Transcription and microRNA Profiling of Cultured Human Tympanic Membrane Epidermal Keratinocytes.

The human tympanic membrane (TM) has a thin outer epidermal layer which plays an important role in TM homeostasis and ear health. The specialised cells of the TM epidermis have a different physiology compared to normal skin epidermal keratinocytes, displaying a dynamic and constitutive migration that maintains a clear TM surface and assists in regeneration. Here, we characterise and compare molecular phenotypes in keratinocyte cultures from TM and normal skin. TM keratinocytes were isolated by enzymatic digestion and cultured in vitro. We compared global mRNA and microRNA expression of the cultured cells with that of human epidermal keratinocyte cultures. Genes with either relatively higher or lower expression were analysed further using the biostatistical tools g:Profiler and Ingenuity Pathway Analysis. Approximately 500 genes were found differentially expressed. Gene ontology enrichment and Ingenuity analyses identified cellular migration and closely related biological processes to be the most significant functions of the genes highly expressed in the TM keratinocytes. The genes of low expression showed a marked difference in homeobox (HOX) genes of clusters A and C, giving the TM keratinocytes a strikingly low HOX gene expression profile. An in vitro scratch wound assay showed a more individualised cell movement in cells from the tympanic membrane than normal epidermal keratinocytes. We identified 10 microRNAs with differential expression, several of which can also be linked to regulation of cell migration and expression of HOX genes. Our data provides clues to understanding the specific physiological properties of TM keratinocytes, including candidate genes for constitutive migration, and may thus help focus further research.

Optical Coherence Tomography of the Tympanic Membrane and Middle Ear: A Review.

Objective To evaluate the recent developments in optical coherence tomography (OCT) for tympanic membrane (TM) and middle ear (ME) imaging and to identify what further development is required for the technology to be integrated into common clinical use. Data Sources PubMed, Embase, Google Scholar, Scopus, and Web of Science. Review Methods A comprehensive literature search was performed for English language articles published from January 1966 to January 2018 with the keywords "tympanic membrane or middle ear,""optical coherence tomography," and "imaging." Conclusion Conventional imaging techniques cannot adequately resolve the microscale features of TM and ME, sometimes necessitating diagnostic exploratory surgery in challenging otologic pathology. As a high-resolution noninvasive imaging technique, OCT offers promise as a diagnostic aid for otologic conditions, such as otitis media, cholesteatoma, and conductive hearing loss. Using OCT vibrometry to image the nanoscale vibrations of the TM and ME as they conduct acoustic waves may detect the location of ossicular chain dysfunction and differentiate between stapes fixation and incus-stapes discontinuity. The capacity of OCT to image depth and thickness at high resolution allows 3-dimensional volumetric reconstruction of the ME and has potential use for reconstructive tympanoplasty planning and the follow-up of ossicular prostheses. Implications for Practice To achieve common clinical use beyond these initial discoveries, future in vivo imaging devices must feature low-cost probe or endoscopic designs and faster imaging speeds and demonstrate superior diagnostic utility to computed tomography and magnetic resonance imaging. While such technology has been available for OCT, its translation requires focused development through a close collaboration between engineers and clinicians.

Scaffolds for tympanic membrane regeneration in rats.

Tympanic membrane (TM) perforations lead to significant hearing loss and result in possible infection of the middle ear. Myringoplasty is commonly performed to repair chronic perforations. Although various grafts and materials have been used to promote TM regeneration, all have associated limitations. The aim of this study was to evaluate the efficacy and feasibility of two graft materials, silk fibroin scaffold (SFS) and porcine-derived acellular collagen type I/III scaffold (ACS), compared with two commonly used graft materials (paper patch and Gelfoam) for the promotion of TM regeneration. These scaffolds were implanted using on-lay myringoplasty in an acute TM perforation rat model. Surface morphology of the scaffolds was observed with scanning electron microscopy. The morphology of the TM was assessed at various time points postimplantation using otoscopy, light and electron microscopy, and functional outcomes by auditory brainstem responses. We found that SFS and ACS significantly accelerated the TM perforation closure, obtained optimal TM thickness, and resulted in better trilaminar morphology with well-organized collagen fibers and early restoration of hearing. However, paper patch and Gelfoam lost their scaffold function in the early stages and showed an inflammatory response, which may have contributed to delayed healing. This study indicates that compared with paper patch and Gelfoam, SFS and ACS are more effective in promoting an early TM regeneration and an improved hearing, suggesting that these scaffolds may be potential substitutes for clinical use.

