Otology and audiological medicine

A prominent aspect of ear disease regenerative medicine is the potential use of stem cells to regenerate irreversible hearing loss in sensorineural disease. Sensorineural hearing loss is a result of inner-ear cochlear dysfunction, and involves the loss of sensory hair cells and/or a secondary degeneration of spiral ganglion neurons. It is the commonest disability in developed countries, with a considerable socioeconomic impact.Reference Hu and Ulfendahl¹¹

Ageing is a major factor in hearing decline,Reference Roth, Hanebuth and Probst¹² with an estimated 63.1 per cent of individuals aged 70 years and older living with hearing loss in the USA.Reference Lin¹³ Hearing loss is associated with a number of health issues, including delayed cognitive skill development, depression,Reference Monzani, Galeazzi, Genovese, Marrara and Martini¹⁴ cognitive decline and dementia.Reference Lin^13, Reference Lin, Metter, O'Brien, Resnick, Zonderman and Ferrucci¹⁵ These issues will become more concerning with population ageing and the increased exposure to loud environments, particularly in the younger demographic.Reference Daniel¹⁶

Milestone technological advances, such as cochlear implantation and bone-anchored hearing aids, have radically improved the quality of life for many patients with sensorineural hearing loss. The next step would be the regeneration of neural and cochlear tissue to restore hearing.Reference Ibekwe, Ramma and Chindo¹⁷ The two main regenerative strategies currently being researched are: differentiation of endogenous stem cells into new cochlear hair cells, and the introduction of exogenous cells into the inner ear to replace injured hair cells and/or neurons.Reference Hu and Ulfendahl¹¹

The first approach involves inducing resident cochlear cells to proliferate and differentiate into new hair cells and spiral ganglion neurons, to replace lost cells. This would most likely involve targeting stem cells within the inner ear that maintain the ability to differentiate. It is important to note that humans and other mammals do not have the innate ability to regenerate lost sensory cells in the cochlea once development is complete.Reference Rubel, Furrer and Stone¹⁸ Therefore, current research efforts have focused on determining the existence of stem cells in the cochlea that could be exploited for endogenous regeneration strategies. Thus far, there has been little evidence that these cells exist in the mature cochlea,Reference Okano and Kelley¹⁹ although there are some (in vitro) data to suggest the presence of a limited number of cells possessing favourable characteristics including limited proliferation capabilityReference Rask-Andersen, Boström, Gerdin, Kinnefors, Nyberg and Engstrand²⁰ and expression of adult stem cell markers.Reference Chai, Xia, Wang, Jan, Hayashi and Bermingham-McDonogh²¹ Alternatively, there remains the possibility of inducing regeneration by utilising the fully differentiated cells of the inner ear. This could be achieved by: de-differentiating the cells into a more naive state normally only found during development, in order to regenerate sensory cells; direct transdifferentiation of the cells to the target cell type of interest; or using reprogramming strategies.Reference Jopling, Boue and Izpisua Belmonte²²

The second approach involves the transplantation of exogenous cells, and can take advantage of an array of stem cell sources, such as embryonic stem cells, induced pluripotent stem cells, mesenchymal stem cells from the bone marrow, or adipose tissue and amniotic fluid-derived stem cells. There are a number of challenges to this approach. First, there is the potential tumourigenic risk that is intrinsic to all cell-based therapies. Second, there is the issue of controlling differentiation of these cells to ensure that the sensory cells are produced without contamination from other cell types that could interfere with the transplantation of the sensory cells or the function of the organ. Finally, there is the issue of ensuring the survival and integration of exogenous cells into the cochlear sensory epithelium, given the relatively hostile biochemical and immunological environment, the complex architecture of the organ of Corti, and the repair mechanisms of the cochlea, which result in closure of any lesions in the sensory epithelium that arise from hair cell loss. There is also the added technical challenge of delivering the cells to the tissue, although a surgical approach could be used to expose the cochlea in order to implant or inject therapeutic cells into the perilymph (via the scala vestibuli and scala tympani), the endolymph (in the scala media) and the modiolus.Reference Pau, Fagan and Oleskevich²³ A number of promising studies have investigated how various stem cells could be differentiated into sensorineural cells.Reference Chen, Jongkamonwiwat, Abbas, Eshtan, Johnson and Kuhn^24–Reference Oshima, Shin, Diensthuber, Peng, Ricci and Heller²⁶ However, the research is still in its early stages and translation to the clinical setting is distant whilst the above issues are still being addressed.

