Paracrine Activity from Adipose-Derived Stem Cells on In Vitro Wound Healing in Human Tympanic Membrane Keratinocytes.

Stem cell therapies for tympanic membrane repair have shown initial experimental success using mesenchymal stem cells in rat models to promote healing; however, the mechanisms providing this benefit are not known. We investigated in vitro the paracrine effects of human adipose-derived stem cells (ADSCs) on wound healing mechanisms for human tympanic membrane-derived keratinocytes (hTM) and immortalized human keratinocytes (HaCaT). ADSC conditioned media (CMADSC) were assessed for paracrine activity on keratinocyte proliferation and migration, with hypoxic conditions for ADSC culture used to generate contrasting effects on cytokine gene expression. Keratinocytes cultured in CMADSC showed a significant increase in cell number compared to serum-free cultures and further significant increases in hypoxic CMADSC. Assessment of ADSC gene expression on a cytokine array showed a range of wound healing cytokines expressed and under stringent hypoxic and serum-free conditions was upregulated (VEGF A, MMP9, Tissue Factor, PAI-1) or downregulated (CXCL5, CCL7, TNF-α). Several of these may contribute to the activity of conditioned media on the keratinocytes with potential applications in TM perforation repair. VEGFA protein was confirmed by immunoassay to be increased in conditioned media. Together with gene regulation associated with hypoxia in ADSCs, this study has provided several strong leads for a stem cell-derived approach to TM wound healing.

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.

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.

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.

Tympanic membrane repair using silk fibroin and acellular collagen scaffolds.

OBJECTIVES/HYPOTHESIS: 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. STUDY DESIGN: Experimental animal research. METHODS: Seventy-two albino guinea pigs underwent perforation of the right TM and were divided into four experimental groups (n = 18). The perforations were repaired with SFS, ACS, and paper patch using onlay myringoplasty, or they were allowed to heal spontaneously (control). An additional group of 10 guinea pigs without perforation or scaffold was allocated as a normal TM group. Guinea pigs in each experimental group (n = 6) were evaluated at 7, 14, and 28 days following surgery. TM structural healing was evaluated by otomicroscopy and histology, and functional hearing was analyzed by auditory brainstem responses (ABR). Prior to the study, mechanical properties of SFS and ACS were investigated. RESULTS: Tensile strength and elasticity of SFS and ACS were within the known range for human TM. Based on otologic and histologic evaluation, TMs treated with SFS or ACS showed complete closure of the perforation at an earlier stage, with a trilaminar structure and more uniform thickness compared to paper patch and control treated groups. ABR assessment demonstrated that SFS or ACS treatment facilitated a faster restoration of hearing function compared to paper patch and control groups. CONCLUSION: The results of this study show that SFS and ACS are effective graft materials and may be utilized as alternatives to current grafts for TM repair.

TGF-α/HA complex promotes tympanic membrane keratinocyte migration and proliferation via ErbB1 receptor.

Tympanic membrane perforations are common and represent a management challenge to clinicians. Current treatments for chronic perforations involve a graft surgery and require general anaesthesia, including associated costs and morbidities. Bioactive molecules (e.g. growth factors, cytokines) play an important role in promoting TM wound healing following perforation and the use of growth factors as a topical treatment for tympanic membrane perforations has been suggested as an alternative to surgery. However, the choice of bioactive molecules best suited to promote wound healing has yet to be identified. We investigated the effects of hyaluronic acid, vitronectin, TGF-α, IL-24 and their combinations on migration, proliferation and adhesion of cultured human tympanic membrane-derived keratinocytes (hTM), in addition to their possible mechanisms of action. We found that TGF-α, TGF-α/HA and TGF-α/IL-24 promoted wound healing by significantly increasing both migration and proliferation. TGF-α and/or HA treated cells showed comparable cell-cell adhesion whilst maintaining an epithelial cell phenotype. With the use of receptor binding inhibitors for ErbB1 (AG1478) and CD44 (BRIC235), we revealed that the activation of ErbB1 is required for TGF-α/HA-mediated migration and proliferation. These results suggest factors that may be incorporated into a tissue-engineered membrane or directly as topical treatment for tympanic membrane perforations and hence reduce the need for a surgery.

Tissue engineering of the tympanic membrane.

