Objective: Tissue engineering has recently been identified as a suitable tool to develop replacements for the tympanic membrane. This study aimed at investigating PEOT/PBT copolymer scaffolds obtained via electrospinning as potential eardrum substitutes using an in vitro approach. Study design: PEOT/PBT copolymer ultrafine fibre meshes were manufactured and characterized for morphology and pore features. The scaffolds were cultured with human mesenchymal stromal cells (MSCs) under a dynamic flow regimen to improve cell infiltration and viability. The expression of basic extracellular matrix molecules was evaluated and compared to that of the human eardrum. Finally, the interaction between human tympanic membrane keratinocytes and the scaffolds was investigated. Results: The electrospun scaffolds had a fibre diameter of 1.9 +/- 0.9 mm, thickness of 220 +/- 56 mm, and porosity of 80% +/- 0.8%. The macroporous meshes were suitable for cell infiltration, since 83.3% of relative void volume was from pores of 3-300-mm size. Over a four week culture, the bioreactor always increased the viability of human MSCs in the scaffolds with respect to traditional multi-well plate cultures. Viability was best at two weeks in culture, so this time-point was selected for further morphological and histochemical analyses. In static cultures, the human MSCs interacted with the top surface of the fibrous scaffold, while in dynamic cultures they infiltrated the mesh up to 240-mm depth. Adherent human MSCs maintained a non-differentiated phenotype, as shown by a very low production of glycosaminoglycans and glycoproteins, which are conversely highly expressed in the eardrum connective tissue layer. Human tympanic membrane keratinocytes adhered to the scaffold surface and were viable after 48 h. Conclusion: Using electrospinning combined with bioreactor culture appeared an efficient approach to develop biohybrid eardrum replacements in vitro, suitable for re-epithelialization in vivo.