Understanding and enhancing repair of the tympanic membrane

Hearing as one of the five human senses is important not only for communication but also for our quality of life and integration into society, impacting on speech and language skills. We are able to hear due the unique and complex hearing organ that is the ear. The ear is divided into three parts, the external ear (pinna and ear canal), middle ear, with its three little bones, and inner ear, where the hair cells sense vibrations and transfer information to the brain. In between the external and middle ear sits the ear drum, a thin transparent membrane that converts sound waves into vibrations. This membrane is very susceptible to damage caused by ear infections, pressure changes or head trauma. Its thought that 30% of children will suffer from breaks in their ear drum caused by acute ear inflammation (glue ear or otitis media). Most holes in the membrane heal rapidly without any intervention. Some holes, however, do not heal leading to ear pain, ringing in the ears (tinnitus), ear infections, and hearing loss. These perforation that fail to heal are called chronic and may be due to the size or position of the initial damage, or the presence of infectious agents. Such chronic perforations have to be corrected using surgery with tissue from elsewhere in the body grafted into the ear to bridge the gap.
How the ear drum is able to heal is unclear. It has been proposed that adult stem cells reside in the ear and are able to aid in regeneration and healing of holes. Research suggests that these stem cells are located in distinct parts of the ear drum, at the border (the annulus) and in the centre where the ear drum meets the middle ear bones (manubrium). Addition of stem cells, or signaling molecules that can stimulate repair pathways have been suggested as potential treatment for chronic perforations.
We plan to use modern imaging techniques, transgenic mouse models, and novel culture techniques to address some of the questions surrounding repair of ear drum perforations. The lead applicant, Prof Tucker, is an expert on the middle ear, while her collaborator, Prof Paul Martin, provides his experience of epithelial wound healing, to create a unique research project that tackles a serious clinical problem.
We first aim to determine the mechanisms at play during repair by following stem cell populations and their progeny in the ear after injury, and by perturbing some of the processes (cell migration, proliferation, vascularization) that are thought to play a role in repair. This will be followed by an analysis of the signals that control the ear drum stem cells, concentrating on the role of Wnt signaling. Wnts are a family of signaling molecules with essential role in stem cell maintenance and repair in many other tissues in the body. Wnt activity is found in the putative ear drum stem cell niche but nothing is currently known about the role of this pathway in the ear drum. We will look at the effect of enhancing and inhibiting Wnt signaling on the ability of the ear drum to heal.
Finally we will use our explant culture model to understand how current therapeutics, such as topical application of Fgf2, actually work.
The project takes a new approach (explant culture of the ear drum) to allow visualization of the ear as it heals and aims to provide the groundwork to create new and innovative ways to prevent chronic perforations in the future.

Ear drum perforations have been studied in a number of animal models. In each of the cases with animal models the perforation has been performed in vivo, with the healing followed up in a number of different animals. No live imaging of healing has been described due to the problems of accessing the ear. In this proposal we introduce a novel explant culture method in the mouse allowing accurate perforations to be made outside of the mouse and the healing process to be followed in situ in the same ear. This technique also allows us to take advantage of a suite of transgenic mice to follow clones of cells and specific stem/progenitor populations to address previously unanswerable questions. It also avoids the use of multiple animals sacrificed at different time points and invasive in vivo surgery.
To track cells live in culture we will use TdTomato transgenic mice crossed to a range of Cre reporter mice: putative stem cells and progeny (Sox2creERT2), neural crest (Wnt1cre), Epithelium (K14cre and K14creERT2), Endoderm (Sox17icre), mesoderm derived vasculature (Mesp1cre), combined with the use of DiI and DiO. To follow changes in proliferation in specific tissues during repair we will utilise R26Fucci2A mice, crossed to these various Cres. To perturb cellular processes (proliferation, vascularisation, cytoskeleton arrangement) we will add various small molecule inhibitors to the culture system. To understand how the putative stem cells niche is controlled we will investigate the role of Wnt signalling, through the use of pharmacological stimulators and inhibitors of this pathway and through the use of transgenice mice where the pathway is stimulated (Axin2lacZhom, B-catenin gain of function) or repressed (Wntless/GRP177). Finally we will utilise our model to focus on understanding the mechanisms behind the current clinical use of Fgf ligands to treat perforations.

Who will benefit from this research?

In addition to the academic beneficiaries the intention is that this research will also benefit a wider audience including ENT surgeons and their patients. The general public will also benefit in the shape of diverse public engagement activities focuses on an understanding of adult stem cells, while the postdoc employed will benefit from training in a new area, and access to a diverse set of colleagues, collaborators and the lay public.

How will they benefit?

With less well-known diseases such as chronic tympanic membrane perforations, patient charities providing support in the form of forums, groups and leaflets are indispensable to patients. We aim to engage with charities such as Action on Hearing Loss and use such platforms to disseminate our research updates and findings to patients in an appropriate manner.
This will provide patients with increased information about ear drum problems helping them understand their condition and the reasons why they suffer from this problem. Our research will therefore impact on patients and their understanding of their hearing problems.

The ultimate aim for the research is that the results should be integrated into clinical practice. For example, providing new information about how to enhance ear drum healing, and valuable insight into the factors that influence susceptibility to developing chronic non-healing perforations. The project therefore has potential to change surgical planning and procedure and to reduce the incidence of myringoplasty (complicated ear drum reconstructive surgery). In the long term therefore there is a real possibility of this work contributing to national health.

The public will benefit from access to clear, insightful and thought provoking activities (at Festivals and through the Museum of Life Sciences) to strengthen their understanding of stem cells in the body, and the ability of the body to heal.

The postdoc employed will also benefit hugely from being given this opportunity to learn a selection of new cutting edge techniques. This will be an important part of his/her training, and will provide key insight to how to tackle research.