The generation of new hair cells and auditory neurons is particularly challenging because of the importance of hair cell alignment and tonotopic organisation. Regenerated hair cells must be correctly orientated within the sensory epithelium to restore functionality, and must be orientated in the same polarity as native hair cells to transduce relevant stimuli. Auditory hair cells vary in terms of biophysiology depending on their position along the tonotopic axis of the cochlea, and thus newly generated cells must reflect the location-dependent properties of native cells.Reference Geleoc and Holt²⁷

The in vitro generation of hair-cell-like cells and auditory neurons from embryonic stem cells, induced pluripotent stem cells and fetal auditory stem cells has been a promising area of research.Reference Chen, Johnson, Marcotti, Andrews, Moore and Rivolta²⁸ Most recently, Koehler et al. created a stepwise protocol differentiating inner-ear sensory epithelia from mouse embryonic stem cells in three-dimensional (3D) culture.Reference Koehler, Mikosz, Molosh, Patel and Hashino²⁵ Using an in vivo 3D culture in combination with bone morphogenetic protein activation and transforming growth factor β inhibition, they stimulated sequential formation of sensory epithelia (non-neural, pre-placodal then otic placode-like epithelia). The sensory epithelia with hair cells possessed functional and structural properties of native hair cells. Koehler's paper highlights the most recent advance in stem cell therapy to treat sensorineural deafness. However, there is a considerable amount of research into the use of genetic and molecular therapies for this condition.Reference Geleoc and Holt²⁷

Surgical intervention to treat middle-ear diseases can disrupt the middle-ear mucosa (an epithelium that serves to maintain a functional middle-ear environment), which can lead to retraction of the tympanic membrane causing further disease or even hearing loss. Little research has been carried out to address this issue. The most promising potential intervention appears to be the use of transplanted cells to replace the lost mucosal epithelium.Reference Yaguchi, Wada, Uchimizu, Tanaka, Kojima and Moriyama^29, Reference Wada, Tanaka, Kojima, Inamatsu, Yoshizato and Moriyama³⁰ For ossicle reconstruction, prosthetics are typically used, but there has been some work looking at using tissue engineering for this purpose, which involves culturing mesenchymal stem cells under osteogenic conditions on bioresorbable 3D scaffolds.Reference Danti, D'Alessandro, Pietrabissa, Petrini and Berrettini^31, Reference Luers, Beutner and Huttenbrink³²

Repair of tympanic membrane perforation is an important target for the use of regenerative techniques, especially in chronic perforation. Whilst the majority of the research is based on animal models of acute tympanic membrane perforation, regenerative therapy would probably be targeted at patients with chronic perforation, where currently available interventions have already failed.Reference Hong, Bance and Gratzer³³ Of note, Kanemaru et al. randomised 63 patients with chronic tympanic membrane perforations to receive basic fibroblast growth factor on a gelatine sponge with fibrin glue, or gelatine sponge plus fibrin glue alone.Reference Kanemaru, Umeda, Kitani, Nakamura, Hirano and Ito³⁴ The biomolecular intervention significantly improved healing rates and mean hearing level compared to controls. Other biomolecules under current investigation include hyaluronic acid, epidermal growth factor and platelet-derived growth factor.Reference Hong, Bance and Gratzer³³

Bioscaffolds, such as acellular dermis, have been used to repair the tympanic membrane. A randomised, unblinded trial of acellular dermis versus temporalis fascia for tympanoplasty found that acellular dermis was associated with shorter operative time and less post-operative pain.Reference Raj, Sayal, Rathore and Meher³⁵ This trial was limited by methodology and sample size (n = 42), but is one of few human trials using this technique at the time of writing.

