Kirsten Peters

Growth of human cells on a non-woven silk fibroin net: a potential for use in tissue engineering

We have examined a novel biomaterial consisting of a non-woven fibroin net produced from silk (Bombyx mori) cocoons for its ability to support the growth of human cells. Various human cells of different tissue and cell types (endothelial, epithelial, fibroblast, glial, keratinocyte, osteoblast) were examined for adherence and growth on the nets by confocal laser microscopy after staining of the cells with calcein-AM and by electron microscopy. All the cells readily adhered and spread over the individual fibers of the nets. Most of the cells were able to grow and survive on the nets for at least 7 weeks and growth not only covered the individual fibers of the net but generally bridged the gaps between individual fibers forming tissue-like structures. Scanning electron microscopic examination of the nets demonstrated a tight association of individual cells with the fibers and nets examined after removal of cells showed no evidence that the growth of cells in any way changed the structure of the fibers. Thus, silk fibroin nets are highly human cell-compatible and should be a useful new scaffolding biomaterial applicable for a wide range of target tissues in addition to supporting endothelial cells required for the vascularization of the newly formed tissue.

Molecular basis of endothelial dysfunction in sepsis

Sepsis is one of the major causes of mortality in critically ill patients and develops as a result of the host response to infection. A complex network of events is set into motion in the body by the infection and results in the pathogenesis of sepsis. This review article focuses on the molecular mechanisms and components involved in the pathogenesis of sepsis with a major emphasis on the endothelium. This includes sepsis-inducing bacterial components (e.g. endotoxins), cellular targets of these molecules and their responses, host reactions, intracellular and cytokine networks, individual susceptibility and new therapeutic targets in sepsis treatment.

Lung epithelial cell lines in coculture with human pulmonary microvascular endothelial cells: development of an alveolo-capillary barrier in vitro.

We have established a coculture system of human distal lung epithelial cells and human microvascular endothelial cells in order to study the cellular interactions of epithelium and endothelium at the alveolocapillary barrier in both pathogenesis and recovery from acute lung injury. The aim was to determine conditions for the development of functional cellular junctions and the formation of a tight epithelial barrier similar to that observed in vivo. The in vitro coculture system consisted of monolayers of human lung epithelial cell lines (A549 or NCI H441) and primary human pulmonary microvascular endothelial cells (HPMEC) on opposite sides of a permeable filter membrane. A549 failed to show sufficient differentiation with respect to formation of a tight epithelial barrier with intact cell–cell junctions. Stimulated with dexamethasone, the cocultures of NCI H441 and HPMEC established contact-inhibited differentiated monolayers, with NCI H441 showing a continuous, circumferential immunostaining of the tight junctional protein, ZO-1 and the adherens junction protein, E-cadherin. The generation of a polarized epithelial cell monolayer with typical junctional structures was confirmed by transmission electron microscopy. Dexamethasone treatment resulted in average transbilayer electrical resistance (TER) values of 500 Ω cm2 after 10–12 days of cocultivation and correlated with a reduced flux of the hydrophilic permeability marker, sodium-fluorescein. In addition, basolateral distribution of the proinflammatory cytokine tumour necrosis factor-alpha caused a significant reduction of TER-values after 24 h exposure. This decrease in TER could be re-established to control level by removal of the cytokine within 24 h. Thus, the coculture system of the NCI H441 with HPMEC should be a suitable in vitro model system to examine epithelial and endothelial interactions in the pathogenesis of acute lung injury, infectious lung diseases and toxic lung injury. In addition, it could be used to improve techniques of lung drug delivery that also requires a functional barrier.

Tissue response and biomaterial integration: the efficacy of in vitro methods

Implantation involves tissue trauma, which evokes an inflammatory response, coupled to a wound healing reaction, involving angiogenesis, fibroblast activation and matrix remodelling. Until now the type and extent of such reactions to give optimal integration of various biomaterials are practically unknown. Three principal fields of research can yield useful data to understand these phenomena better: studies on explanted biomaterials, animal models and relevant in vitro techniques. This paper will present examples of the latter field and the application of endothelial cell (EC) culture systems to study the effects of important tissue (e.g. pro-inflammatory cytokines, chemokines) and material (e.g. metal ions, particulate debris) factors on the regulation of the inflammatory and angiogenic response. A central feature is the use of microvascular endothelial cells (MEC), which can be used in both 2-and 3-dimensional (3-D) assays. We have also used genetic manipulation to develop a permanent MEC line from the human lung (HPMEC-ST1), which is being tested for its suitability to study cell-biomaterial interactions. In addition, suitable in vitro techniques are being developed in order to investigate drug delivery systems (DDS). Of particular interest is the targeting of the central nervous system, our approach being to establish a human model of the blood-brain barrier (BBB). A mainstay of our scientific philosophy is that such in vitro methods can make an important contribution to understanding biological reactions at the tissue-biomaterial interface and thus further a causal approach to tissue engineering (TE) and drug delivery applications.

