Permeability studies in biomimetic glycosaminoglycan-hydrogel membranes
Author(s)Mattern, Kristin J. (Kristin Julie)
Massachusetts Institute of Technology. Dept. of Chemical Engineering.
William M. Deen.
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The rates of water and solute transport tend to be lower in fibrous materials than in bulk solution. This phenomenon of "hindered transport" is caused by steric, hydrodynamic, and electrostatic interactions between the solvent, the solute, and the fibers. In this research the effect of these interactions were studied using charged, fibrous agarose-glycosaminoglycan (GAG) membranes. The work was motivated by current research into the role of the glomerular capillary wall (GCW) in ultrafiltering blood plasma, which is the first step in the processing of blood by the kidney. The GCW is composed of three layers in series: an endothelium, a basement membrane, and an epithelium. Intreasing evidence from experimental results and theoretical models of the GCW indicate that the endothelial layer and its associated glycocalyx may significantly limit the transport of macromolecules across the glomerular barrier. The glycocalyx is primarily composed of proteoglycans, a fibrous mixtures of proteins and anionic GAG. GAG fibers are present in many other biological materials, such as basement membranes and cartilage, making the current studies in agarose-GAG relevant to a variety of biological systems. Agarose-GAG membranes were synthesized by using 1-cyano-4-(dimethylamino)pyridinium tetrafluoroborate (CDAP) to create reactive sites in thin agarose hydrogels. Chondroitin sulfate GAG was then covalently bound to the reactive sites via their terminal amine group. By manipulating the temperature and duration of key reaction steps, the synthesis was optimized to provide high bound GAG yields and a spatially uniform distribution of GAG throughout the membrane. Models of the coupling reaction were developed to guide the synthesis conditions, resulting in 70-115 [mu]m-thick membranes composed of 2-4 v% agarose and 0-0.4 v% GAG.(cont.) The Darcy (or hydraulic) permeabilities of the membranes with variable GAG content were measured with buffer solutions over a range of ionic strengths. In 3 v% agarose gels, the addition of even a small amount of GAG (0.4 v%) resulted in a two-fold reduction in the Darcy permeability. Electrokinetic coupling, caused by the flow of ions past the charged GAG fibers, resulted in an additional two-fold reduction in the opencircuit hydraulic permeability when the solution ionic strength was decreased from 1 M to 0.011 M. A microstructural model was used to understand these phenomena, accounting for the charge of the GAG fibers, heterogeneities in the agarose gels, and the mixture of agarose and GAG fibers. Several "mixing rules" from the literature were compared to predict the permeability of a mixture of fibers from structural models for a single fiber type. A fiber volume-weighted averaging of each fiber resistivity was found to be reasonably reliable, with a root-mean-squared error of 24% for 64 cases of fiber mixtures with differing radii, orientation, and/or charge. The microstructural model, using this mixing rule, accurately predicted the Darcy permeability when charge effects were suppressed at high ionic strengths; however, this model underestimated the reduction in permeability at lower ionic strengths when the effects of the GAG charge were significant. A macroscopic approach to electrokinetic effects using Donnan equilibria better captured the decrease in Darcy permeability with decreasing ionic strength. Studies of equilibrium partitioning and sieving were performed with BSA (an anionic globular protein) and Ficoll (an uncharged spherical polysaccharide). The effects of charge were studied by varying the ionic strength in experiments with BSA; the effects of solute size were examined by using Ficolls with radii ranging from 2.7 to 5.9 nm.(cont.) Solute permeability studies were performed in 4 v% agarose gels with 0 or 0.2 v% GAG. Partition coefficients (F) for BSA were measured for ionic strengths of 0.5 to 0.011 M. For BSA in agarose gels with no GAG, F = 0.65 ± 0.02 (standard error) and did not vary with ionic strength. In gels with 0.2 v% GAG, F = 0.54 ± 0.02 at ionic strengths = 0.2 M, but decreased by nearly two-fold at 0.011 M. For the same Stokes-Einstein radius (3.5-3.6 nm), the partition coefficients of BSA at neutral conditions and of Ficoll were similar in blank agarose gels, but differed by 15% in agarose-GAG gels. The partition coefficients for Ficolls decreased with increasing solute radius. A microstructural model for partitioning in fibrous materials was evaluated against the experimental observations. The experimental data were most consistent models that had a nearly homogeneous fiber density. The model was in good agreement for partition coefficients of Ficolls with various radii. The decrease in BSA partition coefficient at low ionic strengths was well captured by both microstructural and Donnan models of charge effects. The sieving coefficient (T), or ratio of downstream to upstream solute concentrations, was measured at moderately high Péclet number where T = FKc, where Kc is the convective hindrance factor. It has been hypothesized by others that Kc is independent of charge, such than any charge effects in T are caused by F. Sieving coefficients were measured under similar conditions as partition coefficients. Like partitioning, ionic strength had little effect on the sieving of BSA through blank agarose, but T was decreased by over half from 0.1 M to 0.011 M in gels with 0.2 v% GAG. In these agarose-GAG gels, there was not a statistically significant effect of ionic strength on Kc. Models used for agarose-GAG membranes were applied to a simple model of the glomerular endothelial glycocalyx.(cont.) The composition and structure of the glycocalyx are not well characterized, but some of its properties can be inferred from the properties of the entire capillary wall and the other capillary layers. Models of the hydraulic permeability of the endothelium suggest that the glycocalyx may be up to several hundred nanometers thick, but the GAG density is probably less than 4 v%. To determine if sieving through such a layer would contribute to glomerular selectivity, improved models for hindered transport coefficients are needed for fibrous systems where the fiber spacing is on the same scale as the solute size.
Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2008.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.Includes bibliographical references (p. 272-280).
DepartmentMassachusetts Institute of Technology. Dept. of Chemical Engineering.
Massachusetts Institute of Technology