There is a relative paucity of evidence for the use of stem cells in tympanic membrane perforation. There are a number of key studies in animal models that have shown promise. Rahman and colleagues applied mesenchymal stem cells to rats with chronic tympanic membrane perforations and found that the stem cells enhanced healing and reduced the stiffness of the healed tympanic membrane, leading to enhanced restoration.Reference Rahman, Olivius, Dirckx, Von Unge and Hultcrantz³⁶

Outer-ear reconstruction typically uses autologous costal cartilage grafts from the patient. This method involves multistage surgery and permanent loss of cartilage from the donor site. A good alternative would be a stem cell source combined with a compatible scaffold to mimic cartilage.Reference Nayyer, Patel, Esmaeili, Rippel, Birchall and O'Toole³⁷ Recent work using adipose-derived stem cells taken from abdominal tissue, grown on a polyhedral oligomeric silsesquioxane modified polycarbonate urea-urethane (‘POSS-PCU’) bioscaffold, shows great promise for clinical use.Reference Guasti, Vagaska, Bulstrode, Seifalian and Ferretti³⁸ If cost-effectiveness, therapeutic equivalence and safety can be demonstrated, tissue engineering approaches may provide longer-lasting, more effective repairs, without the need to create a donor site. However, although the results of many studies are encouraging, more human clinical trials are required that directly compare tissue engineering with conventional (autologous grafting) techniques. Such techniques may be especially useful in revision cases where autologous graft material may be lacking.

Rhinology

Unlike in otology and laryngology, rhinology plays a greater role in the provision of stem cells rather than in the receipt. The nose contains several populations of relatively easy to access autogenous stem cells that have great potential for use in the regeneration of nerve and cartilage elsewhere. In the olfactory epithelium, olfactory receptor neurons are supported by olfactory ensheathing cells, a form of glial cell with the potential to support neural regeneration within the olfactory epitheliumReference Au and Roskams³⁹ and when transplanted into other tissues such as the brain and spinal cord.Reference Lindsay, Riddell and Barnett^40–Reference Vats and Birchall⁴² De Corgnol and colleagues investigated the in vivo use of olfactory ensheathing cells in rats to assist surgical reinnervation of the vocal folds following vagus nerve section.Reference de Corgnol, Guerout, Duclos, Verin and Marie⁴³ Rats with the olfactory ensheathing cell intervention had improved reinnervation results compared to controls in terms of post-operative vocal fold function.

Choi et al. prospectively analysed nasal biopsies taken during transsphenoidal surgery.Reference Choi, Li, Law, Powell and Raisman⁴⁴ This revealed that the nasal septal mucosa, particularly the superior-posterior area, was a rich source of olfactory ensheathing cells. There has also been some suggestion of the existence of a mesenchymal stem cell population in the olfactory epitheliumReference Murrell, Féron, Wetzig, Cameron, Splatt and Bellette^45, Reference Nivet, Vignes, Girard, Pierrisnard, Baril and Devèze⁴⁶ and respiratory epithelium.Reference Jakob, Hemeda, Janeschik, Bootz, Rotter and Lang⁴⁷ The nasal septum is also rich in chondrogenic cells. Cartilage is a much sought after tissue in the wider regenerative surgery field, with particular prominence in regenerative orthopaedics procedures, and especially in reconstruction of the airways.Reference Elliott, De Coppi, Speggiorin, Roebuck, Butler and Samuel^10, Reference Gómez-Barrena, Rosset, Müller, Giordano, Bunu and Layrolle^48, Reference Wu, Cai, Zhang, Karperien and Lin⁴⁹ In addition, chondrogenic cells harvested from the cranium and airways are capable of healing, unlike adult musculoskeletal cartilage.

In a recent observational first-in-human clinical trial, five patients received tissue-engineered cartilage implants for nasal reconstructive surgery.Reference Birchall and Seifalian^50, Reference Fulco, Miot, Haug, Barbero, Wixmerten and Feliciano⁵¹ The investigators reported restoration of contour and nasal airflow in all five patients studied. In addition, at 12 months, no adverse events were recorded, and patients were satisfied with the aesthetic and functional outcomes. Similar composite tissue-engineered tissues are likely to be useful in situations where other options have been exhausted and where tissue for reconstruction is lacking.

