First report of multinodular pulmonary fibrosis associated with equine herpesvirus 5 in Belgium

A 20-year-old horse was evaluated for symptoms of weight loss, anorexia, fever and lethargy. Clinical examination revealed tachypnea, poor body condition and increased breath sounds on auscultation. Ultrasound showed multiple consolidations on the lungs. Thoracic radiography revealed a severe nodular pattern. The horse was treated with antibiotics, corticoids and supportive medication. Since no improvement was observed, the horse was euthanized. At necropsy, numerous coalescing fibrous nodules were present in the lungs. Histology revealed diffuse interstitial fibrosis and macrophages containing abundant eosinophilic cytoplasm and oval eosinophilic to amphophilic intranuclear inclusion bodies. Tissue samples tested positive for the presence of equine herpes virus 5 (EHV 5) on the basis of the polymerase chain reaction (PCR) test. A diagnosis of equine multinodular pulmonary fibrosis (EMPF) was made. This is the first report of EMPF in Belgium. EMPF can be suspected based on the ultrasonographic, radiographic and histological changes. EMPF is associated with EHV 5, but the etiological role of EHV 5 still remains to be proven

Bioprinting of vascularized bone tissue equivalents

Bone tissue is one of the most frequently transplanted tissues. Since procedures like the transplantation of autologous bone bear risks, though, regenerative medicine and tissue engineering reach to face those problems by engineering bone substitutes by using suitable materials and living cells. A crucial factor is the vascularization of the constructed tissue to ensure supply of the included cells with nutrients and oxygen. For the fabrication of such bone tissue equivalents, evolving manufacturing techniques like bioprinting can be used to construct geometrically defined three-dimensional structures. Please click Additional Files below to see the full abstract

Aspects of Quantum Gravity in de Sitter Spaces

In these lectures we give a review of recent attempts to understand quantum gravity on de Sitter spaces. In particular, we discuss the holographic correspondence between de Sitter gravity and conformal field theories proposed by Hull and by Strominger, and how this may be reconciled with the finite-dimensional Hilbert space proposal by Banks and Fischler. Furthermore we review the no-go theorems that forbid an embedding of de Sitter spaces in string theory, and discuss how they can be circumvented. Finally, some curious issues concerning the thermal nature of de Sitter space are elucidated.Comment: 36+1 pages, 5 Postscript figures, introduction and section 6 extended, further references, final version to appear in JCA

Microstructured layers of biofunctionalized nanoparticles as threedimensional affinity-matrices for protein-detection on microarrays

Nanopartikel mit einer Schale aus Proteinen stellen ein Material mit extrem großer, biofunktioneller Oberfläche dar. Dieser vielseitige Werkstoff wurde in der vorliegenden Arbeit einem breiten Anwendungsspektrum verfügbar gemacht. Kern-Schale-Nanopartikel aus einem anorganischen SiO2-Kern und einer organischen Schale aus funktionellen Silanen wurden verwendet, um daran als Fängerelemente Proteine mit spezifischen Bindeeigenschaften zu immobilisieren. Die Funktionalisierung der Partikeloberflächen konnte mit großer Flexibilität an verschiedene Anforderungen angepasst werden: Fänger-Moleküle konnten in zufälliger Orientierung kovalent oder auch gerichtet auf den Partikeloberflächen immobilisiert werden. Die biofunktionalisierten Nanopartikel wurden anschließend mittels lithografischer Techniken oder Kontakt-Druck-Verfahren in Form von mikrostrukturierten Schichten stabil auf aktivierten Glasoberflächen abgeschieden. Die partikelgebundenen Fänger-Proteine wurden stabilisiert, sodass ihre biologische Funktionalität innerhalb der trockenen Partikelschichten erhalten blieb. Auf diese Weise wurden dreidimensionale Affinitätsoberflächen im Microarrayformat erzeugt, die dauerhaft lagerfähig waren. Die hohe Bindekapazität der dreidimensionalen Microspots resultierte beim Nachweis von Proteinen in einem dynamischen Bereich, der fünf Größenordnungen überspannte. Nanopartikel-Microarrays waren sowohl mit Fluoreszenzdetektion kompatibel als auch mit MALDI-Massenspektrometrie (MALDI-MS), den Standard-Ausleseverfahren der modernen Molekularbiologie und der Proteomforschung. Die hohe Bindekapazität und große Fängerdichte machen Biochips aus Nanopartikel-Microspots besonders relevant für Anwendungen, die für den empfindlichen Nachweis eines Analyten möglichst viele der in der Probe vorhandenen Analytmoleküle auf die Sensor-Oberfläche aufkonzentrieren wollen. Der modulare Aufbau der Chips ermöglicht die Trennung von Kopplungschemie und Microstrukturierung, sodass für die Entwicklung von Hybrid-Tests unterschiedliche Fängerelemente maßgeschneidert auf einer Sensoroberfläche immobilisiert werden können.In the presented work biochip surfaces of layered nanoparticles were developed. Core-Shell nanoparticles with a core of anorganic silicon dioxide and a shell of organic silanes were used to covalently bind distinct proteins as specific capture molecules to particle surfaces. These biofunctionalized nanoparticles were precipitated as microstructured layers on substrate surfaces, generating three-dimensional microspots with high binding capacity for specific target molecules

