Rebecca H. Li

The present studies were undertaken to evaluate the in vitro gel stability of the hydrogels alginate and agarose. Gel strength (of alginate and agarose) and protein diffusion (of alginate only) were shown to correlate with gel stability and to be useful techniques to monitor gel stability over time. The gel strengths of alginate and agarose were followed for a 90-day period using gel strength as a measure of gel stability. The gel strength of agarose diminished in the presence of cells because the cells likely interfered with the hydrogen bond formation required for agarose gelation. In the presence of cells, the gel strength of agarose decreased by an average of 25% from time 0 to 60 days, thereafter maintaining that value to 90 days. The gel strength of calcium- or barium-crosslinked alginate decreased over 90 days, with an equilibrium gel strength being achieved after 30 days. The presence of cells did not further decrease alginate gel strength. The gel strengths of calcium- and barium-crosslinked alginates were similar at 60 days-350 +/- 20 g and 300 +/- 60 g, respectively-indicating equivalence in their stability. The stability of calcium-crosslinked sodium alginate gels over a 60-day time period was monitored by diffusion of proteins ranging in molecular weight from 14.5 to 155 kD. From these diffusion measurements, the average pore size of the calcium-crosslinked alginate gels was estimated, using a semi-empirical model, to increase from approximately 176 to 289 A over a period of 60 days. (c) 1996 John Wiley & Sons, Inc.

Current membrane-based bioartificial organs consist of three basic components: (1) a synthetic membrane, (2) cells that secrete the product of interest, and (3) an encapsulated matrix material. Alginate and agarose have been widely used to encapsulate cells for artificial organ applications. It is important to understand the degree of transport resistance imparted by these matrices in cell encapsulation to determine if adequate nutrient and product fluxes can be obtained. For artificial organs in xenogeneic applications, it may also be important to determine the extent of immunoprotection offered by the matrix material. In this study, diffusion coefficients were measured for relevant solutes [ranging in size from oxygen to immunoglobulin G (IgG)] into and out of agarose and alginate gels. Alginate gels were produced by an extrusion/ionic crosslinking process using calcium while agarose gels were thermally gelled. The effect of varying crosslinking condition, polymer concentration, and direction of diffusion on transport was investigated. In general, 2-4% agarose gels offered little transport resistance for solutes up to 150 kD, while 1.5-3% alginate gels offered significant transport resistance for solutes in the molecular weight range 44-155 kD-lowering their diffusion rates from 10- to 100-fold as compared to their diffusion in water. Doubling the alginate concentration had a more significant effect on hindering diffusion of larger molecular weight species than did doubling the agarose concentration. Average pore diameters of approximately 170 and 147 A for 1.5 and 3% alginate gels, respectively, and 480 and 360 A for 2 and 4% agarose gels, respectively, were estimated using a semiempirical correlation based on diffusional transport of different-size solutes. The method developed for measuring diffusion in these gels is highly reproducible and useful for gels crosslinked in the cylindrical geometry, relevant for studying transport through matrices used in cell immobilization in the hollow fiber configuration. (c) 1996 John Wiley & Sons, Inc.

Differentiation of osteoprogenitor cells into osteoblasts is a pivotal step during the normal development and repair of bone. Upregulation of endogenous cellular alkaline phosphatase activity (AP) is a commonly used intracellular marker for the assessment of osteoprogenitor cell differentiation into the osteoblastic phenotype. Current methods for assaying AP involve colorimetric detection of the enzyme's activity using the synthetic substrate p-nitrophenol phosphate. In this paper, we explored an alternative method of detecting AP using the chemiluminescent substrate disodium 3-(4-methoxyspiro[1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.1(3,7)]decan]-4-yl) phenyl phosphate (CSPD) for enhanced AP sensitivity and a more simplified assay. Using calf intestinal alkaline phosphatase as a standardizing enzyme, we determined that the chemiluminescent detection system was four orders of magnitude more sensitive than the standard colorimetric method of detection. Moreover, the chemiluminescent assay was faster and markedly simpler to perform. To maximize the utility of this assay system, two osteoprogenitor cell lines were compared for their ability to generate alkaline phosphatases in vitro when exposed to recombinant human bone morphogenetic protein (rhBMP-2). The W20-17 cell line was substantially more sensitive to rhBMP-2 than the C3H10T1/2 cell line, where each cell line produced detectable increases in AP after exposure to rhBMP-2 levels of 5 and 25 ng/ml, respectively. The experimental design for AP responsiveness to rhBMP-2 was further optimized for chemiluminescent detection with the W20-17 cell line by comparing the effects of reporter cell seeding density and the day of assay. In summary, the data presented in this paper demonstrate a faster, simpler, and more sensitive chemiluminescent method to monitor changes in AP levels during osteodifferentiation.

Poly(vinyl alcohol) (PVA) foams were used as scaffolds in hollow fiber membrane-based cell encapsulation devices. The surrounding permselective membrane serves as an immunoisolation barrier while allowing metabolites and other small molecules to be freely transported. The internal matrix defines the microenvironment for the encapsulated cells. PC12 cell-containing devices represent one possible strategy for safe transplantation of dopamine-secreting cells for the treatment of dopamine-deficient diseases such as Parkinson's disease. PC12 cells--a dopamine-secreting cell line--were encapsulated with PVA foam as a matrix material in the lumen of these hollow fibers. In this work, we demonstrate the presence of the PVA matrix increased the catecholamine secretion efficiency of the cells as compared to devices containing a chitosan matrix. Devices were implanted in vivo into rodent striatum and device output of catecholamines was measured preimplant and post-explant. Evoked stores of dopamine remained constant (preimplant vs explant) for devices encapsulated with the foam matrix and increased with devices encapsulated with chitosan matrix. Cell proliferation within devices was inhibited in the presence of the foam matrix. Cell viability and distribution was significantly improved with the inclusion of the foam matrix in both in vitro and in vivo studies. In comparison to chitosan--a typical matrix material for PC12 cells--addition of a foam-type matrix altered the encapsulated cell microenvironment and resulted in more efficient secretion of catecholamines and improved distribution within the device resulting in smaller necrotic regions and a lower rate of cell proliferation.