Replacing the Draize eye test: Impedance spectroscopy as a 3R method to discriminate between all GHS categories for eye irritation.

Highly invasive animal based test procedures for risk assessment such as the Draize eye test are under increasing criticism due to poor transferability for the human organism and animal-welfare concerns. However, besides all efforts, the Draize eye test is still not completely replaced by alternative animal-free methods. To develop an in vitro test to identify all categories of eye irritation, we combined organotypic cornea models based on primary human cells with an electrical readout system that measures the impedance of the test models. First, we showed that employing a primary human cornea epithelial cell based model is advantageous in native marker expression to the primary human epidermal keratinocytes derived models. Secondly, by employing a non-destructive measuring system based on impedance spectroscopy, we could increase the sensitivity of the test system. Thereby, all globally harmonized systems categories of eye irritation could be identified by repeated measurements over a period of 7 days. Based on a novel prediction model we achieved an accuracy of 78% with a reproducibility of 88.9% to determine all three categories of eye irritation in one single test. This could pave the way according to the 3R principle to replace the Draize eye test.

A multilayered electrospun graft as vascular access for hemodialysis.

Despite medical achievements, the number of patients with end-stage kidney disease keeps steadily raising, thereby entailing a high number of surgical and interventional procedures to establish and maintain arteriovenous vascular access for hemodialysis. Due to vascular disease, aneurysms or infection, the preferred access-an autogenous arteriovenous fistula-is not always available and appropriate. Moreover, when replacing small diameter blood vessels, synthetic vascular grafts possess well-known disadvantages. A continuous multilayered gradient electrospinning was used to produce vascular grafts made of collagen type I nanofibers on luminal and adventitial graft side, and poly-ɛ-caprolactone as medial layer. Therefore, a custom-made electrospinner with robust environmental control was developed. The morphology of electrospun grafts was characterized by scanning electron microscopy and measurement of mechanical properties. Human microvascular endothelial cells were cultured in the graft under static culture conditions and compared to cultures obtained from dynamic continuous flow bioreactors. Immunofluorescent analysis showed that endothelial cells form a continuous luminal layer and functional characteristics were confirmed by uptake of acetylated low-density-lipoprotein. Incorporation of vancomycin and gentamicin to the medial graft layer allowed antimicrobial inhibition without exhibiting an adverse impact on cell viability. Most striking a physiological hemocompatibility was achieved for the multilayered grafts.

Catch-up validation study of an in vitro skin irritation test method based on an open source reconstructed epidermis (phase II).

To replace the Draize skin irritation assay (OECD guideline 404) several test methods based on reconstructed human epidermis (RHE) have been developed and were adopted in the OECD test guideline 439. However, all validated test methods in the guideline are linked to RHE provided by only three companies. Thus, the availability of these test models is dependent on the commercial interest of the producer. To overcome this limitation and thus to increase the accessibility of in vitro skin irritation testing, an open source reconstructed epidermis (OS-REp) was introduced. To demonstrate the capacity of the OS-REp in regulatory risk assessment, a catch-up validation study was performed. The participating laboratories used in-house generated OS-REp to assess the set of 20 reference substances according to the performance standards amending the OECD test guideline 439. Testing was performed under blinded conditions. The within-laboratory reproducibility of 87% and the inter-laboratory reproducibility of 85% prove a high reliability of irritancy testing using the OS-REp protocol. In addition, the prediction capacity was with an accuracy of 80% comparable to previous published RHE based test protocols. Taken together the results indicate that the OS-REp test method can be used as a standalone alternative skin irritation test replacing the OECD test guideline 404.

Development and application of three-dimensional skin equivalents for the investigation of percutaneous worm invasion.

