Static and dynamic fibroblast seeding and cultivation in porous PEO/PBT scaffolds

The present study aims at optimizing dermal fibroblast seeding and cultivat ion in Polyactive scaffolds in order to limit the biopsy size needed for au tologous treatment of full-thickness skin defects and chronic wounds. Three methods for seeding and cultivation of fibroblasts in porous scaffolds wer e compared: dynamic seeding followed by static cultivation (DS), static see ding followed by static cultivation (SS) and dynamic seeding followed by dy namic cultivation (DD). Human dermal fibroblasts isolated from cultured exp lants were seeded in porous PEO/PBT (Polyactive) scaffolds. Samples were ta ken from 6 h to 21 days post-seeding for both histological analysis (cell d istribution and extracellular matrix (ECM) formation), and quantitative cel l number assay. The seeding efficiency 24 h post-seeding was 76% (+/- 3.6%) for dynamically seeded matrices, whereas it was only 30% (+/- 5%) for stat ically seeded matrices (p<0.001). ECM formation was abundant in DS samples already at day 10, while even after 21 days ECM formation was less pronounc ed in SS samples. Surprisingly, cells detached from DD samples as aggregate s, starting from day 10. Cell numbers as assayed quantitatively correlated with the histological results. At all timepoints cell numbers found for DS samples were significantly higher as compared to SS samples. At day 21, DS samples contained approximately twofold more cells as compared to SS and DD samples and comprised ECM consisting of collagen types I and III. Our resu lts indicate that the combination of dynamic seeding and static cultivation assures efficient utilization of isolated fibroblasts and improved neoderm is formation, thereby allowing a reduction in the skin biopsy size needed f or the engineering of living skin substitute. (C) 1999 Kluwer Academic Publ ishers.