In vitro cellular uptake and neuroprotective efficacy of poly-arginine-18 (R18) and poly-ornithine-18 (O18) peptides: critical role of arginine guanidinium head groups for neuroprotection.

We have previously demonstrated that Cationic Arginine-Rich Peptides (CARPs) and in particular poly-arginine-18 (R18; 18-mer of arginine) exhibit potent neuroprotective properties in both in vitro and in vivo neuronal injury models. Based on the current literature, there is a consensus that arginine residues by virtue of their positive charge and guanidinium head group is the critical element for imparting CARP neuroprotective properties and their ability to traverse cell membranes. This study examined the importance of guanidinium head groups in R18 for peptide cellular uptake, localization, and neuroprotection. This was achieved by using poly-ornithine-18 (O18; 18-mer of ornithine) as a control, which is structurally identical to R18, but possesses amino head groups rather than guanidino head groups. Epifluorescence and confocal fluorescence microscopy was used to examine the cellular uptake and localization of the FITC-conjugated R18 and O18 in primary rat cortical neurons and SH-SY5Y human neuroblastoma cell cultures. An in vitro cortical neuronal glutamic acid excitotoxicity model was used to compare the effectiveness of R18 and O18 to inhibit cell death and intracellular calcium influx, as well as caspase and calpain activation. Fluorescence imaging studies revealed cellular uptake of both FITC-R18 and FITC-O18 in neuronal and SH-SY5Y cells; however, intracellular localization of the peptides differed in neurons. Following glutamic acid excitotoxicity, only R18 was neuroprotective, prevented caspases and calpain activation, and was more effective at reducing neuronal intracellular calcium influx. Overall, this study demonstrated that for long chain cationic poly-arginine peptides, the guanidinium head groups provided by arginine residues are an essential requirement for neuroprotection but are not required for entry into neurons.

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.

Enhancing Resistance of Silk Fibroin Material to Enzymatic Degradation by Cross-Linking Both Crystalline and Amorphous Domains.

Silk fibroin (SF) membranes are finding widespread use as biomaterial scaffolds in a range of tissue engineering applications. The control over SF scaffold degradation kinetics is usually driven by the proportion of SF crystalline domains in the formulation, but membranes with a high ß-sheet content are brittle and still contain amorphous domains, which are highly susceptible to enzymatic degradation. In this work, photo-cross-linking of SF using a ruthenium-based method, and with the addition of glycerol, was used to generate robust and flexible SF membranes for long-term tissue engineering applications requiring slow degradation of the scaffolds. The resulting mechanical properties, protein secondary structure, and degradation rate were investigated. In addition, the cytocompatibility and versatility of porous micropatterning of SF films were assessed. The photo-cross-linking reduced the enzymatic degradation of SF in vitro without interfering with the ß-sheet content of the SF material, while adding glycerol to the composition grants flexibility to the membranes. By combining these methods, the membrane resistance to protease degradation was significantly enhanced compared to either method alone, and the SF mechanical properties were not impaired. We hypothesize that photo-cross-linking protects the SF amorphous regions from enzymatic degradation and complements the natural protection offered by ß-sheets in the crystalline region. Overall, this approach presents broad utility in tissue engineering applications that require a long-term degradation profile and mechanical support.

3D Printing of Silk Particle-Reinforced Chitosan Hydrogel Structures and Their Properties.

Hydrogel bioprinting is a major area of focus in the field of tissue engineering. However, 3D printed hydrogel scaffolds often suffer from low printing accuracy and poor mechanical properties because of their soft nature and tendency to shrink. This makes it challenging to process them into structural materials. In this study, natural chitosan hydrogel scaffolds were, for the first time, reinforced with milled silk particles and fabricated by 3D printing. Compared with pure chitosan scaffolds, the addition of silk particles resulted in up to a 5-fold increase in compressive modulus as well as significantly better printing accuracy and improved scaffold stability. The chitosan/silk inks flowed well during printing; loading of up to 300% silk (w/w) resulted in only minor changes in the rheological properties of the ink. Particle loading also enabled tuning of the surface roughness of the scaffolds and improved scaffolds' biodegradability. The printed composite hydrogel scaffolds showed no cytotoxicity and supported adherence and growth of human fibroblast cells.

