Assessment of Melanogenesis in a Pigmented Human Tissue-Cultured Skin Equivalent.

BACKGROUND: Organotypic tissue-cultured skin equivalents are used for a broad range of applications either as possible substitute for animal tests or for transplantation in patient-centered care. AIMS: In this study, we implemented melanocytes in a tissue-cultured full-thickness skin equivalent, consisting of epidermis and dermis. The versatility of this skin-like model with respect to pigmentation and morphological criteria was tested. MATERIALS AND METHODS: Pigmented skin equivalents were morphologically characterized, and melanogenesis was evaluated after treatment with kojic acid - a tyrosinase inhibitor and forskolin - a well-known activator of the cyclic adenosine 3,5-monophosphate pathway. Pigmentation was measured either by determination of the extinction at 400 nm after melanin extraction with KOH correlated to a melanin standard curve or by reflectance colorimetric analysis, monitoring reflectance of 660 nm and 880 nm emitting diodes. RESULTS: The morphological analysis revealed characteristic epidermal stratification with melanocytes located at the basal layer. Stimulation with forskolin increased the pigmentation, whereas treatment with kojic acid caused bleaching. CONCLUSION: The present study demonstrates that the herein-introduced organotypic tissue-cultured skin equivalent is comparable to the normal human skin and its versatility in tests regarding skin pigmentation. Therefore, this model might help understand diseases with dysfunctional pigmentation such as melasma, vitiligo, and postinflammatory hyperpigmentation.

Influence of Biodentine® - A Dentine Substitute - On Collagen Type I Synthesis in Pulp Fibroblasts In Vitro.

Preserving a patient's own teeth-even in a difficult situation-is nowadays preferable to surgical intervention and therefore promotes development of suitable dental repair materials. Biodentine®, a mineral trioxide aggregate substitute, has been used to replace dentine in a bioactive and biocompatible manner in both the dental crown and the root. The aim of our study was to evaluate the influence of Biodentine® on pulp fibroblasts in vitro. For this study, one to five Biodentine® discs with a diameter of 5.1mm were incubated in DMEM. To obtain Biodentine® suspensions the media were collected and replaced with fresh medium every 24h for 4 days. Primary pulp cells were isolated from freshly extracted wisdom teeth of 20-23 year old patients and incubated with the Biodentine® suspensions. Proliferation, cell morphology, cell integrity and cell viability were monitored. To evaluate the effect of Biodentine® on collagen type I synthesis, the secretion of the N-terminal domain of pro-collagen type I (P1NP) and the release of transforming growth factor-ß1 (TGF-ß1) were quantified. None of the Biodentine® suspensions tested influenced cell morphology, proliferation or cell integrity. The cell viability varied slightly depending on the suspension used. However, the concentrations of P1NP of all pulp fibroblast cultures treated for 24h with the moderate to high Biodentine® concentration containing suspensions of day 1 were reduced to 5% of the control. Furthermore, a significant TGF-ß1 reduction was observed after treatment with these suspensions. It could be shown that Biodentine® is biocompatible. However, dissolved particles of the moderate to high concentrated Biodentine® suspensions 24h after mixing induce a significant reduction of TGF-ß1 release and reduce the secretion of collagen type I of primary pulp fibroblasts.

Vascular endothelial growth factor C-induced lymphangiogenesis decreases tumor interstitial fluid pressure and tumor.

Characteristically, most solid tumors exhibit an increased tumor interstitial fluid pressure (TIFP) that directly contributes to the lowered uptake of macromolecular therapeutics into the tumor interstitium. Abnormalities in the tumor-associated lymph vessels are a central brick in the development and prolonged sustaining of an increased TIFP. In the current study, vascular endothelial growth factor C (VEGF-C) was used to enhance tumor-associated lymphangiogenesis as a new mechanism to actively reduce the TIFP by increased lymphatic drainage of the tumor tissue. Human A431 epidermoid vulva carcinoma cells were inoculated in NMRI nu/nu mice to generate a xenograft mouse model. Seven days after tumor cell injection, VEGF-C was peritumorally injected to induce lymphangiogenesis. Tumor growth and TIFP was lowered significantly over time in VEGF-C-treated tumors in comparison to control or VEGF-A-treated animals. These data demonstrate for the first time that actively induced lymphangiogenesis can lower the TIFP in a xenograft tumor model and apparently reduce tumor growth. This model represents a novel approach to modulate biomechanical properties of the tumor interstitium enabling a lowering of TIFP in vivo.

Evaluation of beneficial and adverse effects of glucocorticoids on a newly developed full-thickness skin model.

Glucocorticoids (GCs) are highly effective compounds widely used in the treatment of inflammatory diseases; however, they offer distinct adverse effects such as skin thinning in response to long-term topical treatment. Nevertheless it is difficult to deduce the safety of a newly synthesized compound from its structural formula. Efficient assay systems that measure beneficial and adverse effects are needed. In the present study the applicability of a three-dimensional full-thickness skin model (FTSM) is tested to display GC-induced effects regarding anti-inflammation and atrophy. It is shown that topical application of a commercial GC ointment suppresses the ultraviolet (UV)B induced induction of interleukin (IL)-6 and IL-8. Addition of purified betamethasone-17-valerate, prednicarbate and clobetasol-17-propionate to the culture medium for 14 days caused a reduction in the number of epidermal cell-layers corresponding to the atrophic risk found in vivo. Similarly, repeated topical application of five GC creams induced epidermal thinning. Evidence is given that the inhibitory effect on keratinocyte proliferation contributes to this effect. Furthermore, dermal thinning was monitored by measuring type I collagen synthesis; a decreased collagen synthesis similar to the in vivo situation is shown. The present study demonstrates the versatility of this FTSM in the validation of effectiveness and safety of GCs.