Software-supported image quantification of angiogenesis in an in vitro culture system: application to studies of biocompatibility

Healing of soft tissue trauma and bone discontinuities following implantation involves acute inflammatory reactions and the formation of blood vessels (angiogenesis). During angiogenesis new capillary vessels arise from the existing vasculature. Endothelial cells (EC) are the major cell type involved in angiogenesis. Corrosion of orthopaedic metallic implant materials (e.g. CoCr alloys) can cause locally high concentrations of heavy metal ions in the peri-implant tissues. Some divalent metal ions (Co2+, Ni2+, Zn2+) lead to the activation of EC in vitro. Upon exposure to these ions. EC release cytokines and chemokines and increase the expression of cell surface adhesion molecules, which represents the pro-inflammatory phenotype. In this study we have examined whether metal ions influence the other endothelial aspect of wound healing, the angiogenic response. Therefore, we utilized an in vitro model of angiogenesis and examined the effects of divalent cobalt ions on the in vitro vessel formation. The quantification of the cobalt/ion-exerted effects on angiogenesis in vitro was performed using a contrast-rich vital staining and analysed by software-supported image quantification.

Paradoxical effects of hypoxia-mimicking divalent cobalt ions in human endothelial cells in vitro

Divalent cobalt ions (Co2+) induce the expression of hypoxia responsive genes and are often used in cell biology to mimic hypoxia. In this in vitro study we compared the effects of hypoxia and Co2+ on human endothelial cells and examined processes that are stimulated in hypoxia in vivo (proliferation and angiogenesis). We analyzed the expression of the hypoxia-inducible factor-1α (HIF-1α) under different hypoxic conditions (3% and nearly 0% O2) and Co2+-concentrations (0.01–0.7 mM). As in hypoxia, the amount of HIF-1α protein was enhanced by exposure to Co2+ (did not correlate with mRNA amount). However, contrary to the results of hypoxia, in vitro-angiogenesis was inhibited after exposure to even low Co2+-concentrations (≥0.01 mM). This led to the conclusion that although hypoxia signaling after Co2+-exposure took place, further yet unknown Co2+-induced event(s) must have occurred. (Mol Cell Biochem 270: 157–166, 2005)

Experimental approaches to study vascularization in tissue engineering and biomaterial applications

The success of tissue engineering and biomaterial applications is not only dependent on the growth and functioning of the organ- or tissue-specific cells on the biomaterial but is entirely dependent in most cases on a successful vascularization after implantation. The process of vascularization involves angiogenesis; the formation of new blood vessels which spread into the implant material and supply the existing cells with the nutrients to survive. We have established in vitro methods using human microvascular endothelial cells to evaluate novel biomaterials for endothelial cell attachment, cytotoxicity, growth, angiogenesis and the effects on gene regulation. These in vitro studies can be used to rapidly evaluate the potential success of a new biomaterial and for the development of matrix scaffolds which will promote a physiological vascularization response.

Induction of apoptosis in human microvascular endothelial cells by divalent cobalt ions. Evidence for integrin-mediated signaling via the cytoskeleton

Wound healing following implantation is characterized by an acute inflammatory reaction and a subsequent reorganizing phase in which angiogenesis is involved. Endothelial cells (EC) participate in both inflammation and angiogenesis. Thus, the effects on functions of EC exerted by implanted materials could affect the progression of wound healing. The corrosion of metallic implants can cause high concentrations of heavy metal ions in the peri-implant tissues. The purpose of the present study was to test the effects of possible corrosion products on the function and viability of human EC in vitro. Long-term exposure of EC to CoCl2 and NiCl2 (3 days, 0.7 mM) leads to a decrease of cell number and changes in cellular morphology. However, the morphological changes between CoCl2- and NiCl2-treated cells differ significantly. The changed morphology of CoCl2-treated EC and the fragmented DNA pattern indicates apoptosis. Nickel-treated cells demonstrated necrosis. The activity of integrins was tested by an assay of cellular adhesion on collagen-coated surfaces. It was shown that the number of adherent cells significantly decreased upon exposure to CoCl2. Our studies suggest that induction of cell death in EC upon exposure to CoCl2 could be attributed to impaired integrin signaling, which leads to a damaged cytoskeleton and culminates in apoptosis.© 2001 Kluwer Academic Publishers