Comparative analysis of paracrine factor expression in human adult mesenchymal stem cells derived from bone marrow, adipose, and dermal tissue.

Human adult mesenchymal stem cells (MSCs) support the engineering of functional tissue constructs by secreting angiogenic and cytoprotective factors, which act in a paracrine fashion to influence cell survival and vascularization. MSCs have been isolated from many different tissue sources, but little is known about how paracrine factor secretion varies between different MSC populations. We evaluated paracrine factor expression patterns in MSCs isolated from adipose tissue (ASCs), bone marrow (BMSCs), and dermal tissues [dermal sheath cells (DSCs) and dermal papilla cells (DPCs)]. Specifically, mRNA expression analysis identified insulin-like growth factor-1 (IGF-1), vascular endothelial growth factor-D (VEGF-D), and interleukin-8 (IL-8) to be expressed at higher levels in ASCs compared with other MSC populations whereas VEGF-A, angiogenin, basic fibroblast growth factor (bFGF), and nerve growth factor (NGF) were expressed at comparable levels among the MSC populations examined. Analysis of conditioned media (CM) protein confirmed the comparable level of angiogenin and VEGF-A secretion in all MSC populations and showed that DSCs and DPCs produced significantly higher concentrations of leptin. Functional assays examining in vitro angiogenic paracrine activity showed that incubation of endothelial cells in ASC(CM) resulted in increased tubulogenic efficiency compared with that observed in DPC(CM). Using neutralizing antibodies we concluded that VEGF-A and VEGF-D were 2 of the major growth factors secreted by ASCs that supported endothelial tubulogenesis. The variation in paracrine factors of different MSC populations contributes to different levels of angiogenic activity and ASCs maybe preferred over other MSC populations for augmenting therapeutic approaches dependent upon angiogenesis.

Hypoxic preconditioning enhances survival of human adipose-derived stem cells and conditions endothelial cells in vitro.

To grow more robust cardiac tissue for implantation in vivo, strategies to improve survival of implanted stem cells are required. Here we report the protective effects of hypoxic preconditioning (HPC) and identify mechanisms for improving survival of adipose-derived stem cells (ASC) in vitro. Human ASC were preconditioned for 24 h with hypoxia and then exposed to simulated ischemia for a further 24 h. HPC significantly increased ASC viability, and reduced cell injury and apoptosis compared with non-preconditioned cells under ischemic conditions, as shown by 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), lactate dehydrogenase-release, and caspase activity assays. Preconditioned ASC increased levels of hypoxia-inducible factor-1 alpha and secreted significantly more of the downstream target vascular endothelial growth factor (VEGF-A; 13-fold) compared with control during the 24 h. Exogenous VEGF (50 ng/mL) increased phosphorylation of Akt without affecting ERK1/2, JNK, or p38 MAPK protein levels. Phospho-Akt was also increased in preconditioned ASC compared with non-preconditioned ASC, an effect that may be mediated via VEGF-A. Importantly, the protective effects of HPC were abolished by a neutralizing antibody against VEGF-A and the phosphoinositol 3-kinase inhibitor LY294002, demonstrating the importance of VEGF-A and Akt in hypoxia-induced ASC survival. Importantly, we showed that media derived from hypoxic preconditioned ASC support endothelial cell survival and endothelial tube formation in vitro. Our in vitro findings indicate that HPC may be a promising strategy to improve survival of ASC and promote angiogenesis in ischemic environments.