF) reduces mortality, yet impact on functional disability remains unclear. Thus, we aimed to determine the influence of IVF on functional outcomes. Methods: This individual participant data meta-analysis pooled 1501 patients from 2 randomized trials and 7 observational studies enrolled during 2004 to 2015. We compared IVF versus standard of care (including placebo) in patients treated with external ventricular drainage due to acute hydrocephalus caused by ICH with intraventricular hemorrhage. The primary outcome was functional disability evaluated by the modified Rankin Scale (mRS; range: 0-6, lower scores indicating less disability) at 6 months, dichotomized into mRS score: 0 to 3 versus mRS: 4 to 6. Secondary outcomes included ordinal-shift analysis, all-cause mortality, and intracranial adverse events. Confounding and bias were adjusted by random effects and doubly robust models to calculate odds ratios and absolute treatment effects (ATE). Results: Comparing treatment of 596 with IVF to 905 with standard of care resulted in an ATE to achieve the primary outcome of 9.3% (95% CI, 4.4-14.1). IVF treatment showed a significant shift towards improved outcome across the entire range of mRS estimates, common odds ratio, 1.75 (95% CI, 1.39-2.17), reduced mortality, odds ratio, 0.47 (95% CI, 0.35-0.64), without increased adverse events, absolute difference, 1.0% (95% CI, -2.7 to 4.8). Exploratory analyses provided that early IVF treatment (<= 48 hours) after symptom onset was associated with an ATE, 15.2% (95% CI, 8.6-21.8) to achieve the primary outcome. Conclusions: As compared to standard of care, the administration of IVF in patients with acute hydrocephalus caused by intracerebral and intraventricular hemorrhage was significantly associated with improved functional outcome at 6 months. The treatment effect was linked to an early time window <48 hours, specifying a target population for future trials