Pooyan Aliuos

Inhibition of fibroblast adhesion by covalently immobilized protein repellent polymer coatings studied by single cell force spectroscopy.

Cochlea implants (CI) restore the hearing in patients with sensorineural hearing loss by electrical stimulation of the auditory nerve via an electrode array. The increase of the impedance at the electrode-tissue interface due to a postoperative connective tissue encapsulation leads to higher power consumption of the implants. Therefore, reduced adhesion and proliferation of connective tissue cells around the CI electrode array is of great clinical interest. The adhesion of cells to substrate surfaces is mediated by extracellular matrix (ECM) proteins. Protein repellent polymers (PRP) are able to inhibit unspecific protein adsorption. Thus, a reduction of cell adhesion might be achieved by coating the electrode carriers with PRPs. The aim of this study was to investigate the effects of two different PRPs, poly(dimethylacrylamide) (PDMAA) and poly(2-ethyloxazoline) (PEtOx), on the strength and the temporal dynamics of the initial adhesion of fibroblasts. Polymers were immobilized onto glass plates by a photochemical grafting onto method. Water contact angle measurements proved hydrophilic surface properties of both PDMAA and PEtOx (45 ± 1° and 44 ± 1°, respectively). The adhesion strength of NIH3T3 fibroblasts after 5, 30, and 180 s of interaction with surfaces was investigated by using single cell force spectroscopy. In comparison to glass surfaces, both polymers reduced the adhesion of fibroblasts significantly at all different interaction times and lower dynamic rates of adhesion were observed. Thus, both PDMAA and PEtOx represented antiadhesive properties and can be used as implant coatings to reduce the unspecific ECM-mediated adhesion of fibroblasts to surfaces.

Dexamethasone released from cochlear implant coatings combined with a protein repellent hydrogel layer inhibits fibroblast proliferation.

The insertion of cochlear implants into the inner ear often causes inflammation and fibrosis inside the scala tympani and thus growth of fibrous tissue on the implant surface. This deposition leads to the loss of function in both electrical and laser-based implants. The design of this study was to realize fibroblast growth inhibition by dexamethasone (Dex) released from the base material of the implant [polydimethylsiloxane (PDMS)]. To prevent cell and protein adhesion, the PDMS was coated with a hydrogel layer [star-shaped polyethylene glycol prepolymer (sPEG)]. Drug release rates were studied over 3 months, and surface characterization was performed. It was observed that the hydrogel slightly smoothened the surface roughened by the Dex crystals. The hydrogel coating reduced and prolonged the release of the drug over several months. Unmodified, sPEG-coated, Dex-loaded, and Dex/sPEG-equipped PDMS filaments were cocultivated in vitro with fluorescent fibroblasts, analyzed by fluorescent microscopy, and quantified by cell counting. Compared to the unmodified PDMS, cell growth on all modified filaments was averagely 95% ±standard deviation (SD) less, while cell growth on the bottom of the culture dishes containing Dex-loaded filaments was reduced by 70% ±SD. Both, Dex and sPEG prevented direct cell growth on the filament surfaces, while drug delivery was maintained for the duration of several months.

One-pot aqueous synthesis of highly strained CdTe/CdS/ZnS nanocrystals and their interactions with cells

In this work, a very simple one-pot synthetic approach was developed to generate aqueous CdTe/CdS/ZnS type-II/type-I red-emitting nanocrystals (NCs). Strain-induced optical properties of CdTe/CdS particles having core(small)/shell(thick) structure with a maximum quantum yield (QYmax) ∼ 57% were further improved with the overgrowth of a ZnS shell, resulting in a core(small)/shell(thick)/shell(small) structure (QYmax ∼ 64%). The spectral properties were tuned further to the near-infrared region as the ZnS shell grew in thickness. X-ray powder diffraction (XRD) analysis and high-resolution transmission electron microscope (HRTEM) images showed the crystalline structure of NCs proving the epitaxial growth of ZnS without crystalline defects. Under continuous UV-irradiation for 5 h, the NCs did not exhibit any photo-degradation but instead displayed a photo-annealing process. These extremely photostable NCs were further characterized in terms of their cytotoxicity and their cell labeling performances. The presence of a ZnS shell was found to reduce the toxicity of the CdTe/CdS NCs. Furthermore, aptamer–conjugated NCs were successfully utilized in targeted cell imaging. Promisingly, the aptamer–NCs bioconjugates were internalized by A549 cells within 2 hours of incubation and retained their fluorescence even after 24 hours of internalization.

Evaluation of single-cell force spectroscopy and fluorescence microscopy to determine cell interactions with femtosecond-laser microstructured titanium surfaces.

