Concentration Independent Modulation of Local Micromechanics in a Fibrin Gel

Methods for tuning extracellular matrix (ECM) mechanics in 3D cell culture that rely on increasing the concentration of either protein or cross-linking molecules fail to control important parameters such as pore size, ligand density, and molecular diffusivity. Alternatively, ECM stiffness can be modulated independently from protein concentration by mechanically loading the ECM. We have developed a novel device for generating stiffness gradients in naturally derived ECMs, where stiffness is tuned by inducing strain, while local mechanical properties are directly determined by laser tweezers based active microrheology (AMR). Hydrogel substrates polymerized within 35 mm diameter Petri dishes are strained non-uniformly by the precise rotation of an embedded cylindrical post, and exhibit a position-dependent stiffness with little to no modulation of local mesh geometry. Here we present the device in the context of fibrin hydrogels. First AMR is used to directly measure local micromechanics in unstrained hydrogels of increasing fibrin concentration. Changes in stiffness are then mapped within our device, where fibrin concentration is held constant. Fluorescence confocal imaging and orbital particle tracking are used to quantify structural changes in fibrin on the micro and nano levels respectively. The micromechanical strain stiffening measured by microrheology is not accompanied by ECM microstructural changes under our applied loads, as measured by confocal microscopy. However, super-resolution orbital tracking reveals nanostructural straightening, lengthening, and reduced movement of fibrin fibers. Furthermore, we show that aortic smooth muscle cells cultured within our device are morphologically sensitive to the induced mechanical gradient. Our results demonstrate a powerful cell culture tool that can be used in the study of mechanical effects on cellular physiology in naturally derived 3D ECM tissues

Non-Linear Elasticity of Extracellular Matrices Enables Contractile Cells to Communicate Local Position and Orientation

Most tissue cells grown in sparse cultures on linearly elastic substrates typically display a small, round phenotype on soft substrates and become increasingly spread as the modulus of the substrate increases until their spread area reaches a maximum value. As cell density increases, individual cells retain the same stiffness-dependent differences unless they are very close or in molecular contact. On nonlinear strain-stiffening fibrin gels, the same cell types become maximally spread even when the low strain elastic modulus would predict a round morphology, and cells are influenced by the presence of neighbors hundreds of microns away. Time lapse microscopy reveals that fibroblasts and human mesenchymal stem cells on fibrin deform the substrate by several microns up to five cell lengths away from their plasma membrane through a force limited mechanism. Atomic force microscopy and rheology confirm that these strains locally and globally stiffen the gel, depending on cell density, and this effect leads to long distance cell-cell communication and alignment. Thus cells are acutely responsive to the nonlinear elasticity of their substrates and can manipulate this rheological property to induce patterning

Food safety and evaluation of intention to practice safe eating out measures during COVID-19: Cross sectional study in Indonesia and Malaysia

Factors contributing to foodborne illnesses in Malaysia were identified as insanitary food handling procedures and lack of hygiene in food preparation area. Food safety at home is a critical point as consumers represent the final step in food preparation and prevention of foodborne diseases. This study aims to investigate the food safety knowledge, attitude and self-reported practices among consumers in Malaysia. An online survey was conducted, and data were analysed using descriptive statistics and exploratory factor analysis. A model linking food safety knowledge and attitude and their direct effects on practices were confirmed using structural equation modelling (SEM). The proposed model fulfilled the goodness of fit indices and is deemed acceptable. Respondents demonstrate good level of food safety knowledge and positive attitudes and self-reported practices. Food safety knowledge has a negative and insignificant relationship with food safety practices (β1 =- 0.284, p>0.05) while attitude significantly affects food safety practices (β1 =0.534, p<0.05). The findings clearly indicate that food safety knowledge does not directly affect food safety practices This is also the first study to provide new empirical findings on thermometer usage among consumers in Malaysia. This study establishes an important point of reference where consumers use visual appearances to determine if food is thoroughly cooked and practice washing raw chicken prior to cooking. Food safety practices at home play a critical role in protecting consumers in reducing risks of foodborne illnesses