(93o) Three-Dimensional Cell Colonization in a Sulfate Rich Environment

The field of tissue engineering offers great potential in rebuilding healthy viable tissues and organs. One of the many hurdles in this field is incomplete understanding of the influence of 3-dimensional environment on cell colonization. This research focused on evaluating the influence of differently charged 3-D and 2-D scaffolds on colonization of fibroblasts. Chitosan, a positively charged polymer in solution, was used to make structures, combined with negatively charged dextran sulfate (5kD, 10kD, and 500kD), a synthetic glycosaminoglycan-analogue. 2D matrices were formed by air drying chitosan solution and then immobilizing DS. 3D matrices were formed inside 24-well culture plate using 300?ÝL of chitosan solution in each well and frozen at -20 degrees C for at least 4 hours prior to lyophilizing overnight at -86 degrees C. Two methods were used to form the negatively charged scaffolds: i) chitosan structures were formed first and then allowed to react with dextran sulfate; ii) dextran sulfate and chitosan in solution and then scaffolds were formed. Various volumes, 10, 100, and 500 ?ÝL of dextran sulfate (DS) were used to determine the optimum combination in both cases. Analysis of pore structures with a scanning electron microscope showed increased surface roughness and open pore architecture in-DS-chitosan matrices. Further, matrices formed with 50 ?ÝL of DS solution showed optimum porosity and mechanical stability. Analysis for the quantity and stable immobilization of DS by Toluidine blue assay indicated significant presence of DS in the 3D matrices even after seven days of incubation in phosphate buffered saline solution. Next, 25,000 fibroblasts per matrix were seeded onto 3-D matrices and analyzed for proliferation by MTT-formazan assay, and cytoskeletal organization by actin staining. These results showed MW of DS-dependent cell growth (with low MW more favorable) and organized actin distribution in 3-D matrices. Analysis of fibronectin binding by ELISA showed negligible binding to all the DS-containing matrices. In summary, results show significant difference in the influence of sulfate charge on cell colonization.