Laryngology

Regenerative medicine for laryngology, to replace parts or the whole of the airway, oesophagus and larynx, requires a much larger scale that is best suited to a tissue engineering approach. A combination of a scaffold with the right physical and functional properties, seeded with cells that promote regeneration and allow function, that can be used for transplanting or grafting, has been the primary aim.

Airway reconstruction is indicated in patients with extensive tracheal stenosis following malignancy, trauma or in cases of congenital malformations.Reference Lange, Fishman, Elliott, De Coppi and Birchall⁴ In cases where tracheal resection and primary anastomosis is not possible, such as after extensive burns, trauma or tumour resection, tracheal replacement may be indicated.Reference Fishman, De Coppi, Elliott, Atala, Birchall and Macchiarini⁵² Both cadaveric and synthetic tracheal transplants have been used, but both convey considerable morbidity, including foreign body reactions and fibrotic stenosis. Fresh, viable tracheal allografts usually require considerable immunosuppressive therapy; they are rapidly rejected by the host without such therapy, leading to necrosis and fibrotic stenosis.Reference Tojo, Niwaya and Sawabata⁵³

In 2008, the first stem cell based tissue-engineered neo-trachea was successfully utilised in a patient.Reference Macchiarini, Jungebluth, Go, Asnaghi, Rees and Cogan⁵⁴ The trachea was generated by an in vitro tissue engineering process using a donor trachea, which was decellularised, then readily colonised by the recipient's epithelial cells and mesenchymal stem cell derived chondrocytes. Importantly, the nature of the patient's disease in this case made in vitro (thus delayed) preparation of the tissue-engineered organ possible with subsequent implantation. Five-year follow-up data have recently been reported, indicating positive long-term outcomes.Reference Gonfiotti, Jaus, Barale, Baiguera, Comin and Lavorini⁵⁵ Following this success, Elliot and colleagues utilised an in vivo approach to develop a tissue-engineered neo-trachea for a child requiring emergent airway reconstruction.Reference Elliott, De Coppi, Speggiorin, Roebuck, Butler and Samuel¹⁰ By using a decellularised cadaveric trachea seeded with autologous mesenchymal stem cells and exposed to topical biochemical inducers of differentiation, the airway was successfully reconstructed with total functionality at two years post-operation.

These significant advances in the generation of stem cell derived airway grafts and the development of advanced bioreactors has led to frontier work on the construction of a tissue-engineered larynx.Reference Fishman, Wiles, Lowdell, De Coppi, Elliott and Atala⁵⁶ The larynx, unlike the trachea, is a dynamic organ. This adds considerable complexity to the generation of laryngeal reconstructions, something that is also observed in the reconstruction of other dynamic organs, such as the oesophagus. A neo-larynx therefore requires functional muscle tissue with appropriate reinnervation.

Decellularised skeletal muscle matrices have been described as a potential scaffold for the generation of the muscular activity required for an engineered larynx.Reference Fishman, Lowdell, Ansari, Sibbons, De Coppi and Birchall^57, Reference Fishman, Lowdell, Urbani, Ansari, Burns and Turmaine⁵⁸ Fishman et al. implanted decellularised laryngeal muscle scaffolds into a rabbit model to determine the in vivo effects on scaffold biodegradation time and immunogenicity following implantation.Reference Fishman, Lowdell, Ansari, Sibbons, De Coppi and Birchall^57, Reference Fishman, Lowdell, Urbani, Ansari, Burns and Turmaine⁵⁸ They found that their decellularisation process resulted in total DNA clearance and down-regulation of major histocompatibility complex expression, whilst maintaining the scaffold's structural integrity. This improved the longevity of the xenogeneic bioscaffold by reducing the cell-mediated immune response, which also increased its neo-angiogenic potential. The in situ implantation of such a scaffold in combination with stem cells and growth factors has been described in the reconstruction of vocal folds in an animal model.Reference Johnson, Fox, Chen and Thibeault⁵⁹ This has yielded promising results in terms of graft survival, functionality and safety, but further research in humans is warranted.

