Organotypic soft-tissue co-cultures: Morphological changes in microvascular endothelial tubes after incubation with iodinated contrast media.

INTRODUCTION: Clinical complications like thrombosis or anaphylaxis have been described to go along with the intra-venous or intra-arterial injection of iodinated contrast media (CM). It has been suggested that the administration of CM affects rheological parameters and thereby causes reduced blood velocity in microvessels. In vitro studies revealed significant buckling of endothelial cells after exposure to CM reducing the lumen of vessels. The aim of this study was to test the influence of CM on three-dimensional microvascular tubules with open lumina within an organotypic soft-tissue co-culture assay in vitro. This model, which is based on the co-culture of endothelial cells and fibroblasts, allows the analysis and quantitation of different parameters of microvascular endothelial capillary structures. MATERIAL AND METHODS: Human dermal fibroblasts and human dermal microvascular endothelial cells were co-cultured for 10 days. Fibroblasts were adapted to the endothelial cell medium before co-culture and allowed to proliferate as well as produce extracellular matrix. The co-cultures were exposed to three different CM, i.e., Iomeprol (Imeron 400MCT), Iodixanol (Visipaque 320) or Iohexol (Accupaque 350) for 1.5 minutes or 5.0 minutes, respectively. For this, a mixture of CM and cell culture medium in a ratio of 30% CM by volume was prepared. After fixation in methanol/acetone, the endothelial cells were immunolabeled with the endothelial marker anti-CD31 and the tubular structures were assessed morphometrically. RESULTS: In the organotypic soft-tissue co-cultures with fibroblasts, the endothelial cells developed three-dimensional capillary-like structures which expanded via sprouting branches. After incubation with the different CM, the numbers of endothelial tubes (p = 0.001) and their lengths (p = 0.003) were significantly lower after the 5 minutes incubation time, when compared to the 1.5 minutes incubation time. The tubular diameters were significantly reduced after 5 minutes (p < 0.001), when compared to the 1.5 minutes incubation duration. Interestingly, Iomeprol and Iodixanol induced an elongation of the tubular branches during incubation duration of 1.5 minutes (p = 0.015). However, after 5 minutes incubation, the tubular branches were drastically shorter in the presence of Iomeprol and Iodixanol than the tubular branches of the control (p = 0.007). SUMMARY AND CONCLUSION: All CM exerted a negative effect on the parameters of in vitro blood vessel development.

The effect of endothelialization on the epidermal differentiation in human three-dimensional skin constructs - A morphological study.

INTRODUCTION: Inducing vascularization in three-dimensional skin constructs continues to be difficult. In this study, two variations of human full-thickness skin constructs were examined. Type KCFB consists of keratinocytes (epidermal equivalent) and fibroblasts that were embedded in a collagen matrix (dermal equivalent). Type KCFB-EC consists of keratinocytes as well as fibroblasts and vascular endothelial cells. The epidermal equivalent of KCFB-EC constructs underwent cellular alterations in their differentiation possibly induced by the presence of endothelial cells. The objective of the study was to assess the effect of endothelial cells, i.e., endothelialization of the dermal equivalent on the differentiation of keratinocytes by comparing the morphology and ultrastructure of the two types of skin constructs, as well as to excised normal human skin. HYPOTHESIS: The differentiation of keratinocytes is influenced by the presence of endothelial cells. METHODS, PATIENTS, MATERIAL: KCFB constructs (keratinocytes, fibroblasts) and KCFB-EC skin constructs(kera-tinocytes, fibroblasts, endothelial cells) were prepared according to Küchler et al. [25]. After two weeks, the skin constructs were processed for analysis by light microscopy (LM) and electron microscopy (TEM), followed by quantitative, semi-quantitative as well as qualitative assessment. For comparison, analysis by LM and TEM of excised normal human skin was also performed. RESULTS: Both KCFB and KCFB-EC skin constructs and the human skin had all strata of stratified soft-cornified epidermis present. The comparison of the respective layers of the skin constructs brought the following characteristics to light: The KCFB-EC constructs had significantly more mitotic cells in the stratum spinosum, more cell layers in the stratum granulosum and more keratohyalin granules compared to KCFB skin constructs. Additionally, the epidermal architecture was unorganized in the endothelialized constructs and features of excessive epidermal differentiation appeared in KCFB-EC skin constructs. CONCLUSION: The endothelialization of the dermal equivalent caused changes in the differentiation of the epidermis of KCFB-EC skin constructs that may be interpreted as an unbalanced, i.e., uncontrolled or enhanced maturation process.