A Model for Studying the Biomechanical Effects of Varying Ratios of Collagen Types I and III on Cardiomyocytes.

PURPOSE: To develop a novel model composed solely of Col I and Col III with the lower and upper limits set to include the ratios of Col I and Col III at 3:1 and 9:1 in which the structural and mechanical behavior of the resident CM can be studied. Further, the progression of fibrosis due to change in ratios of Col I:Col III was tested. METHODS: Collagen gels with varying Col I:Col III ratios to represent a healthy (3:1) and diseased myocardial tissue were prepared by manually casting them in wells. Absorbance assay was performed to confirm the gelation of the gels. Rheometric analysis was performed on each of the collagen gels prepared to determine the varying stiffnesses and rheological parameters of the gels made with varying ratios of Col I:Col III. Second Harmonic Generation (SHG) was performed to observe the 3D characterization of the collagen samples. Scanning Electron microscopy was used for acquiring cross sectional images of the lyophilized collagen gels. AC16 CM (human) cell lines were cultured in the prepared gels to study cell morphology and behavior as a result of the varying collagen ratios. Cellular proliferation was studied by performing a Cell Trace Violet Assay and the applied force on each cell was measured by means of Finite Element Analysis (FEA) on CM from each sample. RESULTS: Second harmonic generation microscopy used to image Col I, displayed a decrease in acquired image intensity with an increase in the non-second harmonic Col III in 3:1 gels. SEM showed a fiber-rich structure in the 3:1 gels with well-distributed pores unlike the 9:1 gels or the 1:0 controls. Rheological analysis showed a decrease in substrate stiffness with an increase of Col III, in comparison with other cases. CM cultured within 3:1 gels exhibited an elongated rod-like morphology with an average end-to-end length of 86 ± 28.8 µm characteristic of healthy CM, accompanied by higher cell growth in comparison with other cases. Finite element analysis used to estimate the forces exerted on CM cultured in the 3:1 gels, showed that the forces were well dispersed, and not concentrated within the center of cells, in comparison with other cases. CONCLUSION: This study model can be adopted to simulate various biomechanical environments in which cells crosstalk with the Collagen-matrix in diseased pathologies to generate insights on strategies for prevention of fibrosis.

Hydrogel scaffolds with elasticity-mimicking embryonic substrates promote cardiac cellular network formation.

Hydrogels are a class of biomaterials used for a wide range of biomedical applications, including as a three-dimensional (3D) scaffold for cell culture that mimics the extracellular matrix (ECM) of native tissues. To understand the role of the ECM in the modulation of cardiac cell function, alginate was used to fabricate crosslinked gels with stiffness values that resembled embryonic (2.66 ± 0.84 kPa), physiologic (8.98 ± 1.29 kPa) and fibrotic (18.27 ± 3.17 kPa) cardiac tissues. The average pore diameter and hydrogel swelling were seen to decrease with increasing substrate stiffness. Cardiomyocytes cultured within soft embryonic gels demonstrated enhanced cell spreading, elongation, and network formation, while a progressive increase in gel stiffness diminished these behaviors. Cell viability decreased with increasing hydrogel stiffness. Furthermore, cells in fibrotic gels showed enhanced protein expression of the characteristic cardiac stress biomarker, Troponin-I, while reduced protein expression of the cardiac gap junction protein, Connexin-43, in comparison to cells within embryonic gels. The results from this study demonstrate the role that 3D substrate stiffness has on cardiac tissue formation and its implications in the development of complex matrix remodeling-based conditions, such as myocardial fibrosis.

Dynamic extracellular matrix stiffening induces a phenotypic transformation and a migratory shift in epithelial cells.

Soft tissue tumors, including breast cancer, become stiffer throughout disease progression. This increase in stiffness has been shown to correlate to malignant phenotype and epithelial-to-mesenchymal transition (EMT) in vitro. Unlike current models, utilizing static increases in matrix stiffness, our group has previously created a system that allows for dynamic stiffening of an alginate-matrigel composite hydrogel to mirror the native dynamic process. Here, we utilize this system to evaluate the role of matrix stiffness on EMT and metastasis both in vitro and in vivo. Epithelial cells were seen to lose normal morphology and become protrusive and migratory after stiffening. This shift corresponded to a loss of epithelial markers and gain of mesenchymal markers in both the cell clusters and migrated cells. Furthermore, stiffening in a murine model reduced tumor burden and increased migratory behavior prior to tumor formation. Inhibition of FAK and PI3K in vitro abrogated the morphologic and migratory transformation of epithelial cell clusters. This work demonstrates the key role extracellular matrix stiffening has in tumor progression through integrin signaling and, in particular, its ability to drive EMT-related changes and metastasis.