One goal in biomaterials research is to limit the formation of connective tissue around the implant. Antiwetting surfaces are known to reduce ability of cells to adhere. Such surfaces can be achieved by special surface structures (lotus effect). Aim of the study was to investigate the feasibility for creating antiwetting surface structures on titanium and to characterize their effect on initial cell adhesion and proliferation. Titanium microstructures were generated using femtosecond- (fs-) laser pulses. Murine fibroblasts served as a model for connective tissue cells. Quantitative investigation of initial cell adhesion was performed using atomic force microscopy. Fluorescence microscopy was used for the characterization of cell-adhesion pattern, cell morphology, and proliferation. Water contact angle (WCA) measurements evinced antiwetting properties of laser-structured surfaces. However, the WCA was decreased in serum-containing medium. Initial cell adhesion to microstructured titanium was significantly promoted when compared with polished titanium. Microstructures did not influence cell proliferation on titanium surfaces. However, on titanium microstructures, cells showed a flattened morphology, and the cell orientation was biased according to the surface topography. In conclusion, antiwetting properties of surfaces were absent in the presence of serum and did not hinder adhesion and proliferation of NIH 3T3 fibroblasts.

New, Non‐quinone Fluorogeldanamycin Derivatives Strongly Inhibit Hsp90

Streptomyces hygroscopicus is a natural producer of geldanamycin. Mutasynthetic supplementation of an AHBA‐blocked mutant with all possible monofluoro 3‐aminobenzoic acids provided new fluorogeldanamycins. These showed strong antiproliferative activity and inhibitory effects on human heat shock protein Hsp90. Binding to Hsp90 in the low nanomolar range was determined from molecular modelling, AFM analysis and by calorimetric studies.

Dissociated Neurons and Glial Cells Derived from Rat Inferior Colliculi after Digestion with Papain

The formation of gliosis around implant electrodes for deep brain stimulation impairs electrode–tissue interaction. Unspecific growth of glial tissue around the electrodes can be hindered by altering physicochemical material properties. However, in vitro screening of neural tissue–material interaction requires an adequate cell culture system. No adequate model for cells dissociated from the inferior colliculus (IC) has been described and was thus the aim of this study. Therefore, IC were isolated from neonatal rats (P3_5) and a dissociated cell culture was established. In screening experiments using four dissociation methods (Neural Tissue Dissociation Kit [NTDK] T, NTDK P; NTDK PN, and a validated protocol for the dissociation of spiral ganglion neurons [SGN]), the optimal media, and seeding densities were identified. Thereafter, a dissociation protocol containing only the proteolytic enzymes of interest (trypsin or papain) was tested. For analysis, cells were fixed and immunolabeled using glial- and neuron-specific antibodies. Adhesion and survival of dissociated neurons and glial cells isolated from the IC were demonstrated in all experimental settings. Hence, preservation of type-specific cytoarchitecture with sufficient neuronal networks only occurred in cultures dissociated with NTDK P, NTDK PN, and fresh prepared papain solution. However, cultures obtained after dissociation with papain, seeded at a density of 2×104 cells/well and cultivated with Neuro Medium for 6 days reliably revealed the highest neuronal yield with excellent cytoarchitecture of neurons and glial cells. The herein described dissociated culture can be utilized as in vitro model to screen interactions between cells of the IC and surface modifications of the electrode.

Polymer Coatings of Cochlear Implant Electrode Surface - An Option for Improving Electrode-Nerve-Interface by Blocking Fibroblast Overgrowth.

Overgrowth of connective tissue and scar formation induced by the electrode array insertion increase the impedance and, thus, diminish the interactions between neural probes as like cochlear implants (CI) and the target tissue. Therefore, it is of great clinical interest to modify the carrier material of the electrodes to improve the electrode nerve interface for selective cell adhesion. On one side connective tissue growth needs to be reduced to avoid electrode array encapsulation, on the other side the carrier material should not compromise the interaction with neuronal cells. The present in vitro-study qualitatively and quantitatively characterises the interaction of fibroblasts, glial cells and spiral ganglion neurons (SGN) with ultrathin poly(N,N-dimethylacrylamide) (PDMAA), poly(2-ethyloxazoline) (PEtOx) and poly([2-methacryloyloxy)ethyl]trimethylammoniumchlorid) (PMTA) films immobilised onto glass surfaces using a photoreactive anchor layer. The layer thickness and hydrophilicity of the polymer films were characterised by ellipsometric and water contact angle measurement. Moreover the topography of the surfaces was investigated using atomic force microscopy (AFM). The neuronal and non-neuronal cells were dissociated from spiral ganglions of postnatal rats and cultivated for 48 h on top of the polymer coatings. Immunocytochemical staining of neuronal and intermediary filaments revealed that glial cells predominantly attached on PMTA films, but not on PDMAA and PEtOx monolayers. Hereby, strong survival rates and neurite outgrowth were only found on PMTA, whereas PDMAA and PEtOx coatings significantly reduced the SG neuron survival and neuritogenesis. As also shown by scanning electron microscopy (SEM) SGN strongly survived and retained their differentiated phenotype only on PMTA. In conclusion, survival and neuritogenesis of SGN may be associated with the extent of the glial cell growth. Since PMTA was the only of the polar polymers used in this study bearing a cationic charge, it can be assumed that this charge favours adhesion of both glial cells and SG neurons glial cells and SGN.

