Mathematical Simulation And Quantitative Control Of Polyvinyl Alcohol Hydrogel For Cell Culture In Vitro

In recent years, the effect of micro-mechanical environment on the biologicalbehaviors of cells attracted widespread attention. A variety of cell biological behaviors,such as cell morphological adjustment, migration orientation changes, proliferation anddifferentiation changes, were influenced by rigidity of adhesion substrate for most tissuecells which were anchorage dependent in vivo. These biological behaviors were closelyrelated to many physiological and pathological processes. Biochemical environment andbiophysical environment were two key problems needed to be solved in constructing acell culture model in vitro to simulate the cell growth environment. The former part hadbeen gradually improved by biologists and medical scientists, and substrate rigidity wasmost notably of effects of biophysical factors. Mathematical simulation and quantitativecontrol of substrate for cell culture in vitro would be the core of this work.Hydrogels are water-soluble polymers which have the very similar water contentto macromolecular compounds in vivo, they have been widely used as biomedicalmaterials as their better biocompatibility. A polyvinylalcohol （PVA） hydrogel whichused as substrate material for cell culture in vitro to study respond rules of cell tomechanical environment was prepared by chemical cross-linking method.Glutaraldehyde was used in this study as the crosslinker to prepare saturated PVAhydrogel which improved the mechanical strength and thermal stability of the film, anda small amount of glutaraldehyde had the function of sterilization and disinfection.Glutaraldehyde cross-linked PVA hydrogel had a broad application prospect in cellculture in vitro.Known by the Flory-Huggins theory, the cross-linking density determinesequilibrium water content（EWC）of the material，EWC is the direct factor in affectingthe Young’s modulus. Since the difficulties in measuring cross-linking density afterreaction, the conventional method used the molar ratio between cross-linking agentand hydroxyl groups monomer （d） instead of the cross-linking density, there was alarge deviation between calculated value and the actual measurement value. Combined,the gravity deformation of water in hydrogel would seriously interfere with the accuratedetermination of the modulus. So far, scatter diagrams with no mathematical laws wereused to control the mechanical properties of hydrogel, which lead to large deviation andthe operation was not easy. Improvements of this paper were mainly lie in: first, changed the traditionalpractice of simply use the molar ratio between cross-linking agent and hydroxylgroups monomer （d） instead of the cross-linking density. The initial concentration （C₀）and d affect the cross-linking density, how did C₀and d influence E and EWC valuehad been discussed, and a mathematical model was established by regression analysis.Second, whether objective law could be presented in the test and the accuracy of thedata samples taken were key factors for mathematical simulation. The Young’s modulus（E） was measured by improved stretching method in this paper to gain more accurateresults. The results show that the two improvements made the model could be used forsimulating the relationship between E and the factors of C₀, d and EWC, and forcontrolling the mechanical properties of the material.Choose3.6kPa,10kPa and30kPa to simulate the rigidity of the liver tissue inthe different stages of liver fibrosis, the effects of substrate stiffness on L02cell shapeadjustment and skeleton development were also studied. The Image J software was usedto measure the spreading area of the cells and cell conform factor （CF）. The resultsshowed that cells tend to spread on the substrate with high stiffness, while cellmorphology became round on the softer substrate. The immunofluorescence observationof cytoskeleton conformation on substrate with different rigidity showed thatcytoskeleton assembly was complete, fiber bundle was formed inside, and around thecells grew stress fibers. While on soft substrate, F-actin distributed mainly along thecytoderm. Cell skeleton tensity was regulated by substrate stiffness, and was higher withthe increase of substrate stiffness.