Overcoming barriers: a review on innovations in drug delivery to the middle and inner ear.

Despite significant advances in the development of therapeutics for hearing loss, drug delivery to the middle and inner ear remains a challenge. As conventional oral or intravascular administration are ineffective due to poor bioavailability and impermeability of the blood-labyrinth-barrier, localized delivery is becoming a preferable approach for certain drugs. Even then, localized delivery to the ear precludes continual drug delivery due to the invasive and potentially traumatic procedures required to access the middle and inner ear. To address this, the preclinical development of controlled release therapeutics and drug delivery devices have greatly advanced, with some now showing promise clinically. This review will discuss the existing challenges in drug development for treating the most prevalent and damaging hearing disorders, in particular otitis media, perforation of the tympanic membrane, cholesteatoma and sensorineural hearing loss. We will then address novel developments in drug delivery that address these including novel controlled release therapeutics such as hydrogel and nanotechnology and finally, novel device delivery approaches such as microfluidic systems and cochlear prosthesis-mediated delivery. The aim of this review is to investigate how drugs can reach the middle and inner ear more efficiently and how recent innovations could be applied in aiding drug delivery in certain pathologic contexts.

Isolation of Epidermal Progenitor Cells from Rat Tympanic Membrane.

The eardrum is an important structural component for hearing, but it is delicate and subject to traumatic injury and disease. Healing mechanisms are activated after injury but sometimes healing fails and chronic perforations develop, requiring surgical intervention. To model the wound healing responses we established a simple method for isolating keratinocytes and progenitors from individual eardrums. The central region of the eardrum contains epidermal proliferative centers that produce keratinocytes which migrate to cover the eardrum surface. We dissected out the central region and explanted it to the plastic membrane of a culture well insert. Epidermal cells grew from the explant onto the surface of the insert membrane. The cells could be serially harvested and passaged for continuous culture and characterization. Magnetic immunoseparation methods were used to enrich for epithelial cells with stem cell-like characteristics. Proliferation and migration in vitro was demonstrated, and the cells were shown suitable for tissue engineering applications.

Tympanic Membrane Derived Stem Cell-Like Cultures for Tissue Regeneration.

Epidermal cells with stem cell-like characteristics have been identified in the tympanic membrane (TM) and localized specifically to the umbo and annulus regions. While they have been proposed to play a role in the regeneration of both acute and chronic TM perforations, evidence for the mechanism and regulation of their contribution is not yet described. Indeed, the behavior of these putative stem cells is largely unknown, in part due to a lack of refined methods for efficient cell isolation. In this study, we compared different explant techniques using normal and perforated rat TM tissues and investigated their ex vivo characteristics. TM after perforation in vivo showed increased staining for epidermal stem cell markers integrin ß1 and cytokeratin (CK) 19, and for proliferation Ki-67, indicating activation of the proliferative centers. A mixed population of fibroblasts and epithelial cells were isolated from explant cultures. Excised TM umbo implanted on a culture well insert was the most effective technique. Explants made from perforated TM produced cells before those from unperforated TM. More importantly, the implanted TM umbo organoid was capable of producing cells in a continuous manner, allowing subsequent harvest using trypsin. Primary rat TM epithelial cell cultures positive for pancytokeratin had colony forming activity and could be enriched for CK 19-positive cells that were capable of culture expansion by proliferation and cell migration when subject to a wound assay. Taken together, trauma to the TM activated the proliferative centers and prompted early cell production from TM umbo organoid cultures, which produced TM stem cell-like cultures that proved suitable for tissue engineering of the TM.

Mechanical behaviour of alginate-gelatin hydrogels for 3D bioprinting.

Hydrogels comprised of alginate and gelatin have demonstrated potential as biomaterials in three dimensional (3D) bioprinting applications. However, as with all hydrogel-based biomaterials used in extrusion-based bioprinting, many parameters influence their performance and there is limited data characterising the behaviour of alginate-gelatin (Alg-Gel) hydrogels. Here we investigated nine Alg-Gel blends by varying the individual constituent concentrations. We tested samples for printability and print accuracy, compressive behaviour and change over time, and viability of encapsulated mesenchymal stem cells in bioprinted constructs. Printability tests showed a decrease in strand width with increasing concentrations of Alg-Gel. However due to the increased viscosity associated with the higher Alg-Gel concentrations, the minimum width was found to be 0.32mm before blends became too viscous to print. Similarly, printing accuracy was increased in higher concentrations, exceeding 90% in some blends. Mechanical properties were assessed through uniaxial compression testing and it was found that increasing concentrations of both Alg and Gel resulted in higher compressive modulus. We also deemed 15min crosslinking in calcium chloride to be sufficient. From our data, we propose a blend of 7%Alg-8%Gel that yields high printability, mechanical strength and stiffness, and cell viability. However, we found the compressive behaviour of Alg-Gel to reduce rapidly over time and especially when incubated at 37°C. Here we have reported relevant data on Alg-Gel hydrogels for bioprinting. We tested for biomaterial properties and show that these hydrogels have many desirable characteristics that are highly tunable. Though further work is needed before practical use in vivo can be achieved.

