Plasma-assisted hydrophilization of cochlear implant electrode array surfaces enables adhesion of neurotrophin-secreting cells.

OBJECTIVE: To evaluate plasma treatment for enhancing the biocompatibility of cochlear implant (CI) silicone surfaces, thus allowing colonization with human adipose-derived stem cells (hASCs) that are known to provide neurotrophic support. METHODS: Silicone samples and CI electrode arrays were treated with 4 low-pressure plasmas of different characteristics. The hydrophilicity of plasma-treated and control surfaces as well as the adherence and morphology of hASCs were assessed. Finally, the insertion forces of electrode arrays were determined and the colonization potential of the electrode arrays with hASCs were tested. RESULTS: The hydrophilicity of the silicone surfaces was significantly enhanced after plasma treatment, as was the adherence of hASCs. The characteristic morphology of hASCs was observed when grown on plasma-treated but not on untreated silicone surfaces. The insertion forces of plasma-treated electrode arrays were similar to those of untreated arrays, and the colonization of plasma-treated electrode arrays with hASCs was feasible. CONCLUSION: Plasma treatment of CI electrode arrays enhances their biocompatibility and allows for the colonization with hASCs that are known to produce neurotrophic factors.

Online Measurement System for Dynamic Flow Bioreactors to Study Barrier Integrity of hiPSC-Based Blood-Brain Barrier In Vitro Models.

Electrochemical impedance spectroscopy (EIS) is a noninvasive, reliable, and efficient method to analyze the barrier integrity of in vitro tissue models. This well-established tool is used most widely to quantify the transendothelial/epithelial resistance (TEER) of Transwell-based models cultured under static conditions. However, dynamic culture in bioreactors can achieve advanced cell culture conditions that mimic a more tissue-specific environment and stimulation. This requires the development of culture systems that also allow for the assessment of barrier integrity under dynamic conditions. Here, we present a bioreactor system that is capable of the automated, continuous, and non-invasive online monitoring of cellular barrier integrity during dynamic culture. Polydimethylsiloxane (PDMS) casting and 3D printing were used for the fabrication of the bioreactors. Additionally, attachable electrodes based on titanium nitride (TiN)-coated steel tubes were developed to perform EIS measurements. In order to test the monitored bioreactor system, blood-brain barrier (BBB) in vitro models derived from human-induced pluripotent stem cells (hiPSC) were cultured for up to 7 days. We applied equivalent electrical circuit fitting to quantify the electrical parameters of the cell layer and observed that TEER gradually decreased over time from 2513 Ω·cm2 to 285 Ω·cm2, as also specified in the static control culture. Our versatile system offers the possibility to be used for various dynamic tissue cultures that require a non-invasive monitoring system for barrier integrity.

Dose determination using alanine detectors in a mixed neutron and gamma field for boron neutron capture therapy of liver malignancies.

UNLABELLED: Boron Neutron Capture Therapy for liver malignancies is being investigated at the University of Mainz. One important aim is the set-up of a reliable dosimetry system. Alanine dosimeters have previously been applied for dosimetry of mixed radiation fields in antiproton therapy, and may be suitable for measurements in mixed neutron and gamma fields. MATERIAL AND METHODS: Two experiments have been carried out in the thermal column of the TRIGA Mark II reactor at the University of Mainz. Alanine dosimeters have been irradiated in a phantom and in liver tissue. RESULTS: For the interpretation and prediction of the dose for each pellet, beside the results of the measurements, calculations with the Monte Carlo code FLUKA are presented here. For the phantom, as well as for the liver tissue, the measured and calculated dose and flux values are in good agreement. DISCUSSION: Alanine dosimeters, in combination with flux measurements and Monte Carlo calculations with FLUKA, suggest that it is possible to establish a system for monitoring the dose in a mixed neutron and gamma field for BNCT and other applications in radiotherapy.

Surface activation with oxygen plasma promotes osteogenesis with enhanced extracellular matrix formation in three-dimensional microporous scaffolds.

Various types of synthetic polyesters have been developed as biomaterials for tissue engineering. These materials commonly possess biodegradability, biocompatibility, and formability, which are preferable properties for bone regeneration. The major challenge of using synthetic polyesters is the result of low cell affinity due to their hydrophobic nature, which hinders efficient cell seeding and active cell dynamics. To improve wettability, plasma treatment is widely used in industry. Here, we performed surface activation with oxygen plasma to hydrophobic copolymers, poly(l-lactide-co-trimethylene carbonate), which were shaped in 2D films and 3D microporous scaffolds, and then we evaluated the resulting surface properties and the cellular responses of rat bone marrow stem cells (rBMSC) to the material. Using scanning electron microscopy and Fourier-transform infrared spectroscopy, we demonstrated that short-term plasma treatment increased nanotopographical surface roughness and wettability with minimal change in surface chemistry. On treated surfaces, initial cell adhesion and elongation were significantly promoted, and seeding efficiency was improved. In an osteoinductive environment, rBMSC on plasma-treated scaffolds exhibited accelerated osteogenic differentiation with osteogenic markers including RUNX2, osterix, bone sialoprotein, and osteocalcin upregulated, and a greater amount of collagen matrix and mineral deposition were found. This study shows the utility of plasma surface activation for polymeric scaffolds in bone tissue engineering.

