Robert J. Marano

Dendrimer delivery of an anti-VEGF oligonucleotide into the eye : a long-term study into inhibition of laser- induced CNV, distribution, uptake and toxicity

We have performed a long-term study into the use of a lipophilic amino-acid dendrimer to deliver an anti-vascular endothelial growth factor (VEGF) oligonucleotide (ODN-1) into the eyes of rats and inhibit laser-induced choroidal neovascularization (CNV). In addition, the uptake, distribution and retinal tolerance of the dendrimer plus oligonucleotide conjugates were examined. Analysis of fluorescein angiograms of laser photocoagulated eyes revealed that dendrimer plus ODN-1 significantly inhibited (P<0.05) the development of CNV for 4–6 months by up to 95% in the initial stages. Eyes similarly injected with ODN-1 alone showed no significant difference (P>0.05) in mean severity score at 2 months (2.86±0.09), 4 months (2.15±0.17) or 6 months (2.7±0.12) compared to the vehicle-injected controls. Furthermore, we showed that intravitreally injected ODN-1 tagged with 6-fam was absorbed by a wide area of the retina and penetrated all of the retinal cell layers to the retinal pigment epithelium. Ophthalmological examinations indicated that the dendrimers plus ODN-1 conjugates were well tolerated in vivo, which was later confirmed using immunohistochemistry, which showed no observable increase in antigens associated with inflammation. We conclude that the use of such dendrimers may provide a viable mechanism for the delivery of therapeutic oligonucleotides for the treatment of angiogenic eye diseases.

Minireview: Extrapituitary Prolactin: An Update on the Distribution, Regulation, and Functions

Prolactin (PRL) is an important hormone with many diverse functions. Although it is predominantly produced by lactrotrophs of the pituitary there are a number of other organs, cells, and tissues in which PRL is expressed and secreted. The impact of this extrapituitary PRL (ePRL) on localized metabolism and cellular functions is gaining widespread attention. In 1996, a comprehensive review on ePRL was published. However, since this time, there have been a number of advancements in ePRL research. This includes a greater understanding of the components of the control elements located within the superdistal promoter of the ePRL gene. Furthermore, several new sites of ePRL have been discovered, each under unique control by a range of transcription factors and elements. The functional role of ePRL at each of the expression sites also varies widely leading to gender and site bias. This review aims to provide an update to the research conducted on ePRL since the 1996 review. The focus is on new data concerning the sites of ePRL expression, its regulation, and its function within the organs in which it is expressed.

Syntheses of polycationic dendrimers on lipophilic peptide core for complexation and transport of oligonucleotides

Synthesis of novel polycationic lipophilic peptide core(s) was accomplished and these agents successfully transfected human retinal pigment epithelium cells with ODN1 upon complexation with the oligonucleotide. The level of transfection was indirectly measured by the decreased production of the protein hVEGF (human vascular endothelial growth factor) in comparison to the transfection agent cytofectin GSV.

Preliminary results of the application of a silk fibroin scaffold to otology

The surgical treatment to repair chronic tympanic membrane perforations is myringoplasty. Although multiple autologous grafts, allografts, and synthetic graft materials have been used over the years, no single graft material is superior for repairing all perforation types. Recently, the remarkable properties of silk fibroin protein have been studied, with biomedical and tissue engineering applications in mind, across a number of medical and surgical disciplines. The present study examines the use of silk fibroin for its potential suitability as an alternative graft in myringoplasty surgery by investigating the growth and proliferation of human tympanic membrane keratinocytes on a silk fibroin scaffold in vitro. Light microscopy, immunofluorescent staining, and confocal imaging all reveal promising preliminary results. The biocompatibility, transparency, stability, high tensile strength, and biodegradability of fibroin make this biomaterial an attractive option to study for this utility.

A review on the use of hyaluronic acid in tympanic membrane wound healing

Introduction: Tympanic membrane perforation represents a significant morbidity, especially if it occurs during a childs speech and language development. Recently, there has been an increased interest in hyaluronic-acid-related research and products as a potential therapeutic option for tympanic membrane perforation repair. Areas covered: This review describes the physical and chemical properties of hyaluronic acid and examines the role of hyaluronic acid in wound healing, in particular on the tympanic membrane. It also reviews the safety and efficacy of hyaluronic acid and its derivatives in animal studies as well as in clinical trials. Finally, it considers the potential future clinical applications in tympanic membrane perforation repair. Expert opinion: Hyaluronic acid has been found to accelerate tympanic membrane perforation closure, shorten the period of healing, produce a better quality neo-membrane and improve hearing. More importantly, hyaluronic acid is biodegradable, safe and biocompatible in the ear. Recently, there has been a trend towards the use of modified hyaluronic acid. However, there is a lack of higher-level evidence to support the use of hyaluronic acid in tympanic membrane perforations in the clinical setting. More large-scale randomised control trials are warranted before these bio-devices will be used routinely.

Tympanic membrane repair using silk fibroin and acellular collagen scaffolds

To evaluate the efficacy of silk fibroin scaffolds (SFS) and acellular collagen scaffolds (ACS) for the repair of tympanic membrane (TM) in a guinea pig acute perforation model.

