Synthesis of Soft Polysiloxane-urea Elastomers for Intraocular Lens Application.

This study discusses a synthesis route for soft polysiloxane-based urea (PSU) elastomers for their applications as accommodating intraocular lenses (a-IOLs). Aminopropyl-terminated polydimethylsiloxanes (PDMS) were previously prepared via the ring-chain equilibration of the cyclic siloxane octamethylcyclotetrasiloxane (D4) and 1,3-bis(3-aminopropyl)-tetramethyldisiloxane (APTMDS). Phenyl groups were introduced into the siloxane backbone via the copolymerization of D4 and 2,4,6,8-tetramethyl-2,4,6,8-tetraphenyl-cyclotetrasiloxane (D4Me,Ph). These polydimethyl-methyl-phenyl-siloxane-block copolymers were synthesized for increasing the refractive indices of polysiloxanes. For applications as an a-IOL, the refractive index of the polysiloxanes must be equivalent to that of a young human eye lens. The polysiloxane molecular weight is controlled by the ratio of the cyclic siloxane to the endblocker APTMDS. The transparency of the PSU elastomers is examined by the transmittance measurement of films between 200 and 750 nm, using a UV-Vis spectrophotometer. Transmittance values at 750 nm (upper end of the visible spectrum) are plotted against the PDMS molecular weight, and > 90% of the transmittance is observed until a molecular weight of 18,000 g·mol-1. Mechanical properties of the PSU elastomers are investigated using stress-strain tests on die-cut dog-bone-shaped specimens. For evaluating mechanical stability, mechanical hysteresis is measured by repeatedly stretching (10x) the specimens to 5% and 100% elongation. Hysteresis considerably decreases with the increase in the PDMS molecular weight. In vitro cytotoxicity of some selected PSU elastomers is evaluated using an MTS cell viability assay. The methods described herein permit the synthesis of a soft, transparent, and noncytotoxic PSU elastomer with a refractive index approximately equal to that of a young human eye lens.

A standardized method based on pigmented epidermal models evaluates sensitivity against UV-irradiation.

To protect the human skin from extensive solar radiation, melanocytes produce melanin and disperse it via melanosomes to keratinocytes in the basal and suprabasal layers of the human epidermis. Moreover, melanocytes are associated with pathological skin conditions such as vitiligo and psoriasis. Thus, an in vitro skin model that comprises a defined cutaneous pigmentation system is highly relevant in cosmetic, pharmaceutical and medical research. Here, we describe how the epidermal-melanin-unit can be established in vitro. Therefore, primary human melanocytes are implemented in an open source reconstructed epidermis. Following 14 days at the air liquid interface, a differentiated epidermis was formed and melanocytes were located in the basal layer. The functionality of the epidermal-melanin-unit could be shown by the transfer of melanin to the surrounding keratinocytes, and a significantly increased melanin content of models stimulated with either UV-radiation or the melanin precursor dihydroxyphenylalanine. Additionally, an UV50 assay was developed to test the protective effect of melanin. In analogy to the IC50 value in risk assessment, the UV50 value facilitates a quantitative investigation of harmful effects of natural UV-radiation to the skin in vitro. Employing this test, we could demonstrate that the melanin content correlates with the resilience against simulated sunlight, which comprises 2.5 % UVB and 97.5 % UVA. Besides demonstrating the protective effect of melanin in vitro, the assay was used to determine the protective effect of a consumer product in a highly standardized setup.

Antimicrobial effect and biocompatibility of novel metallic nanocrystalline implant coatings.

AIM: The present in vitro study was designed to evaluate the surface characteristics, biocompatibilities and antimicrobial effects of experimental titanium implant surfaces, coated by nanocrystalline silver, copper, and bismuth. Biocompatible and antimicrobial implant modifications could result in reduced biofilm formation on implant surfaces and therefore in less periimplant inflammation. FINDINGS: Titanium discs (thickness 1 mm and 12 mm in diameter) were coated by pulsed magnetron-sputtering of nanocrystalline metals (bismuth, copper, and silver). Bismuth coatings revealed higher surface roughness values in comparison to silver and copper coatings via atomic force microscopy. Ion release after 168 h in culture medium was analyzed by inductively coupled plasma-mass spectrometry and showed significant different amounts of released copper (>120 000 µg/L), silver (550 µg/L) or bismuth (80 µg/L). No cytotoxic effect on HaCaT cell proliferation was detected on the uncoated Ti/TiO2 reference surfaces, the bismuth coatings and silver coatings. In contrast, copper-coated discs showed a strong cytotoxic effect. All three coatings exhibited antimicrobial effects by trend in the fluorometric Resazurin testing and significant localized antibacterial effects in live/dead microscopy after incubation of the specimens for 150 min in bacterial solution of S. epidermidis. CONCLUSIONS: The tested metallic implant coatings (silver and bismuth) allowed surface modifications that may improve therapeutic approaches to biofilm prevention on dental implants. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2015. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 1571-1579, 2016.

Cell death stages in single apoptotic and necrotic cells monitored by Raman microspectroscopy.

