Sabine Fuchs

The effect of human osteoblasts on proliferation and neo-vessel formation of human umbilical vein endothelial cells in a long-term 3D co-culture on polyurethane scaffolds.

Angiogenesis is a key element in early wound healing and is considered important for tissue regeneration and for directing inflammatory cells to the wound site. The improvement of vascularization by implementation of endothelial cells or angiogenic growth factors may represent a key solution for engineering bone constructs of large size. In this study, we describe a long-term culture environment that supports the survival, proliferation, and in vitro vasculogenesis of human umbilical vein endothelial cells (HUVEC). This condition can be achieved in a co-culture model of HUVEC and primary human osteoblasts (hOB) employing polyurethane scaffolds and platelet-rich plasma in a static microenvironment. We clearly show that hOB support cell proliferation and spontaneous formation of multiple tube-like structures by HUVEC that were positive for the endothelial cell markers CD31 and vWF. In contrast, in a monoculture, most HUVEC neither proliferated nor formed any apparent vessel-like structures. Immunohistochemistry and quantitative PCR analyses of gene expression revealed that cell differentiation of hOB and HUVEC was stable in long-term co-culture. The three-dimensional, FCS-free co-culture system could provide a new basis for the development of complex tissue engineered constructs with a high regeneration and vascularization capacity.

Contribution of outgrowth endothelial cells from human peripheral blood on in vivo vascularization of bone tissue engineered constructs based on starch polycaprolactone scaffolds

In the present study we assessed the potential of human outgrowth endothelial cells (OEC), a subpopulation within endothelial progenitor cell cultures, to support the vascularization of a complex tissue engineered construct for bone. OEC cultured on starch polycaprolactone fiber meshes (SPCL) in monoculture retained their endothelial functionality and responded to angiogenic stimulation by VEGF (vascular endothelial growth factor) in fibrin gel-assays in vitro. Co-culture of OEC with human primary osteoblasts (pOB) on SPCL, induced an angiogenic activation of OEC towards microvessel-like structures achieved without additional supplementation with angiogenic growth factors. Effects of co-cultures with pOB on the vascularization process by OEC in vivo were tested by subcutaneous implantation of Matrigel plugs containing both, OEC and pOB, and resulted in OEC-derived blood vessels integrated into the host tissue and anastomosed to the vascular supply. In addition, morphometric analysis of the vascularization process by OEC indicated a better performance of OEC in the co-cultures with primary osteoblasts compared to monocultures of OEC. The contribution of OEC to vascular structures and the beneficial effect of the co-culture with primary human osteoblasts on the vascularization in vivo was additionally proven by subcutaneous implantation of pre-cellularized and pre-cultured SPCL constructs. OEC contributed to the vascular structures, by generating autogenic vessels or by incorporation into chimeric vessels consisting of both, human and mouse endothelial cells. The current data highlight the vasculogenic potential of OEC for bone tissue engineering applications and indicate a beneficial influence of constructs including both osteoblasts and endothelial cells for vascularization strategies.

Co-culture systems for vascularization — Learning from nature

The endothelial cell (EC) is practically ubiquitous in the human body and forms the inner cellular lining of the entire cardiovascular system. Following tissue injury, the microcirculation becomes the stage for both the inflammatory response and the subsequent healing reaction to restore physiological function to the damaged tissue. The advent of the multidisciplinary field of Regenerative Medicine (RegMed), of which Tissue Engineering (TE) and drug delivery using modern stimuli-responsive or interactive biomaterials are important components, has opened up new approaches to the acceleration of the healing response. A central and rate-limiting role in the latter is played by the process of vascularization or neovascularization, so that it is not surprising that in RegMed concepts have been developed for the drug- and gene-delivery of potent stimuli such as vascular-endothelial growth factor (VEGF) to promote neovessel development. However, not all of these novel materials can be tested in vivo, and in vitro co-culture model systems using human primary cells are being developed to pre-evaluate and determine which of the RegMed concepts exhibit the most promising potential for success after implantation. This review describes some of the growing number of in vitro co-cultures model systems that are being used to study cell-cell and cell-material interactions at the cellular and molecular levels to determine which materials are best suited to integrate into the host, promote a rapid vascularization and fit into the regenerative process without disturbing or slowing the normal healing steps.