Dose calculation in biological samples in a mixed neutron-gamma field at the TRIGA reactor of the University of Mainz.

To establish Boron Neutron Capture Therapy (BNCT) for non-resectable liver metastases and for in vitro experiments at the TRIGA Mark II reactor at the University of Mainz, Germany, it is necessary to have a reliable dose monitoring system. The in vitro experiments are used to determine the relative biological effectiveness (RBE) of liver and cancer cells in our mixed neutron and gamma field. We work with alanine detectors in combination with Monte Carlo simulations, where we can measure and characterize the dose. To verify our calculations we perform neutron flux measurements using gold foil activation and pin-diodes. Material and methods. When L-α-alanine is irradiated with ionizing radiation, it forms a stable radical which can be detected by electron spin resonance (ESR) spectroscopy. The value of the ESR signal correlates to the amount of absorbed dose. The dose for each pellet is calculated using FLUKA, a multipurpose Monte Carlo transport code. The pin-diode is augmented by a lithium fluoride foil. This foil converts the neutrons into alpha and tritium particles which are products of the (7)Li(n,α)(3)H-reaction. These particles are detected by the diode and their amount correlates to the neutron fluence directly. Results and discussion. Gold foil activation and the pin-diode are reliable fluence measurement systems for the TRIGA reactor, Mainz. Alanine dosimetry of the photon field and charged particle field from secondary reactions can in principle be carried out in combination with MC-calculations for mixed radiation fields and the Hansen & Olsen alanine detector response model. With the acquired data about the background dose and charged particle spectrum, and with the acquired information of the neutron flux, we are capable of calculating the dose to the tissue. Conclusion. Monte Carlo simulation of the mixed neutron and gamma field of the TRIGA Mainz is possible in order to characterize the neutron behavior in the thermal column. Currently we also speculate on sensitizing alanine to thermal neutrons by adding boron compounds.

Nanotopographical Coatings Induce an Early Phenotype-Specific Response of Primary Material-Resident M1 and M2 Macrophages.

Implants elicit an immunological response after implantation that results in the worst case in a complete implant rejection. This biomaterial-induced inflammation is modulated by macrophages and can be influenced by nanotopographical surface structures such as titania nanotubes or fractal titanium nitride (TiN) surfaces. However, their specific impact on a distinct macrophage phenotype has not been identified. By using two different levels of nanostructures and smooth samples as controls, the influence of tubular TiO2 and fractal TiN nanostructures on primary human macrophages with M1 or M2-phenotype was investigated. Therefore, nanotopographical coatings were either, directly generated by physical vapor deposition (PVD) or by electrochemical anodization of titanium PVD coatings. The cellular response of macrophages was quantitatively assessed to demonstrate a difference in biocompatibility of nanotubes in respect to human M1 and M2-macrophages. Depending on the tube diameter of the nanotubular surfaces, low cell numbers and impaired cellular activity, was detected for M2-macrophages, whereas the impact of nanotubes on M1-polarized macrophages was negligible. Importantly, we could confirm this phenotypic response on the fractal TiN surfaces. The results indicate that the investigated topographies specifically impact the macrophage M2-subtype that modulates the formation of the fibrotic capsule and the long-term response to an implant.

An in vitro model mimics the contact of biomaterials to blood components and the reaction of surrounding soft tissue.

The therapeutic efficacy of a medical product after implantation depends strongly on the host-initiated fibrotic response (foreign body reaction). For novel biomaterials, it is of high relevance to understand this fibrotic process. As an alternative to in vivo studies, in vitro models mimic parts of the whole foreign body reaction. Aim of this study was to develop a wound model with key cells and matrix proteins in coculture. This approach combined blood components such as primary macrophages in a plasma-derived fibrin hydrogel, directly exposed to reference biomaterials (PTFE, glass, titanium). The soft tissue reaction is resembled by integrating fibroblasts in a collagen or a fibrin matrix. Those two experimental setups were conducted to show whether a long-term in vitro culture of 13 days is feasible. The response to reference biomaterials was assessed by multi-parametric analyses, comprising molecular profiling (cytokines, collagen I and ß-actin) and tissue remodeling (cell adherence, histological structure, tissue deposition). Polytetrafluorethylene (PTFE) and titanium were tested as references to correlate the in vitro evaluation to previous in vivo studies. Most striking, both model setups evaluated references' fibrotic characteristics as previously reported by in vivo studies. STATEMENT OF SIGNIFICANCE: We present a test platform applied for assessments on the foreign body reaction to biomaterials. This test system consists of blood components - macrophages and plasma-derived fibrin - as well as fibroblasts and collagen, generating a three-dimensional wound microenvironment. By this modular approach, we achieved a suitable test for long-term studies and overcame the limited short-term stability of whole blood tests. In contrast to previous models, macrophages' viability is maintained during the extended culture period and excels the quality of the model. The potential to evaluate a foreign body reaction in vitro was demonstrated with defined reference materials. This model system might be of high potential as a screening platform to identify novel biomaterial candidates.