Characterisation of hyaluronic acid methylcellulose hydrogels for 3D bioprinting.

Hydrogels containing hyaluronic acid (HA) and methylcellulose (MC) have shown promising results for three dimensional (3D) bioprinting applications. However, several parameters influence the applicability bioprinting and there is scarce data in the literature characterising HAMC. We assessed eight concentrations of HAMC for printability, swelling and stability over time, rheological and structural behaviour, and viability of mesenchymal stem cells. We show that HAMC blends behave as viscous solutions at 4°C and have faster gelation times at higher temperatures, typically gelling upon reaching 37°C. We found the storage, loss and compressive moduli to be dependent on HAMC concentration and incubation time at 37°C, and show the compressive modulus to be strain-rate dependent. Swelling and stability was influenced by time, more so than pH environment. We demonstrated that mesenchymal stem cell viability was above 75% in bioprinted structures and cells remain viable for at least one week after 3D bioprinting. The mechanical properties of HAMC are highly tuneable and we show that higher concentrations of HAMC are particularly suited to cell-encapsulated 3D bioprinting applications that require scaffold structure and delivery of cells.

Isolation and Culture of Adipose-Derived Stromal Cells from Subcutaneous Fat.

Cells with mesenchymal stem cell characteristics can be isolated from human adipose tissue stroma. Relative ease of isolation in large numbers and their ability for expansion and differentiation means that they are becoming a preferred cell type for mesenchymal regenerative medicine applications. In addition to expansion and differentiation capacity, they also express valuable paracrine activities which promote tissue formation and wound healing, including pro- and anti-fibrotic mediators. Here we describe a method for routine isolation of adipose stromal cells, culture expansion, and characterization by differentiation and then production of conditioned media.

Tympanic membrane organ culture using cell culture well inserts engrafted with tympanic membrane tissue explants.

Tissue engineering approaches using growth factors and various materials for repairing chronic perforations of the tympanic membrane are being developed, but there are surprisingly few relevant tissue culture models available to test new treatments. Here, we present a simple three-dimensional model system based on micro-dissecting the rat tympanic membrane umbo and grafting it into the membrane of a cell culture well insert. Cell outgrowth from the graft produced sufficient cells to populate a membrane of similar surface area to the human tympanic membrane within 2 weeks. Tissue grafts from the annulus region also showed cell outgrowth but were not as productive. The umbo organoid supported substantial cell proliferation and migration under the influence of keratinocyte growth medium. Cells from umbo grafts were enzymatically harvested from the polyethylene terephthalate (PET) membrane for expansion in routine culture and cells could be harvested consecutively from the same graft over multiple cycles. We used harvested cells to test cell migration properties and to engraft a porous silk scaffold material as proof-of-principle for tissue engineering applications. This model is simple enough to be widely adopted for tympanic membrane regeneration studies and has promise as a tissue-equivalent model alternative to animal testing.

Rat model of chronic tympanic membrane perforation: A longitudinal histological evaluation of underlying mechanisms.

OBJECTIVE: To evaluate histologically the progressive development and underlying mechanisms of chronic tympanic membrane perforation (TMP) in a rat model using a two-weeks ventilation tube (VT) treatment combined with topical application of mitomycin C/dexamethasone (VT-M/D), compared with normal tympanic membrane and acute TMPs. METHODS: Fifty male Sprague-Dawley rats were divided into three experimental groups: a normal control group (n = 5), an acute TMP group (n = 5) (i.e. 3 days post-myringotomy) and a VT-M/D group (n = 40). The TMs were regularly assessed by otoscopy. The normal control animals were sacrificed on day 0 and the acute TMP group was sacrificed 3 days post-myringotomy for histological and immunohistochemical evaluations. The VT-M/D group was sacrificed at various time points - 14 and 17 days, 3, 4, 6, 8 and 10 weeks. RESULTS: On longitudinal histological examination, compared with normal TM and acute TMP, the perforation edges at the later time points illustrated thickened stratified squamous epithelium rimming around the edges, significant increase in keratin and collagen deposition, increased macrophage infiltration as well as reduced cellular proliferation. Three phases of TMP healing process were identified - the acute healing phase (3-17 days), the transition phase (3-4 weeks) and the chronic phase (6-10 weeks). CONCLUSION: Based on the histological results of this study, the progressive development of chronic TMPs appeared to be associated with increased epidermal thickening, collagen and keratin deposition, macrophage infiltration and reduced cellular proliferation. After the 3-4 weeks of transition phase, the TMPs seemed to have transformed into a non-healing chronic TMP between 6 and 10 weeks.

Rat model of chronic tympanic membrane perforation: Ventilation tube with mitomycin C and dexamethasone.