Glycerol-plasticised silk membranes made using formic acid are ductile, transparent and degradation-resistant.

Regenerated silk fibroin membranes tend to be brittle when dry. The use of plasticisers such as glycerol improve membrane ductility, but, when combined with aqueous processing, can lead to a higher degradation rate than solvent-annealed membranes. This study investigated the use of formic acid as the solvent with glycerol to make deformable yet degradation-resistant silk membranes. Here we show that membranes cast using formic acid had low light scattering, with a diffuse transmittance of less than 5% over the visible wavelengths, significantly lower than the 20% transmittance of aqueous derived silk/glycerol membranes. They had 64% ß-sheet content and lost just 30% of the initial silk weight over 6h when tested with an accelerated enzymatic degradation assay, in comparison the aqueous membranes completely degraded within this timeframe. The addition of glycerol also improved the maximum elongation of formic acid derived membranes from under 3% to over 100%. They also showed good cytocompatibility and supported the adhesion and migration of human tympanic membrane keratinocytes. Formic acid based, silk/glycerol membranes may be of great use in medical applications such as repair of tympanic membrane perforation or ocular applications where transparency and resistance to enzymatic degradation are important.

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.

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.

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.

Keratinocyte growth factor 1, fibroblast growth factor 2 and 10 in the healing tympanic membrane following perforation in rats.

The aim of this study was to provide a transcriptome profile of Keratinocyte Growth Factor (KGF)-1, Fibroblast Growth Factor (FGF) 2 and FGF10 (KGF2) in the healing rat tympanic membrane (TM) over 7 days and an immunohistochemical account over 14 days following perforation. KGF1, FGF2, and FGF10 play important roles in TM wound healing. The tympanic membranes of rats were perforated and sacrificed at time points over a 14-day period following perforation. The normalized signal intensities and immunohistochemical protein expression patterns at each time point for KGF1, FGF2, and FGF10 are presented. The primary role of both KGF1 and FGF2 appeared to be in the proliferation and migration of keratinocytes. Whereas the role of KGF1 appeared to be exclusively concerned with increased proliferation and migration at the perforation site, the continued expression of FGF2, beyond perforation closure, suggested it has an additional role to play. FGF10 (KGF2), whilst possessing the highest sequence homologous to KGF1, has a different role in TM wound healing. The effect of FGF10 on keratinocytes in wound healing appeared to emanate from the connective tissue layer.

Structure and properties of biomedical films prepared from aqueous and acidic silk fibroin solutions.

Silk fibroin films are promising materials for a range of biomedical applications. To understand the effects of casting solvents on film properties, we used water (W), formic acid (FA), and trifluoroacetic acid (TFA) as solvents. We characterized molecular weight, secondary structure, mechanical properties, and degradation behavior of cast films. Significant degradation of fibroin was observed for TFA-based film compared to W and TA-based films when analyzed by SDS-PAGE. Fibroin degradation resulted in a significant reduction in tensile strength and modulus of TFA-based films. Compared to water, TFA-based films demonstrated lower water solubility (19.6% vs. 62.5% in 12 h) despite having only a marginal increase in their ß-sheet content (26.9% vs. 23.7%). On the other hand, FA-based films with 34.3% ß-sheet were virtually water insoluble. Following solubility treatment, ß-sheet content in FA-based films increased to 50.9%. On exposure to protease XIV, water-annealed FA-based films lost 74% mass in 22 days compared to only 30% mass loss by ethanol annealed FA films. This study demonstrated that a small variation in the ß-sheet percentage and random coil conformations resulted in a significant change in the rates of enzymatic degradation without alteration to their tensile properties. The film surface roughness changed with the extent of enzymatic hydrolysis.