Investigation of percutaneous helminth infection is generally based on animal models or excised skin. As desirable replacement of animal experiments, tissue-engineered skin equivalents have recently been applied in microbial and viral in vitro infection models. In the present study, the applicability of tissue-engineered skin equivalents for the investigation of percutaneous helminth invasion was evaluated. Epidermal and a full-thickness skin equivalents that suit the requirements for helminth invasion studies were developed. Quantitative invasion assays were performed with the skin-invading larvae of the helminths Strongyloides ratti and Schistosoma mansoni. Both skin equivalents provided a physical barrier to larval invasion of the nematode S. ratti, while these larvae could invade and permeate a cell-free collagen scaffold and ex vivo epidermis. In contrast, the epidermal and full-thickness skin equivalents exhibited a human host-specific susceptibility to larvae of trematode S. mansoni, which could well penetrate. Invasion of S. mansoni in cell-free collagen scaffold was lowest for all experimental conditions. Thus, reconstructed epidermis and full-thickness skin equivalents confirmed a high degree of accordance to native tissue. Additionally, not only tailless schistosomula but also cercariae could permeate the skin equivalents, and thus, delayed tail loss hypothesis was supported. The present study indicates that the limitations in predictive infection test systems for human-pathogenic invading helminths can be overcome by tissue-engineered in vitro skin equivalents allowing a substitution of the human skin for analysis of the interaction between parasites and their hosts' tissues. This novel tissue-engineered technology accomplishes the endeavor to save animal lives.

Impedance spectroscopy for the non-destructive evaluation of in vitro epidermal models.

PURPOSE: Reconstructed human epidermis (RHE) is standardly used for the risk assessment of chemical compounds. However, analysis is dependent on invasive methods such as histological processing or 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) staining. METHODS: As an alternative, we have developed a non-destructive technology to analyze the integrity of epidermal equivalents based on impedance spectroscopy. RHEs were generated and impedance spectra were recorded. from these spectra, we extrapolated electrical characteristics such as the capacitance and the ohmic resistance. Furthermore, the measurable electrical parameters were used to quantify the effects of mechanical and chemical disruption of the epidermal integrity. RESULTS: A fully matured RHE exhibits typical impedance spectra in a frequency ranging between 1 Hz and 100 kHz, which is comparable to the spectra of freshly isolated human epidermal biopsies. We could show that, during RHE maturation, these characteristics change significantly. Thus, capacitance and ohmic resistance can be employed as a criterion for the quality control of skin equivalents. Additionally, our application of impedance spectroscopy reveals sufficient sensitivity to detect a transient decreased ohmic resistance caused by 2-propanol, which is classified as a non-irritant by MTT assays. CONCLUSION: These results indicate that impedance spectroscopy can be employed as a non-destructive complementary method to assess mild irritative effects, which is currently not possible.

[Improving the biocompatibility of silicone implants using spider silk coatings: immunohistochemical analysis of capsule formation]. / Verbesserung der Biokompatibilität von Silikonimplantaten durch Spinnenseidenbeschichtung: Immunhistochemische Untersuchungen zum Einfluss auf die Kapselbildung.

INTRODUCTION: Optimisation of the biocompatibility of silicone implants and reduction of capsule formation around the surface of such implants are in the focus of plastic surgical biomaterial research. In addition to its extraordinary physical and biochemical properties, spider silk shows high biocompatibility. Therefore, the coating of silicone implant surfaces with recombinant spider silk was analysed regarding foreign body reactions. MATERIALS AND METHODS: In the context of a preclinical study, miniaturised silicone implants were implanted in the back of 60 Sprague-Dawley rats. The animals were randomised; 30 animals received a texturised implant coated with the recombinant spider silk protein eADF4(C16) and 30 animals received uncoated implants. 3, 6 and 12 months after implantation, implants together with the surrounding capsules were removed and submitted to histological and immunohistochemical assessment. RESULTS: Coating of silicone implants with the recombinant spider silk protein eADF4(C16) resulted in a delayed and significantly decreased foreign body reaction and a reduced capsule manifestation. CONCLUSION: eADF4(C16) seems to be a promising candidate for the reduction of foreign body-associated capsule formation. Moreover, coating of other medical implants with this recombinant spider silk protein may improve their biocompatibility with little additional effort.

Transport of hop aroma compounds across Caco-2 monolayers.