Anomalous spin-orbit torques in magnetic single-layer films.

The spin Hall effect couples charge and spin transport1-3, enabling electrical control of magnetization4,5. A quintessential example of spin-Hall-related transport is the anomalous Hall effect (AHE)6, first observed in 1880, in which an electric current perpendicular to the magnetization in a magnetic film generates charge accumulation on the surfaces. Here, we report the observation of a counterpart of the AHE that we term the anomalous spin-orbit torque (ASOT), wherein an electric current parallel to the magnetization generates opposite spin-orbit torques on the surfaces of the magnetic film. We interpret the ASOT as being due to a spin-Hall-like current generated with an efficiency of 0.053 ± 0.003 in Ni80Fe20, comparable to the spin Hall angle of Pt7. Similar effects are also observed in other common ferromagnetic metals, including Co, Ni and Fe. First-principles calculations corroborate the order of magnitude of the measured values. This work suggests that a strong spin current with spin polarization transverse to the magnetization can be generated within a ferromagnet, despite spin dephasing8. The large magnitude of the ASOT should be taken into consideration when investigating spin-orbit torques in ferromagnetic/non-magnetic bilayers.

The applicability of furfuryl-gelatin as a novel bioink for tissue engineering applications.

Three-dimensional bioprinting is an innovative technique in tissue engineering, to create layer-by-layer structures, required for mimicking body tissues. However, synthetic bioinks do not generally possess high printability and biocompatibility at the same time. So, there is an urgent need for naturally derived bioinks that can exhibit such optimized properties. We used furfuryl-gelatin as a novel, visible-light crosslinkable bioink for fabricating cell-laden structures with high viability. Hyaluronic acid was added as a viscosity enhancer and either Rose Bengal or Riboflavin was used as a visible-light crosslinker. Crosslinking was done by exposing the printed structure for 2.5 min to visible light and confirmed using Fourier transform infrared spectroscopy and rheometry. Scanning electron microscopy revealed a highly porous networked structure. Three different cell types were successfully bioprinted within these constructs. Mouse mesenchymal stem cells printed within monolayer and bilayer sheets showed viability, network formation and proliferation (∼5.33 times) within 72 h of culture. C2C12 and STO cells were used to print a double layered structure, which showed evidence of the viability of both cells and heterocellular clusters within the construct. This furfuryl-gelatin based bioink can be used for tissue engineering of complex tissues and help in understanding how cellular crosstalk happens in vivo during normal or diseased pathology. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 107B: 314-323, 2019.

A Visible Light-Cross-Linkable, Fibrin-Gelatin-Based Bioprinted Construct with Human Cardiomyocytes and Fibroblasts.

In this study, fibrin was added to a photo-polymerizable gelatin-based bioink mixture to fabricate cardiac cell-laden constructs seeded with human induced pluripotent stem cell-derived cardiomyocytes (iPS-CM) or CM cell lines with cardiac fibroblasts (CF). The extensive use of platelet-rich fibrin, its capacity to offer patient specificity, and the similarity in composition to surgical glue prompted us to include fibrin in the existing bioink composition. The cell-laden bioprinted constructs were cross-linked to retain a herringbone pattern via a two-step procedure including the visible light cross-linking of furfuryl-gelatin followed by the chemical cross-linking of fibrinogen via thrombin and calcium chloride. The printed constructs revealed an extremely porous, networked structure that afforded long-term in vitro stability. Cardiomyocytes printed within the sheet structure showed excellent viability, proliferation, and expression of the troponin I cardiac marker. We extended the utility of this fibrin-gelatin bioink toward coculturing and coupling of CM and cardiac fibroblasts (CF), the interaction of which is extremely important for maintenance of normal physiology of the cardiac wall in vivo. This enhanced "cardiac construct" can be used for drug cytotoxicity screening or unraveling triggers for heart diseases in vitro.

Phenotypic Basis for Matrix Stiffness-Dependent Chemoresistance of Breast Cancer Cells to Doxorubicin.

