Soft materials to treat central nervous system injuries: Evaluation of the suitability of non-mammalian fibrin gels

AbstractPolymeric scaffolds formed from synthetic or natural materials have many applications in tissue engineering and medicine, and multiple material properties need to be optimized for specific applications. Recent studies have emphasized the importance of the scaffolds' mechanical properties to support specific cellular responses in addition to considerations of biochemical interactions, material transport, immunogenicity, and other factors that determine biocompatibility. Fibrin gels formed from purified fibrinogen and thrombin, the final two reactants in the blood coagulation cascade, have long been shown to be effective in wound healing and supporting the growth of cells in vitro and in vivo. Fibrin, even without additional growth factors or other components has potential for use in neuronal wound healing in part because of its mechanical compliance that supports the growth of neurons without activation of glial proliferation. This review summarizes issues related to the use of fibrin gels in neuronal cell contexts, with an emphasis on issues of immunogenicity, and considers the potential advantages and disadvantages of fibrin prepared from non-mammalian sources

Spatially Explicit Burden Estimates of Malaria in Tanzania: Bayesian Geostatistical Modeling of the Malaria Indicator Survey Data

A national HIV/AIDS and malaria parasitological survey was carried out in Tanzania in 2007–2008. In this study the parasitological data were analyzed: i) to identify climatic/environmental, socio-economic and interventions factors associated with child malaria risk and ii) to produce a contemporary, high spatial resolution parasitaemia risk map of the country. Bayesian geostatistical models were fitted to assess the association between parasitaemia risk and its determinants. Bayesian kriging was employed to predict malaria risk at unsampled locations across Tanzania and to obtain the uncertainty associated with the predictions. Markov chain Monte Carlo (MCMC) simulation methods were employed for model fit and prediction. Parasitaemia risk estimates were linked to population data and the number of infected children at province level was calculated. Model validation indicated a high predictive ability of the geostatistical model, with 60.00% of the test locations within the 95% credible interval. The results indicate that older children are significantly more likely to test positive for malaria compared with younger children and living in urban areas and better-off households reduces the risk of infection. However, none of the environmental and climatic proxies or the intervention measures were significantly associated with the risk of parasitaemia. Low levels of malaria prevalence were estimated for Zanzibar island. The population-adjusted prevalence ranges from in Kaskazini province (Zanzibar island) to in Mtwara region. The pattern of predicted malaria risk is similar with the previous maps based on historical data, although the estimates are lower. The predicted maps could be used by decision-makers to allocate resources and target interventions in the regions with highest burden of malaria in order to reduce the disease transmission in the country

A Nonintrusive Method of Measuring the Local Mechanical Properties of Soft Hydrogels Using Magnetic Microneedles

Soft hydrogels serving as substrates for cell attachment are used to culture many types of cells. The mechanical properties of these gels influence cell morphology, growth, and differentiation. For studies of cell growth on inhomogeneous gels, techniques by which the mechanical properties of the substrate can be measured within the proximity of a given cell are of interest. We describe an apparatus that allows the determination of local gel elasticity by measuring the response of embedded micron-sized magnetic needles to applied magnetic fields. This microscope-based four-magnet apparatus can apply both force and torque on the microneedles. The force and the torque are manipulated by changing the values of the magnetic field at the four poles of the magnet using a feedback circuit driven by LABVIEW. Using Hall probes, we have mapped out the magnetic field and field gradients produced by each pole when all the other poles are held at zero magnetic field. We have verified that superposition of these field maps allows one to obtain field maps for the case when the poles are held at arbitrary field values. This allows one to apply known fields and field gradients to a given microneedle. An imaging system is employed to measure the displacement and rotation of the needles. Polyacrylamide hydrogels of known elasticity were used to determine the relationship between the field gradient at the location of the needles and the force acting on the needles. This relationship allows the force on the microneedle to be determined from a known field gradient. This together with a measurement of the displacement of the needle in a given gel allows one to determine the stiffness (F/δ) of the gel and the elastic modulus, provided Poison\u27s ratio is known. Using this method, the stiffness and the modulus of elasticity of type-I collagen gels were found to be 2.64±0.05 nN/µm and 284.6±5.9 Pa, respectively. This apparatus is presently being employed to track the mechanical stiffness of the DNA-cross-linked hydrogels, developed by our group, whose mechanical properties can be varied on demand by adding or removing cross-linker strands. Thus a system that can be utilized to track the local properties of soft media as a function of time with minimum mechanical disturbance in the presence of cells is presented

Hepatic stellate cells require a stiff environment for myofibroblastic differentiation

The myofibroblastic differentiation of hepatic stellate cells (HSC) is a critical event in liver fibrosis and is part of the final common pathway to cirrhosis in chronic liver disease from all causes. The molecular mechanisms driving HSC differentiation are not fully understood. Because macroscopic tissue stiffening is a feature of fibrotic disease, we hypothesized that mechanical properties of the underlying matrix are a principal determinant of HSC activation. Primary rat HSC were cultured on inert polyacrylamide supports of variable but precisely defined shear modulus (stiffness) coated with different extracellular matrix proteins or poly-l-lysine. HSC differentiation was determined by cell morphology, immunofluorescence staining, and gene expression. HSC became progressively myofibroblastic as substrate stiffness increased on all coating matrices, including Matrigel. The degree rather than speed of HSC activation correlated with substrate stiffness, with cells cultured on supports of intermediate stiffness adopting stable intermediate phenotypes. Quiescent cells on soft supports were able to undergo myofibroblastic differentiation with exposure to stiff supports. Stiffness-dependent differentiation required adhesion to matrix proteins and the generation of mechanical tension. Transforming growth factor-β treatment enhanced differentiation on stiff supports, but was not required. HSC differentiate to myofibroblasts in vitro primarily as a function of the physical rather than the chemical properties of the substrate. HSC require a mechanically stiff substrate, with adhesion to matrix proteins and the generation of mechanical tension, to differentiate. These findings suggest that alterations in liver stiffness are a key factor driving the progression of fibrosis

Fibroblast Adaptation and Stiffness Matching to Soft Elastic Substrates

Many cell types alter their morphology and gene expression profile when grown on chemically equivalent surfaces with different rigidities. One expectation of this change in morphology and composition is that the cell's internal stiffness, governed by cytoskeletal assembly and production of internal stresses, will change as a function of substrate stiffness. Atomic force microscopy was used to measure the stiffness of fibroblasts grown on fibronectin-coated polyacrylamide gels of shear moduli varying between 500 and 40,000 Pa. Indentation measurements show that the cells' elastic moduli were equal to, or slightly lower than, those of their substrates for a range of soft gels and reached a saturating value at a substrate rigidity of 20 kPa. The amount of cross-linked F-actin sedimenting at low centrifugal force also increased with substrate stiffness. Together with enhanced actin polymerization and cross-linking, active contraction of the cytoskeleton can also modulate stiffness by exploiting the nonlinear elasticity of semiflexible biopolymer networks. These results suggest that within a range of stiffness spanning that of soft tissues, fibroblasts tune their internal stiffness to match that of their substrate, and modulation of cellular stiffness by the rigidity of the environment may be a mechanism used to direct cell migration and wound repair