Magnetic Beads Enhance Adhesion of NIH 3T3 Fibroblasts: A Proof-of-Principle In Vitro Study for Implant-Mediated Long-Term Drug Delivery to the Inner Ear.

Introduction Long-term drug delivery to the inner ear may be achieved by functionalizing cochlear implant (CI) electrodes with cells providing neuroprotective factors. However, effective strategies in order to coat implant surfaces with cells need to be developed. Our vision is to make benefit of electromagnetic field attracting forces generated by CI electrodes to bind BDNF-secreting cells that are labelled with magnetic beads (MB) onto the electrode surfaces. Thus, the effect of MB-labelling on cell viability and BDNF production were investigated. Materials and Methods Murine NIH 3T3 fibroblasts—genetically modified to produce BDNF—were labelled with MB. Results Atomic force and bright field microscopy illustrated the internalization of MB by fibroblasts after 24 h of cultivation. Labelling cells with MB did not expose cytotoxic effects on fibroblasts and allowed adhesion on magnetic surfaces with sufficient BDNF release. Discussion Our data demonstrate a novel approach for mediating enhanced long-term adhesion of BDNF-secreting fibroblasts on model electrode surfaces for cell-based drug delivery applications in vitro and in vivo. This therapeutic strategy, once transferred to cells suitable for clinical application, may allow the biological modifications of CI surfaces with cells releasing neurotrophic or other factors of interest.

Molecular Survival Strategies of Organisms: HSP and Small Molecules for Diagnostics and Drug Development

Nature has developed evolutionary strategies to synthesize the vast number of what are called secondary metabolites or in short natural products. These include molecules that are able to inhibit heat shock proteins (HSP90). A prominent group of natural products are polyketides whose molecular cores are biosynthesized by the polyketide synthase (PKS) which is a sophisticated catalytic machinery which evolved through evolutionary processes. Manipulation of the PKS based on genetic engineering allows utilizing the biosynthetic machinery in combination with chemically synthesized unnatural building blocks and the initiation of what may be called a chemosynthetic evolution with the aim to obtain new derivatives based on the parental natural product. This review illuminates the progress in the development of such chemoenzymatic approaches exemplified for geldanamycin and how new analogues show promising and improved inhibitory properties towards HSP90 and hence cancer cell growth. Based on the understanding of the molecular interaction of new inhibitors with their target, we discuss methods to redesign target-oriented natural products as well as reasons why susceptibilities towards HSP differ. Moreover, the development of novel screening methods to identify new HSP90 inhibitors and the investigation of their physical properties e.g. by using AFM are presented. This report sheds light on different aspects of targeting HSP90 with geldanamycin as lead structure, a prerequisite to move on towards preclinical studies for in vitro or in vivo fighting of cancer cells.

In Vitro Evaluation of PCL and P(3HB) as Coating Materials for Selective Laser Melted Porous Titanium Implants

Titanium is widely used as a bone implant material due to its biocompatibility and high resilience. Since its Young’s modulus differs from bone tissue, the resulting “stress shielding” could lead to scaffold loosening. However, by using a scaffold-shaped geometry, the Young’s modulus can be adjusted. Also, a porous geometry enables vascularisation and bone ingrowth inside the implant itself. Additionally, growth factors can improve these effects. In order to create a deposit and release system for these factors, the titanium scaffolds could be coated with degradable polymers. Therefore, in the present study, synthetic poly-ε-caprolactone (PCL) and the biopolymer poly(3-hydroxybutyrate) (P(3HB)) were tested for coating efficiency, cell adhesion, and biocompatibility to find a suitable coating material. The underlying scaffold was created from titanium by Selective Laser Melting (SLM) and coated with PCL or P(3HB) via dip coating. To test the biocompatibility, Live Cell Imaging (LCI) as well as vitality and proliferation assays were performed. In addition, cell adhesion forces were detected via Single Cell Force Spectroscopy, while the coating efficiency was observed using environmental scanning electron microscopy (ESEM) and energy-dispersive X-ray (EDX) analyses. Regarding the coating efficiency, PCL showed higher values in comparison to P(3HB). Vitality assays revealed decent vitality values for both polymers, while values for PCL were significantly lower than those for blank titanium. No significant differences could be observed between PCL and P(3HB) in proliferation and cell adhesion studies. Although LCI observations revealed decreasing values in cell number and populated area over time on both polymer-coated scaffolds, these outcomes could be explained by the possibility of coating diluent residues accumulating in the culture medium. Overall, both polymers fulfill the requirements regarding biocompatibility. Nonetheless, since only PCL coating ensured the maintenance of the porous implant structure, it is preferable to be used as a coating material for creating a deposit and release system for growth factors.