High levels of the molecular chaperone Mdg1/ERdj4 reflect the activation state of endothelial cells

Mdg1/ERdj4, a mammalian chaperone that belongs to the HSP40 protein family, has been reported to be located in the endoplasmic reticulum (ER), is induced by ER stress, and protects ER stressed cells from apoptosis. Here we show that under normal physiological conditions, Mdg1/ERdj4 is expressed at various levels in the vasculature due to different activation states of the endothelium. To elucidate the stimuli that induce ER stress and thus upregulate Mdg1/ERdj4, we investigated the effect of several endothelium specific stressors on its expression. Mdg1/ERdj4 mRNA is induced by activated macrophages, by nitric oxide (NO) and heat shock, and during terminal cell differentiation, whereas shear stress does not affect Mdg1/ERdj4 expression levels. While the mRNA stability of BiP/GRP78 is unaffected in ER stressed cells, the stability of Mdg1/ERdj4 mRNA is prolonged during ER stress resulting in rapid increases and high levels of Mdg1/ERdj4 mRNA. Mdg1/ERdj4 protein is localized in the ER under control conditions. While heat shock induces a rapid translocation of Mdg1/ERdj4 to the nucleoli, no translocation could be observed during ER stress. This indicates that Mdg1/ERdj4 protein has diverse mechanisms to protect stressed cells from apoptosis.

Pathomechanismen der gestörten Wundheilung durch metallische Korrosionsprodukte

Hintergrund. Im Dentalbereich werden seit langer Zeit metallische Werkstoffe unterschiedlicher chemischer Zusammensetzung verwendet. Bei einigen Patienten kommt es nach der Implantatinsertion zu Wundheilungsstörungen. In diesem Artikel werden neben Grundlagen der eng verwandten Entzündungs- und Reparationsvorgänge die Pathomechanismen einer gestörten Wundheilung erörtert. Wundheilungsstörungen. Die Modulation der Wundheilung kann über die Effekte der durch Korrosion freigesetzten Metallionen, aber auch durch die durch Abrieb entstandenen Mikropartikel auf die an der Heilung beteiligten Zelltypen (z. B. Endothelzellen) ausgeübt werden. Modelle. In diesem Zusammenhang werden In-vitro-Modelle vorgestellt, mit deren Hilfe die komplexen Geschehnisse der Werkstoff-Gewebe-Grenzfläche in isolierte Aspekte zerlegt werden können. Darüber hinaus werden neu entwickelte, computergestützte Methoden angesprochen, welche die objektive Quantifizierung von biomaterial- bzw. korrosionsprodukt-induzierten Effekten auf komplexe Vorgänge, z. B. die Angiogenese in vitro, erlauben. Wegen der zentralen Bedeutung der Titanimplantate für Anwendungen in der Mund-, Kiefer- und Gesichtschirurgie werden erste experimentelle Ansätze zur Untersuchung möglicher negativer Auswirkungen vorgestellt. Der Beitrag schließt mit einer Diskussion über die Relevanz solcher Studien für die klinische Implantologie. Background. Metallic materials of variable chemical composition have been used in dental practice for a long time. Complications with respect to tissue healing after insertion of implants are well documented. In this paper we present relevant aspects of the related fields of inflammation and repair processes and focus on the pathomechanisms of this impaired healing response. Modulation of wound healing. This latter process is modulated by specific metal ions released by corrosion activity as well as by wear particles, which influence the function of the participating cell types (e.g. endothelial cells). In vitro models. In this context, in vitro models are presented that permit study of isolated aspects of the complex sequence of events at the biomaterial-tissue interface. Furthermore, newly developed, computer-assisted methods allowing an objective quantification of biomaterial/corrosion product-induced effects on complex processes, such as angiogenesis in vitro, are demonstrated. Because of the central importance of titanium implants in maxillofacial surgery, new experimental approaches to study possible negative effects are presented. Finally, the relevance of such studies for clinical implantology is evaluated.