Utilising silk fibroin membranes as scaffolds for the growth of tympanic membrane keratinocytes, and application to myringoplasty surgery

BACKGROUND Chronic tympanic membrane perforations can cause significant morbidity. The term myringoplasty describes the operation used to close such perforations. A variety of graft materials are available for use in myringoplasty, but all have limitations and few studies report post-operative hearing outcomes. Recently, the biomedical applications of silk fibroin protein have been studied. This materials biocompatibility, biodegradability and ability to act as a scaffold to support cell growth prompted an investigation of its interaction with human tympanic membrane keratinocytes. METHODS AND MATERIALS Silk fibroin membranes were prepared and human tympanic membrane keratinocytes cultured. Keratinocytes were seeded onto the membranes and immunostained for a number of relevant protein markers relating to cell proliferation, adhesion and specific epithelial differentiation. RESULTS The silk fibroin scaffolds successfully supported the growth and adhesion of keratinocytes, whilst also maintaining their cell lineage. CONCLUSION The properties of silk fibroin make it an attractive option for further research, as a potential alternative graft in myringoplasty.

The biocompatibility of silk fibroin and acellular collagen scaffolds for tissue engineering in the ear

Recent experimental studies have shown the suitability of silk fibroin scaffold (SFS) and porcine-derived acellular collagen I/III scaffold (ACS) as onlay graft materials for tympanic membrane perforation repair. The aims of this study were to further characterize and evaluate the in vivo biocompatibility of SFS and ACS compared with commonly used materials such as Gelfoam and paper in a rat model. The scaffolds were implanted in subcutaneous (SC) tissue and middle ear (ME) cavity followed by histological and otoscopic evaluation for up to 26 weeks. Our results revealed that SFS and ACS were well tolerated and compatible in rat SC and ME tissues throughout the study. The tissue response adjacent to the implants evaluated by histology and otoscopy showed SFS and ACS to have a milder tissue response with minimal inflammation compared to that of paper. Gelfoam gave similar results to SFS and ACS after SC implantation, but it was found to be associated with pronounced fibrosis and osteoneogenesis after ME implantation. It is concluded that SFS and ACS both were biocompatible and could serve as potential alternative scaffolds for tissue engineering in the ear.

Phenotypic and genotypic profile of human tympanic membrane derived cultured cells

The human tympanic membrane (hTM), known more commonly as the eardrum, is a thin, multi-layered membrane that is unique in the body as it is suspended in air. When perforated, the hTM’s primary function of sound-pressure transmission is compromised. For the purposes of TM reconstruction, we investigated the phenotype and genotype of cultured primary cells derived from hTM tissue explants, compared to epithelial (HaCaT cells) and mesenchymal (human dermal fibroblasts (HDF)) reference cells. Epithelium-specific ets-1 (ESE-1), E-cadherin, keratinocyte growth factor-1 (KGF-1/FGF-7), keratinocyte growth factor-2 (KGF-2/FGF10), fibroblast growth factor receptor 1 (FGFR1), variants of fibroblast growth factor receptor 2 (FGFR2), fibroblast surface protein (FSP), and vimentin proteins were used to assess the phenotypes of all cultured cells. Wholemount and paraffin-embedded hTM tissues were stained with ESE-1 and E-cadherin proteins to establish normal epithelial-specific expression patterns within the epithelial layers. Immunofluorescent (IF) cell staining of hTM epithelial cells (hTMk) demonstrated co-expression of both epithelial- and mesenchymal-specific proteins. Flow cytometry (FCM) analysis further demonstrated co-expression of these epithelial and mesenchymal-specific proteins, indicating the subcultured hTMk cells possessed a transitional phenotype. Gene transcript analysis of hTMk cells by reverse transcriptase polymerase chain reaction (RT-PCR) revealed a down regulation of ESE-1, E-cadherin, FGFR2, variant 1 and variant 2 (FGFR2v1 and FGFR2v2) between low and high passages, and up-regulation of KGF-1, KGF-2, and FGFR1. All results indicate a gradual shift in cell phenotype of hTMk-derived cells from epithelial to mesenchymal.

Prolactin Expression in the Cochlea of Aged BALB/c Mice Is Gender Biased and Correlates to Loss of Bone Mineral Density and Hearing Loss

Prolactin is a versatile hormone with over 300 known functions and predominantly expressed in the pituitary. However, its expression has additionally been found in a number of extrapituitary organs. Recently, we described the expression of prolactin in the inner ear of mice, where it was correlated to age. Previous research has shown prolactin to be linked to abnormal bone metabolism and hearing loss due to changes in morphology of the bony otic capsule. Here we further investigated the relationship between prolactin, hearing loss and cochlea bone metabolism. BALB/c mice were tested for hearing using ABR at 6 and 12 months of age. Bone mineral density of the cochlea was evaluated using microCT scanning. Prolactin expression was calculated using quantitative real time PCR. Expression of the key regulators of bone metabolism, osteoprotegerin and receptor activator of nuclear factor-kappaB ligand were also determined. We found that prolactin expression was exclusive to the female mice. This also correlated to a greater threshold shift in hearing for the females between 6 and 12 months of age. Analyses of the cochlea also show that the bone mineral density was lower in females compared to males. However, no gender differences in expression of osteoprotegerin or receptor activator of nuclear factor-kappaB ligand could be found. Further analysis of cochlea histological sections revealed larger ostocyte lacunae in the females. These results provide a possible mechanism for an age related hearing loss sub-type that is associated with gender and provides clues as to how this gender bias in hearing loss develops. In addition, it has the potential to lead to treatment for this specific type of hearing loss.