Tympanic membrane (TM) perforations are common, with current treatments for chronic perforations involving surgery, using various graft materials, from autologous cartilage or fascia through to paper patch. Recent research developments in this field have begun applying the principles of tissue engineering, with appropriate scaffolds, cells, and bioactive molecules (BMs). This has revolutionized the therapeutic approach due to the availability of a wide range of materials with appropriate compatibility and mechanical properties to regenerate the membrane acoustics and may also represent a paradigm shift in the management of TM perforations in an outpatient setting without surgery. However, many factors need to be considered in the fabrication of a bioengineered TM. This review discusses the issues associated with current treatment and examines TM wound healing relevant to the construction of a bioengineered TM. It also describes the tissue-engineering approach to TM regeneration by summarizing currently used scaffolds, BMs, and cells in TM wound healing. Finally, it considers the design of scaffolds, delivery of BMs, and cell engraftment toward potential clinical application.

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 child's 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.

In vitro cultured primary cells from a human utricle explant possesses hair cell like characteristics.

The utricle is the enlarged portion of the membranous labyrinth of the inner ear and is essential for balance. It comprises of fine hair cells (mechanoreceptors), supporting cells and calcareous otoliths. Utricle cells are considered to be post-mitotic and possess a limited capacity for regeneration. Unlike birds and reptiles, mammalian mechanosensory hair cells do not regenerate. The in vitro culture of primary cells from the utricle and other inner ear structures of mammals have proven difficult. Presented here for the first time is the culture of primary cells derived from an explant of an adult human utricle, without any intervention or manipulation. Cells were proliferative until cellular quiescence occurred during passage six. Cell morphology was atypical of epithelial cells, appearing as a homogenous, slightly elongated population. Analysis of cultured utricle cells by immunofluorescent staining (IF) and reverse transcriptase polymerase chain reaction (RT-PCR) have shown these cells to possess epithelial (Epithelium-specific ets-1 (ESE-1)), supporting hair cell (p27(Kip1)), and hair cell specific (Atoh1 and Myosin VI) markers. Additionally, RT-PCR revealed positive gene expression for the proliferation control marker fibroblast growth factor receptor 1 (FGFR1) and negative gene expression for E-cadherin (CDH1), a vestibular cell differentiation marker.

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.

Novel cationic lipophilic peptides for oligodeoxynucleotide delivery.

In search of new oligodeoxynucleotide (ODN) delivery agents, we evaluated novel peptides derived from core peptide H-GLRILLLKV-OH (CP). CP is a fragment designed from the T-cell antigen receptor (TCR) alpha-chain transmembrane sequence. CP was able to enter cells including T-cells and inhibited interleukin-2 (IL-2) production. To examine the effect of increased lipophilicity on cellular uptake and activity of CP, a lipoamino acid (2-aminododecanoic acid) was incorporated into peptide CP resulting in 2-aminodecanoyl-CP (LP). The toxicity of CP and LP was assessed by measuring the haemolytic activity. Neither compound caused any haemolysis of red blood cells. We have also compared the biological activities of the CP and LP. Using a T-cell antigen presentation assay, the more lipophilic LP caused greater inhibition of IL-2 production than the parent CP in the antigen stimulated T-cells. The LP also showed increased permeability than CP in the Caco-2 cell assay. We utilised the enhanced cell permeability property of LP in oligodeoxynucleotide ODN1 delivery. Isothermal titration calorimetry (ITC) suggested that CP and LP complex with ODN1 in a 12:1 (CP:ODN1) and 15:1 (LP:ODN1) ratio. These complexes were then transfected into human retinal pigment epithelial cells. The level of transfection was measured by the decreased production of the protein human vascular endothelial growth factor (hVEGF). The results revealed greater transfection efficiency for both CP and LP (47%, 55% more inhibition) compared to commercially available transfection agent cytofectin GSV. These results suggested that the CP and particularly its lipophilic analogue LP have the potential to be used as oligodeoxynucleotide delivery systems.

Synthesis of a library of polycationic lipid core dendrimers and their evaluation in the delivery of an oligonucleotide with hVEGF inhibition.

This article follows on from our previous work in the area of non-viral gene delivery using polycationic dendrimers (PCDs). Herein we report on the synthesis and efficacy of a new library of lipid core PCDs in the delivery of the anti-angiogenic oligonucleotide (ODN-1) to retinal pigment epithelial cells. ELISA was used to monitor hVEGF levels in cells transfected with dendriplexes, Cytofectin GSV and control (non-transfected). At 48 h, hVEGF titres had returned to that of the untransfected control for Cytofectin GSV however, a number of dendriplexes continued to exhibit a marked reduction in hVEGF titres.