Tympanic membrane wound healing in rats assessed by transcriptome profiling.

OBJECTIVES/HYPOTHESIS: The aim of this study is to elucidate transcriptional changes that occur in response to tympanic membrane (TM) perforation in rats and to infer key genes and molecular events in the healing process. STUDY DESIGN: A prospective cohort study of 393 male Sprague-Dawley (Rattus norvegicus) rats. METHODS: Sprague-Dawley rats were randomly allocated into either control or perforation groups spanning a 7-day time period. Perforation groups consisted of 12-hour, 24-hour, 36-hour, 2-day, 3-day, 4-day, 5-day, six-day, and 7-day time points. The left TMs of all perforation groups were perforated and the RNA extracted at the specified time point postperforation. Subsequent analysis was performed using Agilent's 4 × 44 k whole rat genome arrays (40 in total) to assess wound-healing gene expression over a 7-day time period. RESULTS: Over a 7-day time course and at nine time points that encompassed the wounding and progression of healing, a total of 3,262 genes were differentially expressed. In this study the transcripts most upregulated occurred at 12 hours. These were Stefin A2 (344-fold), Stefin 2 (143-fold), and Natriuretic peptide precursor type B (222-fold). Those most downregulated also occurred at 12 hours. These were alcohol dehydrogenase 7 (13.1-fold) and gamma-butyrobetaine hydroxylase (10.4-fold). Results were validated by quantitative real-time polymerase chain reaction. CONCLUSIONS: The findings of this study provide a baseline against which to identify disease-related molecular signatures, biomarkers, and to develop new treatments for TM conditions based on molecular evidence.

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.

The role of epidermal growth factor in the healing tympanic membrane following perforation in rats.

Epidermal Growth Factor (EGF) has been identified as playing a critical role in the wound healing process. The objective of this study is to investigate the role that EGF plays in rat tympanic membrane (TM) wound healing using two techniques, microarray and immunohistochemistry. The tympanic membranes of rats were perforated using a sterile needle and sacrificed at time points during 2 weeks following perforation. The normalized signal intensities at the time points for EGF and associated genes are presented. The rat EGF mRNA did not change significantly between time points. Five associated proteins, including heparin-binding EGF-like growth factor were found to be differentially expressed above a two fold threshold at 12 h following perforation. EGF staining was found at low levels in the uninjured TM. Levels of EGF staining increased at 24 h in the basal keratinocyte layer, became diffusely elevated in the specimen at 36 h, before a second peak in staining of the keratinocyte layer at Day 4. The staining of EGF corresponds to its multiple roles in TM wound healing.

Purification of the keratan sulfate proteoglycan expressed in prostatic secretory cells and its identification as lumican.

BACKGROUND: Secretory epithelial cells of human prostate contain a keratan sulfate proteoglycan (KSPG) associated with the prostatic secretory granules (PSGs). The proteoglycan has not been identified, but like the PSGs, it is lost in the early stages of malignant transformation. METHODS: Anion exchange and affinity chromatography were used to purify KSPG from human prostate tissue. Enzymatic deglycosylation was used to remove keratan sulfate (KS). The core protein was isolated using 2D gel electrophoresis, digested in-gel with trypsin, and identified by peptide mass fingerprinting (PMF). RESULTS: The purified proteoglycan was detected as a broad smear on Western blots with an apparent molecular weight of 65-95 kDa. The KS moiety was susceptible to digestion with keratanase II and peptide N-glycosidase F defining it as highly sulfated and N-linked to the core protein. The core protein was identified, following deglycosylation and PMF, as lumican and subsequently confirmed by Western blotting using an anti-lumican antibody. CONCLUSIONS: The KSPG associated with PSGs in normal prostate epithelium is lumican. While the role of lumican in extracellular matrix is well established, its function in the prostate secretory process is not known. It's potential to facilitate packaging of polyamines in PSGs, to act as a tumor suppressor and to mark the early stages of malignant transformation warrant further investigation.