The use of human stem cells as a direct therapy to the larynx, with a view to enhanced healing, is another promising area of research. A small animal study (n = 12) that used injectable mesenchymal stem cells which improved vocal fold healing in rabbits found better viscoelasticity of the folds and enhanced healing following injury.Reference Svensson, Nagubothu, Cedervall, Chan, Le Blanc and Kimura⁶⁰

Ultimately, our understanding of stem cell biology, biomaterials and transplantation immunobiology could lead to airway transplantation without systemic immunosuppression. This, along with new technologies such as 3D printing, a novel technique with the potential to render microscopic control over how cells are incorporated and grown onto the tissue-engineered airway, will have a significant impact on patients with airway disease in the future.Reference Fishman, Wiles, Lowdell, De Coppi, Elliott and Atala^56, Reference Zopf, Hollister, Nelson, Ohye and Green⁶¹

Head and neck

The loss of salivary gland function can result from pharmacological intervention, surgical resection, radiotherapy and autoimmune diseases. It leads to xerostomia, which in turn may predispose an individual to infections, dysphagia and oral mucosal infections. Xerostomia is usually managed with synthetic saliva supplementation.

There has been considerable research into the development of salivary gland regeneration, with an emphasis on using direct stem cell therapy to heal and regenerate the glands following radiotherapy for head and neck cancer, which is a common cause of xerostomia in the developed world.Reference Aframian and Palmon^62, Reference Ogawa, Oshima, Imamura, Sekine, Ishida and Yamashita⁶³ There is comparatively less research to date on the use of tissue engineering techniques used to develop neo-organs to solve this clinical conundrum.

In 2007, Joraku and colleagues successfully regenerated fully functional salivary glands from a single human salivary gland cell using 3D tissue engineering methods in vitro.Reference Joraku, Sullivan, Yoo and Atala⁶⁴ However, at the time of writing, this has not been translated into clinical use. In contrast, the use of direct cellular therapy appears to have had more promising results. Xiong and colleagues exposed rats to radiotherapy to replicate this pathology, and then injected human adipose-derived stem cells into their submandibular salivary glands.Reference Xiong, Shi and Chen⁶⁵ The intervention improved salivary gland flow rate and post-radiotherapy healing compared to controls. These results have been replicated in other studies, but advances in humans remain speculative.Reference Jeong, Baek, Kim, Choi, Lee and Lee^66, Reference Lim, Ra, Shin, Jang, An and Choi⁶⁷

Tissue engineering for the regeneration of neuronal tissue has been applied to several conditions affecting the head and neck, most notably in the regeneration of the facial and recurrent laryngeal nerves. Disease of the facial nerve is usually treated operatively by autologous nerve grafting. However, tissue engineering approaches are currently under investigation, including the use of induced pluripotent stem cells, mesenchymal stem cells and Schwann cells for regeneration of the facial nerve. Research in this field is currently limited to animal models.

Watanbe et al. utilised adipose-derived stem cells, retrieved during liposuction, to regenerate a 7 mm gap in the rat facial nerve, and compared this technique directly with autologous grafting.Reference Watanabe, Sasaki, Matsumine, Yamato and Okano⁶⁸ Some of the adipose-derived stem cells were differentiated into Schwann-like cells and some remained undifferentiated. The adipose-derived stem cells were then embedded onto a collagen scaffold and implanted into the facial nerve defect. Functional recovery was similar for both groups, and both differentiated and undifferentiated adipose-derived stem cells were capable of neuroregeneration.Reference Watanabe, Sasaki, Matsumine, Yamato and Okano⁶⁸

