The effects of substrate composition and topography on the characteristics and growth of cell cultures of cochlear fibrocytes.

Spiral ligament fibrocytes of the cochlea play homoeostatic roles in hearing and their degeneration contributes to hearing loss. Culturing fibrocytes in vitro provides a way to evaluate their functional characteristics and study possible therapies for hearing loss. We investigated whether in vivo characteristics of fibrocytes could be recapitulated in vitro by modifying the culture substrates and carried out proof of concept studies for potential transplantation of culture cells into the inner ear. Fibrocytes cultured from 4 to 5-week old CD/1 mice were grown on 2D substrates coated with collagen I, II, V or IX and, after harvesting, onto or into 3D substrates (hydrogels) of collagen I alone or mixed collagen I and II at a 1:1 ratio. We also assessed magnetic nanoparticle (MNP) uptake. Cell counts, immunohistochemical and ultrastructural studies showed that fibrocytes grown on 2D substrates proliferated, formed both small spindle-shaped and large flat cells that avidly took up MNPs. Of the different collagen coatings, only collagen II had an effect, causing a reduced size of the larger cells. On hydrogels, the cells were plump/rounded with extended processes, resembling native cells. They formed networks over the surface and became incorporated into the gel. In all culture formats, the majority co-expressed caldesmon, aquaporin 1, S-100 and sodium potassium ATPase, indicating a mixed or uncharacterised phenotype. Time-course experiments showed a decrease to ∼50% of the starting population by 4d after seeding on collagen I hydrogels, but better survival (∼60%) was found on collagen I + II gels, whilst TEM revealed the presence of apoptotic cells. Cells grown within gels additionally showed necrosis. These results demonstrate that fibrocytes grown in 3D recapitulate in vivo morphology of native fibrocytes, but have poorer survival, compared with 2D. Therefore hydrogel cultures could be used to study fibrocyte function and might also offer avenues for cell-replacement therapies, but need more optimization for therapeutic use. Fibrocyte function could be modified using MNPs in combination, for example, with gene transfection.

Epigenetic mechanisms in Tendon Ageing.

INTRODUCTION: Tendon is a composite material with a well-ordered hierarchical structure exhibiting viscoelastic properties designed to transfer force. It is recognized that the incidence of tendon injury increases with age, suggesting a deterioration in homeostatic mechanisms or reparative processes. This review summarizes epigenetic mechanisms identified in ageing healthy tendon. SOURCES OF DATA: We searched multiple databases to produce a systematic review on the role of epigenetic mechanisms in tendon ageing. AREAS OF AGREEMENT: Epigenetic mechanisms are important in predisposing ageing tendon to injury. AREAS OF CONTROVERSY: The relative importance of epigenetic mechanisms are unknown in terms of promoting healthy ageing. It is also unknown whether these changes represent protective mechanisms to function or predispose to pathology. GROWING POINT: Epigenetic markers in ageing tendon, which are under-researched including genome-wide chromatin accessibility, should be investigated. AREAS TIMELY FOR DEVELOPING RESEARCH: Metanalysis through integration of multiple datasets and platforms will enable a holistic understanding of the epigenome in ageing and its relevance to disease.