Abstract
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.
Highlights
The tympanic membrane (TM), or eardrum, plays a fundamental role in sound transmission, its unique anatomical and physical features being ideal to function in varying frequency ranges
MO, USA); absolute ethanol was purchased from Carlo Erba (Milan, Italy); Dulbecco’s phosphate buffered saline (D-PBS), penicillin, streptomycin, fluconazole were purchased from Lonza (Milan, Italy); Transforming Growth Factor beta 1 (TGF-β1), ascorbic acid, heat-inactivated fetal bovine serum (FBS), L-Glutamine, Rhodaminate Sytox green, Rhodaminate phalloidin were purchased from Invitrogen (Carlsbad, CA, USA); RNeasy® Plus Mini kit was purchased from Qiagen (Hilden, Germany); SsoFastTM EvaGreen® supermix, iScriptTM cDNA synthesis kit were purchase from Bio-Rad Laboratories (Hercules, CA, USA); neutral buffered formalin, optimal cutting temperature (OCT) medium were purchased from Bio-Optica (Milan, Italy); paraffin Histoplast LP
This study aimed at identifying biochemical, physical, and mechanical signals to induce the differentiation of human mesenchymal stromal cells (hMSCs) into fibroblasts synthesizing collagen type II, in addition to other collagens found in the eardrum, such as types I, III, and IV, which can spontaneously be secreted by other fibroblasts
Summary
The tympanic membrane (TM), or eardrum, plays a fundamental role in sound transmission, its unique anatomical and physical features being ideal to function in varying frequency ranges. TM has been morphologically and functionally described since the 1970s [1,2]. TM is a three-layer structure, consisting of two histo-morphologically distinct areas—pars tensa, the largest, and pars flaccida, the thickest [4,5]. The outer and inner linings of the pars tensa are made up of epithelia, a keratinizing epithelium, and a simple cuboidal epithelium, respectively [4]. In between these two epithelia, a structural layer named lamina propria is located, which is connective tissue proper
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