Joanne M. Belovich

Oxygen Diffusion Through Natural Extracellular Matrices: Implications for Estimating "Critical Thickness" Values in Tendon Tissue Engineering

Oxygen is necessary for maintaining cell proliferation and viability and extracellular matrix (ECM) production in 3-dimensional tissue engineering. Typically, diffusion is the primary mode for oxygen transport in vitro; thus, ensuring an adequate oxygen supply is essential. In this study, we determined the oxygen diffusion coefficients of 3 natural ECMs that are being investigated as construct scaffolds for tendon tissue engineering: small-intestine submucosa (SIS), human dermis (Alloderm), and canine fascia lata. Diffusion coefficients were determined using a standard diffusion cell system. The ranges for each matrix type were: SIS: 7 x 10(-6) - 2 x 10(-5) cm2/s, Alloderm: 1.9 - 3.1 x 10(-5) cm2/s, and canine fascia lata: 1.6 - 4 x 10(-5) cm2/s. We used the experimental oxygen diffusivity data for these natural ECMs in a mathematical model of oxygen diffusion through a cell-seeded scaffold to estimate the critical size of cell-seeded scaffold that can be cultured in vitro.

Retention and Viability Characteristics of Mammalian Cells in an Acoustically Driven Polymer Mesh

A processing approach for the collection and retention of mammalian cells within a high porosity polyester mesh having millimeter‐sized pores has been studied. Cell retention occurs via energizing the mesh with a low intensity, resonant acoustic field. The resulting acoustic field induces the interaction of cells with elements of the mesh or with each other and effectively prevents the entrainment of cells in the effluent stream. Experiments involving aqueous suspensions of polystyrene particles were used to provide benchmark data on the performance of the acoustic retention cell. Experiments using mouse hybridoma cells showed that retention densities of over 1.5 × 108 cell/mL could be obtained. In addition, the acoustic field was shown to produce a negligible effect on cell viability for short‐term exposure.

A distributed model of carbohydrate transport and metabolism in the liver during rest and high-intensity exercise.

A model of reaction and transport in the liver was developed that describes the metabolite concentration and reaction flux dynamics separately within the tissue and blood domains. The blood domain contains equations for convection, axial dispersion, and transport to the surrounding tissue; and the tissue domain consists of reactions representing key carbohydrate metabolic pathways. The model includes the metabolic heterogeneity of the liver by incorporating spatial variation of key enzymatic maximal activities. Simulation results of the overnight fasted, resting state agree closely with experimental values of overall glucose uptake and lactate output by the liver. The incorporation of zonation of glycolytic and gluconeogenic enzyme activities causes the expected increase in glycolysis and decrease in gluconeogenesis along the sinusoid length from periportal to perivenous regions, while fluxes are nearly constant along the sinusoid length in the absence of enzyme zonation. These results confirm that transport limitations are not sufficient to account for the observed tissue heterogeneity of metabolic fluxes. Model results indicate that changes in arterial substrate concentrations and hepatic blood flow rate, which occur in the high-intensity exercise state, are not sufficient to shift the liver metabolism enough to account for the 5-fold increase in hepatic glucose production measured during exercise. Changes in maximal activities, whether caused by exercise-induced changes in insulin, glucagon, or other hormones are shown to be needed to achieve the expected glucose output. This model provides a framework for evaluating the relative importance to hepatic function of various phenomenological changes that occur during exercise. The model can also be used to assess the potential effect of metabolic heterogeneity on metabolism.

Single fiber model of particle retention in an acoustically driven porous mesh

A method for the capture of small particles (tens of microns in diameter) from a continuously flowing suspension has recently been reported. This technique relies on a standing acoustic wave resonating in a rectangular chamber filled with a high-porosity mesh. Particles are retained in this chamber via a complex interaction between the acoustic field and the porous mesh. Although the mesh has a pore size two orders of magnitude larger than the particle diameter, collection efficiencies of 90% have been measured. A mathematical model has been developed to understand the experimentally observed phenomena and to be able to predict filtration performance. By examining a small region (a single fiber) of the porous mesh, the model has duplicated several experimental events such as the focusing of particles near an element of the mesh and the levitation of particles within pores. The single-fiber analysis forms the basis of modeling the overall performance of the particle filtration system.

Novel Hyaluronic Acid Coating for Potential Use in Glucose Sensor Design

Biocompatibility issues such as protein deposition and fibrous capsule formation significantly reduce the sensitivity of implanted glucose sensors. One of the approaches to improve the sensor biocompatibility is to disguise the sensors with coatings that mimic body conditions. We anticipate that a biomimetic coating based on hyaluronic acid (HA) would minimize the problems related to protein deposition and fibrous tissue formation. Diffusion experiments were conducted to assess the transport properties of HA coating on a polyvinyl alcohol (PVA) membrane using a classic diffusion cell. HA was coated on PVA membranes, as cross-linked HA membranes alone have poor mechanical strength. The effective diffusivities of glucose and oxygen in a HA/PVA membrane (95% confidence interval) are 1 +/- 0.26 x 10(-4) and 1.42 +/- 0.34 x 10(-4) cm(2)/min, respectively. The effective diffusivities of glucose and oxygen in HA/PVA membranes were approximately two-thirds when compared with the diffusivities of glucose and oxygen (7.29 x 10(-5) and 2.34 x 10(-4) cm(2)/min, respectively) in pure PVA membranes. The results indicate that the HA/PVA membranes have transport properties similar to the commonly used pure PVA membranes, and thus may find usefulness as a coating for implantable glucose sensors.

Determination of fatty acid methyl esters derived from algae Scenedesmus dimorphus biomass by GC–MS with one-step esterification of free fatty acids and transesterification of glycerolipids

Algae can synthesize, accumulate and store large amounts of lipids in its cells, which holds immense potential as a renewable source of biodiesel. In this work, we have developed and validated a GC-MS method for quantitation of fatty acids and glycerolipids in forms of fatty acid methyl esters derived from algae biomass. Algae Scenedesmus dimorphus dry mass was pulverized by mortar and pestle, then extracted by the modified Folch method and fractionated into free fatty acids and glycerolipids on aminopropyl solid-phase extraction cartridges. Fatty acid methyl esters were produced by an optimized one-step esterification of fatty acids and transesterification of glycerolipids with boron trichloride/methanol. The matrix effect, recoveries and stability of fatty acids and glycerolipids in algal matrix were first evaluated by spiking stable isotopes of pentadecanoic-2,2-d2 acid and glyceryl tri(hexadecanoate-2,2-d2 ) as surrogate analytes and tridecanoic-2,2-d2 acid as internal standard into algal matrix prior to sample extraction. Later, the method was validated in terms of lower limits of quantitation, linear calibration ranges, intra- and inter-assay precision and accuracy using tridecanoic-2,2-d2 acid as internal standard. The method developed has been applied to the quantitation of fatty acid methyl esters from free fatty acid and glycerolipid fractions of algae Scenedesmus dimorphus.