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The tympanic membrane (TM) primes the sound transmission mechanism due to special fibrous layers mainly of collagens II, III, and IV as a product of TM fibroblasts, while type I is less represented. In this study, human mesenchymal stromal cells (hMSCs) were cultured on star-branched poly(ε-caprolactone) (*PCL)-based nonwovens using a TM bioreactor and proper differentiating factors to induce the expression of the TM collagen types. The cell cultures were carried out for one week under static and dynamic conditions. Reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC) were used to assess collagen expression. A Finite Element Model was applied to calculate the stress distribution on the scaffolds under dynamic culture. Nanohydroxyapatite (HA) was used as a filler to change density and tensile strength of *PCL scaffolds. In dynamically cultured *PCL constructs, fibroblast surface marker was overexpressed, and collagen type II was revealed via IHC. Collagen types I, III and IV were also detected. Von Mises stress maps showed that during the bioreactor motion, the maximum stress in *PCL was double that in HA/*PCL scaffolds. By using a *PCL nonwoven scaffold, with suitable physico-mechanical properties, an oscillatory culture, and proper differentiative factors, hMSCs were committed into fibroblast lineage-producing TM-like collagens.

Tympanic Membrane Collagen Expression by Dynamically Cultured Human Mesenchymal Stromal Cell/Star-Branched Poly(ε-Caprolactone) Nonwoven Constructs