The persistence of drug resistant cell populations following chemotherapeutic treatment is a significant challenge in the clinical management of cancer. Resistant subpopulations arise via both cell intrinsic and extrinsic mechanisms. Extrinsic factors in the microenvironment, including neighboring cells, glycosaminoglycans, and fibrous proteins impact therapy response. Elevated levels of extracellular fibrous proteins are associated with tumor progression and cause the surrounding tissue to stiffen through changes in structure and composition of the extracellular matrix (ECM). We sought to determine how this progressively stiffening microenvironment affects the sensitivity of breast cancer cells to chemotherapeutic treatment. MDA-MB-231 triple negative breast carcinoma cells cultured in a 3D alginate-based hydrogel system displayed a stiffness-dependent response to the chemotherapeutic doxorubicin. MCF7 breast carcinoma cells cultured in the same conditions did not exhibit this stiffness-dependent resistance to the drug. This differential therapeutic response was coordinated with nuclear translocation of YAP, a marker of mesenchymal differentiation. The stiffness-dependent response was lost when cells were transferred from 3D to monolayer cultures, suggesting that endpoint ECM conditions largely govern the response to doxorubicin. To further examine this response, we utilized a platform capable of dynamic ECM stiffness modulation to allow for a change in matrix stiffness over time. We found that MDA-MB-231 cells have a stiffness-dependent resistance to doxorubicin and that duration of exposure to ECM stiffness is sufficient to modulate this response. These results indicate the need for additional tools to integrate mechanical stiffness with therapeutic response and inform decisions for more effective use of chemotherapeutics in the clinic.

A Comparative Study of a 3D Bioprinted Gelatin-Based Lattice and Rectangular-Sheet Structures.

3D bioprinting holds great promise in the field of regenerative medicine as it can create complex structures in a layer-by-layer manner using cell-laden bioinks, making it possible to imitate native tissues. Current bioinks lack both high printability and biocompatibility required in this respect. Hence, the development of bioinks that exhibit both properties is needed. In our previous study, a furfuryl-gelatin-based bioink, crosslinkable by visible light, was used for creating mouse mesenchymal stem cell-laden structures with a high fidelity. In this study, lattice mesh geometries were printed in a comparative study to test against the properties of a traditional rectangular-sheet. After 3D printing and crosslinking, both structures were analysed for swelling and rheological properties, and their porosity was estimated using scanning electron microscopy. The results showed that the lattice structure was relatively more porous with enhanced rheological properties and exhibited a lower degradation rate compared to the rectangular-sheet. Further, the lattice allowed cells to proliferate to a greater extent compared to the rectangular-sheet, which initially retained a lower number of cells. All of these results collectively affirmed that the lattice poses as a superior scaffold design for tissue engineering applications.

Observation of spin-orbit effects with spin rotation symmetry.

The spin-orbit interaction enables interconversion between a charge current and a spin current. It is usually believed that in a nonmagnetic metal (NM) or at a NM/ferromagnetic metal (FM) bilayer interface, the symmetry of spin-orbit effects requires that the spin current, charge current, and spin orientation are all orthogonal to each other. Here we demonstrate the presence of spin-orbit effects near the NM/FM interface that exhibit a very different symmetry, hereafter referred to as spin-rotation symmetry, from the conventional spin Hall effect while the spin polarization is rotating about the magnetization. These results imply that a perpendicularly polarized spin current can be generated with an in-plane charge current simply by use of a FM/NM bilayer with magnetization collinear to the charge current. The ability to generate a spin current with arbitrary polarization using typical magnetic materials will benefit the development of magnetic memories.Converting charge to spin currents using spin-orbit interactions has useful applications in spintronics but symmetry constraints can limit the control over spin polarization. Here the authors demonstrate spin-orbit effects with a different symmetry, which could help generate arbitrary spin polarizations.

Extracellular Matrix Stiffening Induces a Malignant Phenotypic Transition in Breast Epithelial Cells.

Tumors are much stiffer than healthy tissue, and progressively stiffen as the cancer develops. Tumor stiffening is largely the result of extracellular matrix (ECM) remodeling, for example, deposition and crosslinking of collagen I. Well established in vitro models have demonstrated the influence of the microenvironment in regulating tissue homeostasis, with matrix stiffness being a particularly influential mediator. Non-malignant MCF10A mammary epithelial cells (MECs) lose their epithelial characteristics and become invasive when cultured in stiff microenvironments, leading to the hypothesis that tumor stiffening could contribute directly to disease progression. However, previous studies demonstrating MCF10A invasion have been performed in gels with constant mechanical properties, unlike the dynamically stiffening tumor microenvironment. Here, we employ a temporally stiffening hydrogel platform to demonstrate that matrix stiffening induces invasion from and proliferation in MCF10A mammary acini. After allowing MCF10A acini to form in soft hydrogels for 14 days, the gels were stiffened to the level of a malignant tumor, giving rise to a proliferative and invasive phenotype. Cells were observed to collectively migrate away from mammary acini while maintaining cell-cell contacts. Small molecule inhibition of PI3K and Rac1 pathways was sufficient to significantly reduce the number and size of invasive acini after stiffening. Our results demonstrate that temporal matrix stiffening can induce invasion from mammary acini and supports the notion that tumor stiffening could be implicated in disease progression and metastasis.

Dynamic phototuning of 3D hydrogel stiffness.