OBJECTIVE: Chronic tympanic membrane perforation (TMP) in a clinical setting may attract surgical intervention. With the advent of modern biomaterials, new options are available for myringoplasty but safety and efficacy need evaluation in a chronic TMP animal model. The aim of this study was to evaluate the efficacy of ventilation tube (VT) insertion in conjunction with topical application of mitomycin C/dexamethasone (M/D) for the creation of chronic TMP in rats. METHODS: Thirty male Sprague-Dawley rats underwent myringotomy of the right tympanic membrane (TM) and were divided into three experimental groups: spontaneous healing (myringotomy control), VT insertion for 2 weeks and VT insertion for 2 weeks in conjunction with topical application of M/D (VT-M/D). All TMs were regularly assessed by otoscopy for 10 weeks and then animals were sacrificed for histological evaluation. RESULTS: In the VT-M/D group, seven out of ten (70%) perforations were patent at 10 weeks (mean patency, 57.9 days; P<0.01). The VT group had two out of ten (20%) perforations patent at 10 weeks (mean patency, 26.5 days; P<0.01), while all TMPs from the myringotomy control group were closed by day 9 (mean patency, 7.2 days). Histologically, the TMPs patent at week 10 had a stratified squamous epithelialized rim, keratinocyte layer thickening around the perforation edge as well as increased collagen deposition and macrophage infiltration. CONCLUSION: Chronic TMP in a rat model was successfully created by VT insertion and the efficacy was increased in combination with topical application of M/D.

Searching for a rat model of chronic tympanic membrane perforation: Healing delayed by mitomycin C/dexamethasone but not paper implantation or iterative myringotomy.

OBJECTIVES: Surgical intervention such as myringoplasty or tympanoplasty is an option in the current clinical management of chronic tympanic membrane perforation (TMP). Animal models of chronic TMP are needed for pre-clinical testing of new materials and to improve existing techniques. We evaluated several reported animal model techniques from the literature for the creation of chronic TMPs. The aim of this study was to evaluate production of chronic TMPs in a rat model using topical mitomycin C/dexamethasone, paper insertion into middle ear cavity (MEC) or re-myringotomy. METHODS: Forty male Sprague-Dawley rats underwent myringotomy of the right tympanic membrane (TM) and were randomly divided into 3 experimental groups: application of topical mitomycin C/dexamethasone, paper insertion into middle ear cavity, or re-myringotomy. Control perforations were allowed to close spontaneously. TMs were assessed regularly with otoscopy for 8 weeks. At the end of 8 weeks, animals were sacrificed for histology. RESULTS: The closure of TMPs was significantly delayed by mitomycin C/dexamethasone (mean patency, 18.9 days; P≤0.01) compared with the control (mean patency, 7 days), but was not significantly delayed in the paper insertion group (mean patency, 9.4 days; P=0.74). Repeated myringotomy of closed perforations (mean number of myringotomies, 8.9 per ear) stimulated acceleration of closure rather than delay. Histologically, the mitomycin C/dexamethasone group had almost normal TM morphology, while the paper insertion group revealed inflammatory and granulomatous responses. The re-myringotomy group had a thickened TM fibrous layer with collagen deposition. CONCLUSIONS: Mitomycin C/dexamethasone delayed TMP closure in rats but the effect was not sufficiently long-lasting to be defined as a chronic TMP. Neither paper insertion into middle ear cavity nor re-myringotomy created chronic TMP in rats.

DNA methylation within the I.4 promoter region correlates with CYPl19A1 gene expression in human ex vivo mature omental and subcutaneous adipocytes.

BACKGROUND: DNA methylation at specific CpG sites within gene promoter regions is known to regulate transcriptional activity in vitro. In human adipose tissue, basal transcription of the aromatase (CYP19A1) gene is driven primarily by the I.4 promoter however the role of DNA methylation in regulating expression in ex vivo mature adipocytes is unknown. This observational study reports the correlation of DNA methylation within the I.4 promoter region of human mature subcutaneous and omental adipocytes with aromatase expression and body composition measures. METHODS: Omental and subcutaneous adipose tissue were collected from 25 obese subjects undergoing bariatric surgery and the mature adipocyte fraction purified. DNA methylation status of 5 CpG sites within a 550 base pair region encompassing the transcription start site (TSS) of promoter I.4 was determined using pyrosequencing. Relative aromatase and I.4 promoter specific mRNA expression was determined by qRT-PCR and whole body DXA performed in 25 participants. RESULTS: Site-specific DNA methylation varied from 21 ± 10% to 81 ± 11%. In omental adipocytes percentage methylation at the I.4.1 and I.4.2 CpG sites, but not other nearby sites, was negatively correlated with relative aromatase mRNA expression (R = - 0.52, P = 0.017 and R = - 0.52, P = 0.015). In contrast subcutaneous adipocytes percentage DNA methylation at the I.4.3 and I.4.5 sites were positively correlated with relative aromatase mRNA expression (R = 0.47, P = 0.022 and R = 0.55, P = 0.004). In a small subset of patients DNA methylation at the I.4.5 site was also positively correlated with whole body lean mass, bone mineral content and density. CONCLUSIONS: In conclusion in mature adipocytes, the primary source of estradiol after menopause, increasing DNA methylation was correlated with aromatase mRNA expression and thus estradiol biosynthesis. These findings support a tissue-specific epigenetic regulation of the basal promoter activity in mature adipocytes; the mechanisms influencing this regulation and its physiological role remain to be elucidated.