Automated real-time monitoring of human pluripotent stem cell aggregation in stirred tank reactors.

The culture of human induced pluripotent stem cells (hiPSCs) at large scale becomes feasible with the aid of scalable suspension setups in continuously stirred tank reactors (CSTRs). Innovative monitoring options and emerging automated process control strategies allow for the necessary highly defined culture conditions. Next to standard process characteristics such as oxygen consumption, pH, and metabolite turnover, a reproducible and steady formation of hiPSC aggregates is vital for process scalability. In this regard, we developed a hiPSC-specific suspension culture unit consisting of a fully monitored CSTR system integrated into a custom-designed and fully automated incubator. As a step towards cost-effective hiPSC suspension culture and to pave the way for flexibility at a large scale, we constructed and utilized tailored miniature CSTRs that are largely made from three-dimensional (3D) printed polylactic acid (PLA) filament, which is a low-cost material used in fused deposition modelling. Further, the monitoring tool for hiPSC suspension cultures utilizes in situ microscopic imaging to visualize hiPSC aggregation in real-time to a statistically significant degree while omitting the need for time-intensive sampling. Suitability of our culture unit, especially concerning the developed hiPSC-specific CSTR system, was proven by demonstrating pluripotency of CSTR-cultured hiPSCs at RNA (including PluriTest) and protein level.

A three-dimensional hybrid pacemaker electrode seamlessly integrates into engineered, functional human cardiac tissue in vitro.

Pacemaker systems are an essential tool for the treatment of cardiovascular diseases. However, the immune system's natural response to a foreign body results in the encapsulation of a pacemaker electrode and an impaired energy efficiency by increasing the excitation threshold. The integration of the electrode into the tissue is affected by implant properties such as size, mechanical flexibility, shape, and dimensionality. Three-dimensional, tissue-like electrode scaffolds render an alternative to currently used planar metal electrodes. Based on a modified electrospinning process and a high temperature treatment, a conductive, porous fiber scaffold was fabricated. The electrical and immunological properties of this 3D electrode were compared to 2D TiN electrodes. An increased surface of the fiber electrode compared to the planar 2D electrode, showed an enhanced electrical performance. Moreover, the migration of cells into the 3D construct was observed and a lower inflammatory response was induced. After early and late in vivo host response evaluation subcutaneously, the 3D fiber scaffold showed no adverse foreign body response. By embedding the 3D fiber scaffold in human cardiomyocytes, a tissue-electrode hybrid was generated that facilitates a high regenerative capacity and a low risk of fibrosis. This hybrid was implanted onto a spontaneously beating, tissue-engineered human cardiac patch to investigate if a seamless electronic-tissue interface is generated. The fusion of this hybrid electrode with a cardiac patch resulted in a mechanical stable and electrical excitable unit. Thereby, the feasibility of a seamless tissue-electrode interface was proven.

Adaption of a PIN-diode detector as an online neutron monitor for the thermal column of the TRIGA research reactor.

A BNCT online neutron monitoring system was tested in a TRIGA reactor, using a silicon PIN-diode with a conversion foil. The setup was tested with different reactor powers at the hot and cold ends of the irradiation channel, using activation foils to compare with measured fluxes. The results demonstrate good reproducibility and show a linear correlation between signal of the PIN-diode and neutron flux at all positions, demonstrating this approach to be suitable for online monitoring of the neutron flux.

A comparative multi-parametric in vitro model identifies the power of test conditions to predict the fibrotic tendency of a biomaterial.

Despite growing effort to advance materials towards a low fibrotic progression, all implants elicit adverse tissue responses. Pre-clinical biomaterial assessment relies on animals testing, which can be complemented by in vitro tests to address the Russell and Burch's 3R aspect of reducing animal burden. However, a poor correlation between in vitro and in vivo biomaterial assessments confirms a need for suitable in vitro biomaterial tests. The aim of the study was to identify a test setting, which is predictive and might be time- and cost-efficient. We demonstrated how sensitive in vitro biomaterial assessment based on human primary macrophages depends on test conditions. Moreover, possible clinical scenarios such as lipopolysaccharide contamination, contact to autologous blood plasma, and presence of IL-4 in an immune niche influence the outcome of a biomaterial ranking. Nevertheless, by using glass, titanium, polytetrafluorethylene, silicone, and polyethylene representing a specific material-induced fibrotic response and by comparison to literature data, we were able to identify a test condition that provides a high correlation to state-of-the-art in vivo studies. Most important, biomaterial ranking obtained under native plasma test conditions showed a high predictive accuracy compared to in vivo assessments, strengthening a biomimetic three-dimensional in vitro test platform.

