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

The present study aims at optimizing dermal fibroblast seeding and cultivation in Polyactive scaffolds in order to limit the biopsy size needed for autologous treatment of full-thickness skin defects and chronic wounds. Three methods for seeding and cultivation of fibroblasts in porous scaffolds were compared: dynamic seeding followed by static cultivation (DS), static seeding followed by static cultivation (SS) and dynamic seeding followed by dynamic cultivation (DD). Human dermal fibroblasts isolated from cultured explants were seeded in porous PEO/PBT (Polyactive) scaffolds. Samples were taken from 6 h to 21 days post-seeding for both histological analysis (cell distribution and extracellular matrix (ECM) formation), and quantitative cell number assay. The seeding efficiency 24 h post-seeding was 76% (+/-3.6%) for dynamically seeded matrices, whereas it was only 30% (+/-5%) for statically 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 pronounced in SS samples. Surprisingly, cells detached from DD samples as aggregates, 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 results indicate that the combination of dynamic seeding and static cultivation assures efficient utilization of isolated fibroblasts and improved neodermis formation, thereby allowing a reduction in the skin biopsy size needed for the engineering of living skin substitute.