Dynamic processes involved in the pre-vascularization of silk fibroin constructs for bone regeneration using outgrowth endothelial cells

For successful bone regeneration tissue engineered bone constructs combining both aspects, namely a high osteogenic potential and a rapid connection to the vascular network are needed. In this study we assessed the formation of pre-vascular structures by human outgrowth endothelial cells (OEC) from progenitors in the peripheral blood and the osteogenic differentiation of primary human osteoblasts (pOB) on micrometric silk fibroin scaffolds. The rational was to gain more insight into the dynamic processes involved in the differentiation and functionality of both cell types depending on culture time in vitro. Vascular tube formation by OEC was assessed quantitatively at one and 4 weeks of culture. In parallel, we assessed the temporal changes in cell ratios by flow cytometry and in the marker profiles of endothelial and osteogenic markers by quantitative real-time PCR. In terms of OEC, we observed an increase in tube length, tube area, number of nodes and number of vascular meshes within a culture period of 4 weeks, but a decrease in endothelial markers in real-time PCR. At the same time early osteogenic markers were downregulated, while marker expression associated with progressing mineralized matrix was upregulated in later stages of the culture. In addition, deposition of matrix components, such as collagen type I, known as a pro-angiogenic substrate for endothelial cells, appeared to increase with time indicated by immunohistochemistry. In summary, the study suggests a progressing maturation of the tissue construct with culture time which seems to be not effected by culture conditions mainly designed for outgrowth endothelial cells.

Retention of a differentiated endothelial phenotype by outgrowth endothelial cells isolated from human peripheral blood and expanded in long-term cultures.

Rapid adequate vascularization by autologous human endothelial cells remains a limiting step in the treatment of ischemic tissues and the generation of new tissues. We have expanded outgrowth endothelial cells (OEC) from human peripheral blood and investigated their phenotypic stability in long-term cultures. Our goal has been to obtain suitable numbers of autologous endothelial cells for pro-angiogenic cell therapies. Mononuclear cells were isolated from human peripheral blood. During culture, cells were characterized for several endothelial and stem cell markers in mono- or in co-culture with mature endothelial cells. In cultures from peripheral blood, we observed cells with a variable ability to assume a differentiated endothelial phenotype. Most of the cells showed markers reported for endothelial progenitor cells or hemangioblasts (CD31, KDR, VE-cadherin, CD34, CD117, CD45) but failed to develop a differentiated phenotype. Caveolin-1 was not detectable in these cells by reverse transcription/polymerase chain reaction (RT-PCR) or immunofluorescence. Another cell type arising from the same cultures expressed a differentiated phenotype and was designated as an OEC. This subset as an OEC was expanded in long-term cultures and analyzed by immunofluorescence, flow-cytometry, and RT-PCR for a stable endothelial phenotype. OEC showed several markers of a differentiated endothelium, such as high levels of caveolin-1 throughout all tested passages, and the ability to form angiogenic sprouts in vitro. Thus, OEC in long-term expansion cultures from blood mononuclear cells are phenotypically highly stable, a feature that is an important prerequisite for using OEC from peripheral blood for autologous endothelial cell therapies.

Rapid vascularization of starch–poly(caprolactone) in vivo by outgrowth endothelial cells in co-culture with primary osteoblasts

The successful integration of in vitro‐generated tissues is dependent on adequate vascularization in vivo. Human outgrowth endothelial cells (OECs) isolated from the mononuclear cell fraction of peripheral blood represent a potent population of circulating endothelial progenitors that could provide a cell source for rapid anastomosis and scaffold vascularization. Our previous work with these cells in co‐culture with primary human osteoblasts has demonstrated their potential to form perfused vascular structures within a starch–poly(caprolactone) biomaterial in vivo. In the present study, we demonstrate the ability of OECs to form perfused vascular structures as early as 48 h following subcutaneous implantation of the biomaterial in vivo. The number of OEC‐derived vessels increased throughout the study, an effect that was independent of the OEC donor. This finding of rapid and thorough OEC‐mediated scaffold vascularization demonstrates the great potential for OEC‐based strategies to promote vascularization in tissue engineering. OECs have the potential to contribute to host‐derived scaffold vascularization, and formed vascular structures at a similar density as those arising from the host. Additionally, immunohistochemical evidence demonstrated the close interaction between OECs and the co‐cultured osteoblasts. In addition to the known paracrine activity osteoblasts have in modulating angiogenesis of co‐cultured OECs, we demonstrate the potential of osteoblasts to provide additional structural support for OEC‐derived vessels, perhaps acting in a pericyte‐like role. Copyright

An impaired alveolar-capillary barrier in vitro: effect of proinflammatory cytokines and consequences on nanocarrier interaction