Treatments for choroidal and retinal neovascularization: a focus on oligonucleotide therapy and delivery for the regulation of gene function.

Blinding eye diseases caused by neovascularization of the retinal tissue are the leading cause of blindness in Western societies. Current treatments, such as laser photocoagulation, are limited in their effectiveness at halting the progression of angiogenesis and are unable to reduce the number of vessels once they have developed. In addition, although complete blindness is often avoided, vision is often permanently impaired by the treatment itself. Several less invasive treatments are being developed and one of these is oligonucleotide gene therapy in which short stretches of nucleotides are being used as inhibitors of key, metabolic processes involved in angiogenesis. Combined with this is the development of new and improved nucleotide chemistries aimed at overcoming many of the problems associated with oligonucleotide gene therapy, such as poor longevity because of endonuclease activity. In addition, advancements in delivery systems have further enhanced the efficacy of oligonucleotide gene therapy by increasing cellular penetration and localizing delivery to specific cell types and organs.

Inhibition of in vitro VEGF expression and choroidal neovascularization by synthetic dendrimer peptide mediated delivery of a sense oligonucleotide.

Ocular neovascularisation is the leading cause of blindness in developed countries and the most potent angiogenic factor associated with neovascularisation is vascular endothelial growth factor (VEGF). We have previously described a sense oligonucleotide (ODN-1) that possesses anti-human and rat VEGF activity. This paper describes the synthesis of lipid-lysine dendrimers and their subsequent ability to delivery ODN-1 to its target and mediate a reduction in VEGF concentration both in vitro and in vivo. Positively charged dendrimers were used to deliver ODN-1 into the nucleus of cultured D407 cells. The effects on VEGF mRNA transcription and protein expression were analysed using RT-PCR and ELISA, respectively. The most effective dendrimers in vitro were further investigated in vivo using an animal model of choroidal neovascularisation (CNV). All dendrimer/ODN-1 complexes mediated in a significant reduction in VEGF expression during an initial 24 hr period (40-60%). Several complexes maintained this level of VEGF reduction during a subsequent, second 24 hr period, which indicated protection of ODN-1 from the effects of endogenous nucleases. In addition, the transfection efficiency of dendrimers that possessed 8 positive charges (x=81.51%) was significantly better (P=0.0036) than those that possessed 4 positive charges (x=56.8%). RT-PCR revealed a correlation between levels of VEGF protein mRNA. These results indicated that the most effective structural combination was three branched chains of intermediate length with 8 positive charges such as that found for dendrimer 4. Dendrimer 4 and 7/ODN-1 complexes were subsequently chosen for in vivo analysis. Fluorescein angiography demonstrated that both dendrimers significantly (P<0.0001) reduced the severity of laser mediated CNV for up to two months post-injection. This study demonstrated that lipophilic, charged dendrimer mediated delivery of ODN-1 resulted in the down-regulation of in vitro VEGF expression. In addition, in vivo delivery of ODN-1 by two of the dendrimers resulted in significant inhibition of CNV in an inducible rat model. Time course studies showed that the dendrimer/ODN-1 complexes remained active for up to two months indicating the dendrimer compounds provided protection against the effects of nucleases.

Discovery of a novel control element within the 5'-untranslated region of the vascular endothelial growth factor. Regulation of expression using sense oligonucleotides.

The regulation of vascular endothelial growth factor (VEGF), a potent stimulator of angiogenesis, is controlled primarily through the interactions of control elements located within the 5'- and 3'-untranslated regions, many of which are yet to be described. In this study we examined the 5'-untranslated region of human VEGF for control elements with the aim of regulating expression both in vitro and in vivo using oligonucleotide gene therapy. A potential control element was located, two sense oligonucleotides (S(1) and S(2)) were designed based on its sequence, and a third oligonucleotide (S(3)) was designed as a control and mapped to the 16 base pairs immediately upstream. Retinal cells cultured in the presence of S(1) and S(2) resulted in a 2-fold increase of VEGF protein and a 1.5-fold increase in mRNA 24 h post-transfection whereas S(3) had no significant effect (p > 0.05) compared with controls. Subsequent reporter gene studies confirmed the necessity of this element for up-regulation by S(1). Further in vivo studies showed that S(1) and S(2) mediated an increase in VEGF protein in a rodent ocular model that resulted in angiogenesis. In addition to providing insight into the regulation of the vascular endothelial growth factor, the use of these oligonucleotides to stimulate vascular growth may prove useful for the treatment of ischemic tissues such as those found in the heart following infarct.

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.