In vitro chemotaxis and tissue remodeling assays quantitatively characterize foreign body reaction.

Surgical implantation of a biomaterial triggers foreign-body-induced fibrous encapsulation. Two major mechanisms of this complex physiological process are (I) chemotaxis of fibroblasts from surrounding tissue to the implant region, followed by (II) tissue remodeling. As an alternative to animal studies, we here propose a process-aligned in vitro test platform to investigate the material dependency of fibroblast chemotaxis and tissue remodeling mediated by material-resident macrophages. Embedded in a biomimetic three-dimensional collagen hydrogel, chemotaxis of fibroblasts in the direction of macrophage-material-conditioned cell culture supernatant was analyzed by live cell imaging. A combination of statistical analysis with a complementary parameterized random walk model allowed quantitative and qualitative characterization of the cellular walk process. We thereby identified an increasing macrophage-mediated chemotactic potential ranking of biomaterials from glass over polytetrafluorethylene to titanium. To address long-term effects of bio-material-resident macrophages on fibroblasts in a three-dimensional microenvironment, we further studied tissue remodeling by applying macrophage-material-conditioned medium on fibrous in vitro tissue models. A high correlation of the in vitro tissue model to state of the art in vivo study data was found. Titanium exhibited a significantly lower tissue remodeling capacity compared to polytetrafluorethylene. With this approach, we identified a material dependency of both chemotaxis and tissue remodeling processes, strengthening knowledge on their specific contribution to the foreign body reaction.

Confirmation of a realistic reactor model for BNCT dosimetry at the TRIGA Mainz.

PURPOSE: In order to build up a reliable dose monitoring system for boron neutron capture therapy (BNCT) applications at the TRIGA reactor in Mainz, a computer model for the entire reactor was established, simulating the radiation field by means of the Monte Carlo method. The impact of different source definition techniques was compared and the model was validated by experimental fluence and dose determinations. METHODS: The depletion calculation code origen2 was used to compute the burn-up and relevant material composition of each burned fuel element from the day of first reactor operation to its current core. The material composition of the current core was used in a mcnp5 model of the initial core developed earlier. To perform calculations for the region outside the reactor core, the model was expanded to include the thermal column and compared with the previously established attila model. Subsequently, the computational model is simplified in order to reduce the calculation time. Both simulation models are validated by experiments with different setups using alanine dosimetry and gold activation measurements with two different types of phantoms. RESULTS: The mcnp5 simulated neutron spectrum and source strength are found to be in good agreement with the previous attila model whereas the photon production is much lower. Both mcnp5 simulation models predict all experimental dose values with an accuracy of about 5%. The simulations reveal that a Teflon environment favorably reduces the gamma dose component as compared to a polymethyl methacrylate phantom. CONCLUSIONS: A computer model for BNCT dosimetry was established, allowing the prediction of dosimetric quantities without further calibration and within a reasonable computation time for clinical applications. The good agreement between the mcnp5 simulations and experiments demonstrates that the attila model overestimates the gamma dose contribution. The detailed model can be used for the planning of structural modifications in the thermal column irradiation channel or the use of different irradiation sites than the thermal column, e.g., the beam tubes.

Physical and chemical characterization of Ag-doped Ti coatings produced by magnetron sputtering of modular targets.

Silver-doped Ti films were produced using a single magnetron sputtering source equipped with a titanium target containing implemented silver modules under variation of bias voltage and substrate temperature. The Ti(Ag) films were characterized regarding their morphology, contact angle, phase composition, silver content and distribution as well as the elution of Ag(+) ions into cell media. SEM and AFM pictures showed that substrate heating during film deposition supported the formation of even and dense surface layers with small roughness values, an effect that could even be enforced, when a substrate bias voltage was applied instead. The deposition of both Ti and Ag was confirmed by X-ray diffraction. ICP-MS and EDX showed a clear correlation between the applied sputtering parameters and the silver content of the coatings. Surface-sensitive XPS measurements revealed that higher substrate temperatures led to an accumulation of Ag in the near-surface region, while the application of a bias voltage had the opposite effect. Additional elution measurements using ICP-MS showed that the release kinetics depended on the amount of silver located at the film surface and hence could be tailored by variation of the sputter parameters.