Although being reported and used as a sedative remedy for several years, the bioactive principle of hop preparations is still not decisively clarified. Understanding absorption and transformation processes of potential physiologically active constituents is essential to evaluate the likeliness of biological effects on humans. Therefore, single hop aroma compounds as well as digestive transformation products thereof have been investigated in view of their human intestinal absorption, applying Caco-2 transport experiments as well as investigations on potential biotransformation processes. Selective and sensitive identification and quantification were thereby achieved by application of two-dimensional high resolution gas chromatography-mass spectrometry in conjunction with stable isotope dilution analysis, leading to the determination of apparent permeability values by different mathematical approaches considering sink and non-sink conditions. Overall, calculated permeability values ranged from 2.6 × 10(-6) to 1.8 × 10(-4) cm s(-1) with all mathematical approaches, indicating high absorption potential and almost complete bioavailability for all tested compounds with hydroxyl-functionalities. Considering this high permeability together with the high lipophilicity of these substances, a passive transcellular uptake route can be speculated. Investigated sesquiterpenes and ß-myrcene showed flat absorption profiles while the investigated esters showed decreasing profiles. In view of the lipophilic and volatile nature of the investigated substances, special attention was paid to recovery and mass balance determination. Furthermore, in the course of the transport experiments of 1-octen-3-ol and 3-methyl-2-buten-1-ol, additional biotransformation products were observed, namely 3-octanone and 3-methyl-2-butenal, respectively. The absence of these additional substances in control experiments strongly indicates an intestinal first-pass metabolism of the α,ß-unsaturated alcohols 1-octen-3-ol and 3-methyl-2-buten-1-ol in Caco-2 cells.

Development of a human three-dimensional organotypic skin-melanoma spheroid model for in vitro drug testing.

Despite remarkable efforts, metastatic melanoma (MM) still presents with significant mortality. Recently, mono-chemotherapies are increasingly replenished by more cancer-specific combination therapies involving death ligands and drugs interfering with cell signaling. Still, MM remains a fatal disease because tumors rapidly develop resistance to novel therapies thereby regaining tumorigenic capacity. Although genetically engineered mouse models for MM have been developed, at present no model is available that reliably mimics the human disease and is suitable for studying mechanisms of therapeutic obstacles including cell death resistance. To improve the increasing requests on new therapeutic alternatives, reliable human screening models are demanded that translate the findings from basic cellular research into clinical applications. By developing an organotypic full skin equivalent, harboring melanoma tumor spheroids of defined sizes we have invented a cell-based model that recapitulates both the 3D organization and multicellular complexity of an organ/tumor in vivo but at the same time accommodates systematic experimental intervention. By extending our previous findings on melanoma cell sensitization toward TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) by co-application of sublethal doses of ultraviolet-B radiation (UVB) or cisplatin, we show significant differences in the therapeutical outcome to exist between regular two-dimensional (2D) and complex in vivo-like 3D models. Of note, while both treatment combinations killed the same cancer cell lines in 2D culture, skin equivalent-embedded melanoma spheroids are potently killed by TRAIL+cisplatin treatment but remain almost unaffected by the TRAIL+UVB combination. Consequently, we have established an organotypic human skin-melanoma model that will facilitate efforts to improve therapeutic outcomes for malignant melanoma by providing a platform for the investigation of cytotoxic treatments and tailored therapies in a more physiological setting.

Schedule-dependent antagonism of paclitaxel and cisplatin in human gastric and ovarian carcinoma cell lines in vitro.

Paclitaxel has demonstrated broad clinical activity in a variety of malignancies both alone and in combination with other chemotherapeutic agents. The in vitro cytotoxicity of paclitaxel and cisplatin alone, in combination and in sequence, were evaluated against established human gastric and ovarian carcinoma cell lines using 2-h drug exposure. The combination of cisplatin and paclitaxel was found to be additive or even synergistic when paclitaxel was given 24 h prior to cisplatin as demonstrated by isobologram analysis. However, when both drugs were given simultaneously or when cisplatin was given prior to paclitaxel, a strong antagonistic interaction was observed. This antagonism was evident for up to 72 h after a 2-h exposure to cisplatin. Pretreatment with cisplatin caused no alteration in [3H]paclitaxel uptake in HM2 gastric carcinoma cells, but resulted in decreased intracellular retention of paclitaxel. Since cisplatin treatment led to a reduction in cellular glutathione content in these cells and reduced levels of glutathione have been associated with protection against cytotoxicity of paclitaxel, cells were pretreated with L-buthionine sulfoximine (L-BSO). However, depletion of glutathione had no influence on the activity of paclitaxel. A significant accumulation of cells in S-phase was observed 24 h after cisplatin, which resolved after 48 h and resulted in a pronounced increase of G2M phase. These data demonstrate that the interactions of paclitaxel and cisplatin are highly schedule-dependent and applications of cisplatin simultaneously with or prior to paclitaxel may result in pronounced antagonism. These findings could have implications for the design of further clinical protocols.