Although apoptosis and necrosis have distinct features, the identification and discrimination of apoptotic and necrotic cell death in vitro is challenging. Immunocytological and biochemical assays represent the current gold standard for monitoring cell death pathways; however, these standard assays are invasive, render large numbers of cells and impede continuous monitoring experiments. In this study, both room temperature (RT)-induced apoptosis and heat-triggered necrosis were analyzed in individual Saos-2 and SW-1353 cells by utilizing Raman microspectroscopy. A targeted analysis of defined cell death modalities, including early and late apoptosis as well as necrosis, was facilitated based on the combination of Raman spectroscopy with fluorescence microscopy. Spectral shifts were identified in the two cell lines that reflect biochemical changes specific for either RT-induced apoptosis or heat-mediated necrosis. A supervised classification model specified apoptotic and necrotic cell death based on single cell Raman spectra. To conclude, Raman spectroscopy allows a non-invasive, continuous monitoring of cell death, which may help shedding new light on complex pathophysiological or drug-induced cell death processes.

Design and analysis of a squamous cell carcinoma in vitro model system.

Tissue-engineered skin equivalents based on primary isolated fibroblasts and keratinocytes have been shown to be useful tools for functional in vitro tests, including toxicological screenings and drug development. In this study, a commercially available squamous cell carcinoma (SCC) cell line SCC-25 was introduced into epidermal and full-thickness skin equivalents to generate human-based disease-in-a-dish model systems. Interestingly, when cultured either in the epidermis or dermis of full-thickness skin equivalents, SCC-25 cells formed hyper-keratinized tumor cell nests, a phenomenon that is frequently seen in the skin of patients afflicted with SCC. Raman spectroscopy was employed for the label-free cell phenotype characterization within the engineered skin equivalents and revealed the presence of differential protein patterns in keratinocytes and SCC-25 cells. To conclude, the here presented SSC disease-in-a-dish approaches offer the unique opportunity to model SSC in human skin in vitro, which will allow further insight into SSC disease progression, and the development of therapeutic strategies.

Setup for investigating gold nanoparticle penetration through reconstructed skin and comparison to published human skin data.

Owing to the limited source of human skin (HS) and the ethical restrictions of using animals in experiments, in vitro skin equivalents are a possible alternative for conducting particle penetration experiments. The conditions for conducting penetration experiments with model particles, 15-nm gold nanoparticles (AuNP), through nonsealed skin equivalents are described for the first time. These conditions include experimental setup, sterility conditions, effective applied dose determination, skin sectioning, and skin integrity check. Penetration at different exposure times (two and 24 h) and after tissue fixation (fixed versus unfixed skin) are examined to establish a benchmark in comparison to HS in an attempt to get similar results to HS experiments presented earlier. Multiphoton microscopy is used to detect gold luminescence in skin sections. λ(ex)=800 nm is used for excitation of AuNP and skin samples, allowing us to determine a relative index for particle penetration. Despite the observed overpredictability of penetration into skin equivalents, they could serve as a first fast screen for testing the behavior of nanoparticles and extrapolate their penetration behavior into HS. Further investigations are required to test a wide range of particles of different physicochemical properties to validate the skin equivalent-human skin particle penetration relationship.

Depth profiling of gold nanoparticles and characterization of point spread functions in reconstructed and human skin using multiphoton microscopy.

Multiphoton microscopy has become popular in studying dermal nanoparticle penetration. This necessitates studying the imaging parameters of multiphoton microscopy in skin as an imaging medium, in terms of achievable detection depths and the resolution limit. This would simulate real-case scenarios rather than depending on theoretical values determined under ideal conditions. This study has focused on depth profiling of sub-resolution gold nanoparticles (AuNP) in reconstructed (fixed and unfixed) and human skin using multiphoton microscopy. Point spread functions (PSF) were determined for the used water-immersion objective of 63×/NA = 1.2. Factors such as skin-tissue compactness and the presence of wrinkles were found to deteriorate the accuracy of depth profiling. A broad range of AuNP detectable depths (20-100 µm) in reconstructed skin was observed. AuNP could only be detected up to ∼14 µm depth in human skin. Lateral (0.5 ± 0.1 µm) and axial (1.0 ± 0.3 µm) PSF in reconstructed and human specimens were determined. Skin cells and intercellular components didn't degrade the PSF with depth. In summary, the imaging parameters of multiphoton microscopy in skin and practical limitations encountered in tracking nanoparticle penetration using this approach were investigated.

Raman spectroscopy: a noninvasive analysis tool for the discrimination of human skin cells.

Noninvasive monitoring of tissue-engineered (TE) constructs during their in vitro maturation or postimplantation in vivo is highly relevant for graft evaluation. However, traditional methods for studying cell and matrix components in engineered tissues such as histology, immunohistochemistry, or biochemistry require invasive tissue processing, resulting in the need to sacrifice of TE constructs. Raman spectroscopy offers the unique possibility to analyze living cells label-free in situ and in vivo solely based on their phenotype-specific biochemical fingerprint. In this study, we aimed to determine the applicability of Raman spectroscopy for the noninvasive identification and spectral separation of primary human skin fibroblasts, keratinocytes, and melanocytes, as well as immortalized keratinocytes (HaCaT cells). Multivariate analysis of cell-type-specific Raman spectra enabled the discrimination between living primary and immortalized keratinocytes. We further noninvasively distinguished between fibroblasts, keratinocytes, and melanocytes. Our findings are especially relevant for the engineering of in vitro skin models and for the production of artificial skin, where both the biopsy and the transplant consist of several cell types. To realize a reproducible quality of TE skin, the determination of the purity of the cell populations as well as the detection of potential molecular changes are important. We conclude therefore that Raman spectroscopy is a suitable tool for the noninvasive in situ quality control of cells used in skin tissue engineering applications.