The alveolar region of the lung is an important target for drug and gene delivery approaches. Treatment with drugs is often necessary under pathophysiological conditions, in which there is acute inflammation of the target organ. Therefore, in vitro models of the alveolar-capillary barrier, which mimic inflammatory conditions in the alveolar region, would be useful to analyse and predict effects of novel drugs on healthy or inflamed tissues. The epithelial cell line H441 was cultivated with primary isolated human pulmonary microvascular endothelial cells (HPMECs) or the endothelial cell line ISO-HAS-1 on opposite sides of a permeable filter support under physiological and inflammatory conditions. Both epithelial and endothelial cell types grew as polarized monolayers in bilayer coculture and were analysed in the presence and absence of the proinflammatory stimuli tumour necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ). In addition, the nanocarrier polyethyleneimine (PEI) was chosen as a model compound to study cell uptake (Oregon Green (OG)-labelled PEI) and gene transfer (PEI–pDNA complex). Upon treatment with TNF-α and IFN-γ, both cocultures exhibited comparable effects on the trans-bilayer electrical resistance, the transport of sodium fluorescein and the increase in secondary cytokine release. Basolateral (endothelial side) exposure to TNF-α or simultaneous exposure to TNF-α and IFN-γ generated an alveolar-capillary barrier with inflammation-like characteristics, impaired barrier function and a local disruption of the continuous apical labelling of the tight junction plaque protein zonula occludens-1 (ZO-1). Although transfection rates of 8 per cent were obtained for H441 cells in non-polarized monocultures, apical–basolateral-differentiated (polarized) H441 in coculture could not be transfected. After basolateral cytokine exposure, uptake of fluorescently labelled PEI in polarized H441 was predominantly detected in those areas with a local disruption of ZO-1 expression. Accordingly, transfected cells were only sparsely found in coculture after basolateral costimulation with TNF-α and IFN-γ. We designed a coculture model that mimics both the structural architecture of the alveolar-capillary barrier and inflammatory mechanisms with consequences on barrier characteristics, cytokine production and nanoparticle interaction. Our model will be suitable to systematically study adsorption, uptake and trafficking of newly synthesized nanosized carriers under different physiological conditions.

Barrier functions and paracellular integrity in human cell culture models of the proximal respiratory unit

Airway epithelial cells provide a barrier to the translocation of inhaled materials. Tight (TJ) and adherens junctions (AJ) play a key role in maintaining barrier functions, and are responsible for the selective transport of various substances through the paracellular pathway. In this study we compared a bronchial cell line (16HBE14o-) and primary bronchial cells (HBEC), both cocultivated with the fibroblast cell line Wi-38, with respect to their structural differentiation and their reaction to cytokine stimulation. HBEC formed a pseudostratified epithelial layer and expressed TJ and AJ proteins after 2 weeks in coculture. Mucus-producing and ciliated cells were found within 24 days. Additionally, a beating activity of the ciliated HBEC (14-19Hz) could be detected. 16HBE14o- in coculture showed a multilayered growth without differentiation to a pseudostratified airway epithelium. Simultaneous exposure to TNF-alpha- and IFN-gamma-induced significant changes in barrier function and paracellular permeability in the cocultures of HBEC/Wi-38 but not in the 16HBE14o-/Wi-38. In summary, HBEC in coculture mimic the structure of native polarized bronchial epithelium showing basal, mucus-producing and ciliated cells. Our system provides an opportunity to examine the factors that influence barrier and mucociliary function of bronchial epithelium within a time frame of 3 weeks up to 3 months in an in vivo-like differentiated model.

Fibronectin-mediated endothelialisation of chitosan porous matrices

Chitosan (Ch) porous matrices were investigated regarding their ability to be colonized by human microvascular endothelial cells (HPMEC-ST1.6R cell line) and macrovascular endothelial cells namely HUVECs. Specifically we assessed if previous incubation of Ch in a fibronectin (FN) solution was effective in promoting endothelial cell (EC) adhesion to Ch matrices with different degrees of acetylation (DAs). Upon FN physiadsorption, marked differences were found between the two DAs investigated, namely DA 4% and 15%. While cell adhesion was impaired on Ch with DA 15%, ECs were able to not only adhere to Ch with DA 4%, but also to spread and colonize the scaffolds, with retention of the EC phenotype and angiogenic potential. To explain the observed differences between the two DAs, protein adsorption studies using (125)I-FN and immunofluorescent labelling of FN cell-binding domains were carried out. In agreement with the higher cell numbers found, scaffolds with DA 4% revealed a higher number of exposed FN cell-binding domains as well as greater ability to adsorb FN and to retain and exchange adsorbed FN in the presence of competitive proteins. These findings suggest that the DA is a key parameter modulating EC adhesion to FN-coated Ch by influencing the adsorbed protein layer.

Outgrowth endothelial cells: sources, characteristics and potential applications in tissue engineering and regenerative medicine.

Endothelial progenitor cells from peripheral blood or cord blood are attracting increasing interest as a potential cell source for cellular therapies aiming to enhance the neovascularization of tissue engineered constructs or ischemic tissues. The present review focus on a specific population contained in endothelial progenitor cell cultures designated as outgrowth endothelial cells (OEC) or endothelial colony forming cells from peripheral blood or cord blood. Special attention will be paid to what is currently known in terms of the origin and the cell biological or functional characteristics of OEC. Furthermore, we will discuss current concepts, how OEC might be integrated in complex tissue engineered constructs based on biomaterial or co-cultures, with special emphasis on their potential application in bone tissue engineering and related vascularization strategies.