Hydrogels are widely used as in vitro culture models to mimic 3D cellular microenvironments. The stiffness of the extracellular matrix is known to influence cell phenotype, inspiring work toward unraveling the role of stiffness on cell behavior using hydrogels. However, in many biological processes such as embryonic development, wound healing, and tumorigenesis, the microenvironment is highly dynamic, leading to changes in matrix stiffness over a broad range of timescales. To recapitulate dynamic microenvironments, a hydrogel with temporally tunable stiffness is needed. Here, we present a system in which alginate gel stiffness can be temporally modulated by light-triggered release of calcium or a chelator from liposomes. Others have shown softening via photodegradation or stiffening via secondary cross-linking; however, our system is capable of both dynamic stiffening and softening. Dynamic modulation of stiffness can be induced at least 14 d after gelation and can be spatially controlled to produce gradients and patterns. We use this system to investigate the regulation of fibroblast morphology by stiffness in both nondegradable gels and gels with degradable elements. Interestingly, stiffening inhibits fibroblast spreading through either mesenchymal or amoeboid migration modes. We demonstrate this technology can be translated in vivo by using deeply penetrating near-infrared light for transdermal stiffness modulation, enabling external control of gel stiffness. Temporal modulation of hydrogel stiffness is a powerful tool that will enable investigation of the role that dynamic microenvironments play in biological processes both in vitro and in well-controlled in vivo experiments.

A comprehensive statewide analysis of seatbelt non-use with injury and hospital admissions: new data, old problem.

OBJECTIVES: To investigate the association of seatbelt nonuse with injury patterns, injury severity, and in-patient hospital admission among adults presenting to emergency departments (EDs) in a statewide, population-based, sample of motor vehicle crashes. METHODS: Using data from the 2002 Crash Outcome Data Evaluation System (CODES) for Wisconsin, 23,920 occupants of motor vehicle crashes, aged 16 years or older, who were treated in an ED, were analyzed. Logistic regression was used to compare the odds ratio of having sustained an injury to specific body regions and of being admitted to an inpatient unit in unbelted individuals compared with those who were belted. RESULTS: Compared with belted occupants presenting to an ED, their unbelted counterparts were more likely to be male (56% vs. 40%) and to have used alcohol (17% vs. 4%). Unbelted occupants were younger (31 years vs. 38 years) and incurred higher ED charges ($681 vs. $509) than belted occupants. Additionally, unbelted occupants have a higher proportion of single-vehicle crashes, such as rollovers (44% vs. 22%), and rural crashes (56% vs. 44%). Unbelted occupants comprised 20% of study patients treated in the ED and discharged, 44% of patients treated in the ED and admitted, and 68% of patients dying in the ED. Unbelted occupants were more likely to be admitted (odds ratio [OR] = 2.6) than belted individuals and were more likely to suffer severe injuries to the head, face, thorax, abdomen, spine, upper and lower extremities (OR ranging from 1.6 to 3.9). CONCLUSIONS: Among patients presenting to an ED after a motor vehicle crash, unbelted occupants are more likely to require inpatient admission and to have sustained a severe injury to numerous body regions than are belted occupants.

Increased risk of death or disability in unhelmeted Wisconsin motorcyclists.

OBJECTIVE: The purpose of this study is to investigate the relationships among motorcycle rider helmet, alcohol use and the full spectrum of health outcomes following crashes. METHODS: Data from the National Highway Traffic Safety Administration-sponsored Crash Outcome Data Evaluation System (CODES) for Wisconsin, 2002, were used to study 2462 motorcycle crash victims. Logistic regression models were used to assess the relationship of helmet and alcohol use with outcomes. RESULTS: Compared to helmeted motorcycle riders, unhelmeted riders were more likely to require inpatient hospitalization (Relative Risk Ratio [RRR] = 1.4; 95% confidence interval [CI]:1.1-1.8) or die (RR = 1.9, 95% CI:1.0-3.7) but equally likely to be treated in emergency departments. Injury patterns differed by helmet use. Unhelmeted riders were more likely to suffer injuries of the head (odds ratio [OR] = 2.3, 95% CI:1.5-3.3) or face (OR = 3.0, 95% CI:2.1-4.2) than helmeted riders. No difference was observed in other injuries, including spine/neck injuries. Reported alcohol use was more prevalent among patients who were inpatients or died, and was associated with higher likelihood of not wearing a helmet (OR = 7.0, 95% CI:4.8-12.9). CONCLUSION: Motorcycle riders who are inpatients or die in a crash are less likely to be helmeted and more likely to sustain head or face injuries. Alcohol use is associated with unhelmeted riding and increased risk of poor outcomes. These findings support policy and educational efforts promoting helmet use, which seek to decrease these tragedies.