Basic fibroblast growth factor promotes the regeneration of peripheral nerve defects by affecting nerve cells, Schwann cells and fibroblasts, and increasing the rate of nerve regeneration, the number of regenerated nerves and the degree of regenerated nerve maturation.Reference Matsumine, Sasaki, Tabata, Matsui, Yamato and Okano⁶⁹ Cui et al. utilised a functional nerve conduit, consisting of a collagen scaffold plus the neurocytokines ciliary-derived neurotropic factor and basic fibroblast growth factor, to repair a considerably larger facial nerve gap of 35 mm in a mini-pig model.Reference Cui, Lu, Meng, Xiao, Hou and Ding⁷⁰ Their rationale for this model was that nerve injuries in humans are often more substantial than what can be replicated in the rat model. At six months post-implantation, they found that the combination of ciliary-derived neurotropic factor and basic fibroblast growth factor promoted facial nerve regeneration (rather than single neurocytokines) in terms of the number of neurons, myelination and functionality.Reference Cui, Lu, Meng, Xiao, Hou and Ding⁷⁰

Treatment of recurrent laryngeal nerve (RLN) palsy is also under scrutiny from a regenerative medicine point of view. There is evidence that the RLN can spontaneously regenerate following trauma, although the exact process by which this occurs is not fully understood.Reference Chen, Chen, Wang, Zhang and Zheng⁷¹ Halum et al. conducted a trial of ciliary-derived neurotropic factor secreting mesenchymal stem cells to attempt to promote this phenomenon.Reference Halum, McRae, Bijangi-Vishehsaraei and Hiatt⁷² Immunohistochemical analysis identified that the mesenchymal stem cells in combination with neurotropic factor selectively enhanced reinnervation and motor neuron regeneration, although the functional outcome on laryngeal muscle was unclear from that study.Reference Halum, McRae, Bijangi-Vishehsaraei and Hiatt⁷²

The use of intravenous stem cell therapy, usually mesenchymal stem cells, has shown promise for the regeneration of neuronal tissue following stroke and spinal cord injury. Lerner et al. performed a pilot randomised trial of intravenous mesenchymal stem cell use to assess RLN recovery following injury in 12 rats.Reference Lerner, Matsushita, Lankford, Radtke, Kocsis and Young⁷³ They found no significant difference between the mesenchymal stem cell group and the control group, although the data were promising for enhanced functional recovery in the experimental arm.

The reconstruction of mandibular defects presents a challenging problem for head and neck surgeons, with current treatment strategies focusing on microvascular free flaps, usually based on the fibula or radius and their respective vascular pedicles. The nature of mandibular defects, usually a result of malignancy, osteonecrosis or trauma, means that the vascular supply of the surrounding tissue is often compromised.Reference Aframian and Palmon⁶² Regenerative techniques, such as the in vitro culture of tissue-engineered mandibular grafts and the in vivo use of cell-signalling factors to stimulate new bone growth de novo, are currently under investigation.

A small case series of patients (n = 14) underwent mandibular reconstruction with bone morphogenetic protein-2 (a bone-promoting cytokine) in a collagen matrix carrier as an alternative to autologous bone graft.Reference Herford and Boyne⁷⁴ All 14 patients developed new bone tissue at the graft site, which was palpable by 3 to 4 months and radiographically evident at 6 months. That study supports the use of regenerative techniques for mandibular reconstruction, although this technique is only suitable for large mandibular defects.

Lee and colleagues developed bespoke synthetic mandibular bioscaffold grafts using 3D printing techniques, which were then infiltrated with rat adipose-derived stem cells and/or bone morphogenetic protein-2.Reference Lee, DeConde, Aghaloo, Lee, Tetradis and St John⁷⁵ These grafts were subsequently implanted into 28 rats with large mandibular defects. The authors found that the bioscaffolds with adipose-derived stem cells, bone morphogenetic protein-2 infiltration and both in combination were capable of producing bone regeneration. There are concerns regarding the risk of malignancy following the use of bone morphogenetic protein, as with many cellular therapies. However, interestingly, a large retrospective series of spinal surgery patients in the USA found no increased risk of cancer following bone morphogenetic protein use, contradicting previous findings.Reference Kelly, Savage, Bentzen, Hsu, Ellison and Anderson⁷⁶