Upon implantation of tissue-engineered scaffolds, hypoxia will occur until neovascularization takes place. In vivo, the temporary fibrin matrix forms a suitable matrix for this process and fibrin variants can influence the extent of neovascularization. In this study, the influence of oxygen tension and naturally occurring fibrinogen variants on adipose tissue-derived mesenchymal stem cell (ASC) expansion and differentiation were determined. ASC proliferated 1.7-fold faster in 1% oxygen and showed reduced cell aging, and their stemness was preserved. The stem cell surface marker expression was similar in 1% and 20% oxygen. The various fibrinogen coatings did not influence ASC expansion and differentiation. Differentiation of ASC toward adipogenic and osteogenic lineages was improved in 20% oxygen, whereas 1% oxygen improved chondrogenic differentiation. In conclusion, optimal oxygen concentrations vary for the intended ASC application, and fibrinogen variants, which can be used to influence neovascularization, do not alter ASC behavior. These data emphasize the importance of oxygen concentrations during stem cell growth and differentiation.

Angiogenesis is essential during development and in pathological conditions such as chronic inflammation and cancer progression. Inhibition of angiogenesis by targeting vascular endothelial growth factor (VEGF) blocks disease progression, but most patients eventually develop resistance which may result from compensatory signaling pathways. In endothelial cells (EC) expression of the pro-angiogenic chemokine CXCL12 is regulated by non-canonical nuclear factor (NF)-κB signaling. Here, we report that NF-κB-inducing kinase (NIK) and subsequent non-canonical NF-κB signaling regulates both inflammation-induced and tumor-associated angiogenesis. NIK is highly expressed in endothelial cells (EC) in tumor tissues and inflamed rheumatoid arthritis synovial tissue. Furthermore, non-canonical NF-κB signaling in human microvascular EC significantly enhanced vascular tube formation, which was completely blocked by siRNA targeting NIK. Interestingly, Nik−/− mice exhibited normal angiogenesis during development and unaltererd TNFα- or VEGF-induced angiogenic responses, whereas angiogenesis induced by non-canonical NF-κB stimuli was significantly reduced. In addition, angiogenesis in experimental arthritis and a murine tumor model was severely impaired in these mice. These studies provide evidence for a role of non-canonical NF-κB signaling in pathological angiogenesis, and identify NIK as a potential therapeutic target in chronic inflammatory diseases and tumor neoangiogenesis.

Wound healing events which occur in humans are difficult to study in animals due to differences in skin physiology. Furthermore there are increasing restrictions in Europe for using animals for testing the therapeutic properties of new compounds. Therefore, in line with the 3Rs (reduction, refinement and replacement of test animals), a number of human in vitro models of different levels of complexity have been developed to investigate cell mobility during wound healing. Keratinocyte, melanocyte, fibroblast and endothelial cell mobility are described, since these are the residential cells which are responsible for restoring the main structural features of the skin. A monolayer scratch assay is used to study random fibroblast and endothelial cell migration in response to EGF and bFGF respectively and a chemotactic assay is used to study directional fibroblast migration towards CCL5. In order to study endothelial sprouting in response to bFGF or VEGF, which involves continuous degradation and resynthesis of a 3D matrix, a fibrin gel is used. Human physiologically relevant tissue-engineered skin models are used to investigate expansion of the stratified, differentiated epidermis (keratinocytes and melanocytes) over a fibroblast populated dermis and also to study migration and distribution of fibroblasts into the dermis. Together these skin models provide a platform for testing the mode of action of novel compounds for enhanced and scar free wound healing.

IntroductionEfficient implementation of peripheral blood-derived endothelial-colony cells (PB-ECFCs) as a therapeutical tool requires isolation and generation of a sufficient number of cells in ex vivo conditions devoid of animal-derived products. At present, little is known how the isolation and expansion procedure in xenogeneic-free conditions affects the therapeutical capacity of PB-ECFCs.ResultsThe findings presented in this study indicate that human platelet lysate (PL) as a serum substitute yields twice more colonies per mL blood compared to the conventional isolation with fetal bovine serum (FBS). Isolated ECFCs displayed a higher proliferative ability in PL supplemented medium than cells in FBS medium during 30 days expansion. The cells at 18 cumulative population doubling levels (CPDL) retained their proliferative capacity, showed higher sprouting ability in fibrin matrices upon stimulation with FGF-2 and VEGF-A than the cells at 6 CPDL, and displayed low β-galactosidase activity. The increased sprouting of PB-ECFCs at 18 CPDL was accompanied by an intrinsic activation of the uPA/uPAR fibrinolytic system. Induced deficiency of uPA (urokinase-type plasminogen activator) or uPAR (uPA receptor) by siRNA technology completely abolished the angiogenic ability of PB-ECFCs in fibrin matrices. During the serial expansion, the gene induction of the markers associated with inflammatory activation such as VCAM-1 and ICAM-1 did not occur or only to limited extent. While further propagation up to 31 CPDL proceeded at a comparable rate, a marked upregulation of inflammatory markers occurred in all donors accompanied by a further increase of uPA/uPAR gene induction. The observed induction of inflammatory genes at later stages of long-term propagation of PB-ECFCs underpins the necessity to determine the right time-point for harvesting of sufficient number of cells with preserved therapeutical potential.ConclusionThe presented isolation method and subsequent cell expansion in platelet lysate supplemented culture medium permits suitable large-scale propagation of PB-ECFC. For optimal use of PB-ECFCs in clinical settings, our data suggest that 15–20 CPDL is the most adequate maturation stage.