Biometrische Knorpelmatrix

Die vorliegende Erfindung betrifft ein Verfahren zur Herstellung einer biomimetischen Knorpelmatrix mit zonaler Strukturierung sowie eine nach dem Verfahren hergestellte biomimetische Knorpelmatrix

Biopolymer-based functional inks for the preparation of artificial cartilage via bioprinting technology

The future vision of implants comprises the generation of artificial tissue generated from the patient’s own cells. Furthermore, sophisticated complex tissue models will help to perform adequate in vitro testing and avoid animal experiments. In order to develop artificial, biomimetic structures which perform as well as natural ones, we need fabrication processes that do not set any limits to the generation of shapes, and materials that allow for tailoring of their physical, chemical, and biological properties. Thereby, biomolecules from the extracellular matrix of native tissues constitute very promising materials as they hold natural signalling motifs for the stimulations of cell adhesion, migration and function. With respect to the generation of artificial tissues by bioprinting technology, for instance articular cartilage, we develop printable and photo-crosslinkable material systems, based on bio-polymers derived from the native extracellular matrix (ECM), e.g. gelatin. Such bioinks can be used for 3D encapsulation of cells and cell printing, thereby constituting biomimetic matrices with adjustable properties for engineering of complex tissue models. In this study we present biopolymer-based biomaterials which are processable by inkjet printing and dispensing technology and can be crosslinked into hydrogels with tunable physico-chemical properties. These biomaterials are used for the biofabrication of artificial articular cartilage with biomimetic hierarchical structure. Photo-crosslinkable derivatives of the ECM bio¬po¬lymers gelatin, chondroitin sulfate, and hyaluronan are prepared by their derivatization with methacrylic an¬hy¬dride [1]. These biopolymer derivatives can be converted into thermo-stable hydrogels by UV-induced radical crosslinking in the presence of a water-soluble photoinitiator [2]. To furthermore achieve printable and dispensable bioinks, the viscous behavior of gelatin precursor solutions is adapted to the re¬quire¬ments of the printing technologies by additional functionalization with acetyl groups [3]. The developed bioinks are then used for bioprinting with porcine articular chondrocytes to proof their cytocompatibility as well as the cytocompatibility of the printing process [2]. For fabrication of zonal cartilage models bioinks with appropriate biopolymer composition for replication of the three cartilage zones (superficial, middle, deep) are determined. Criteria for evaluation are the visco-elastic properties of the resulting hydrogels, such as mechanical strength, swellability, and degradability, as well as their potential to preserve cell viability and functionality. Finally, three-dimensional, zonal cartilage models were fabricated and evaluated for their quality

Modifizierte Gelatine, Verfahren zu ihrer Herstellung und Verwendung

Producing a two- or three-dimensional structure on a substrate, comprises: (a) applying at least a gelatin derivative present in a liquid medium to the substrate and (b) fixing the gelatin derivative applied in step (a) by intermolecular cross-linking of the gelatin derivative. The gelatin derivative comprises at least one artificially introduced non-crosslinking functional group and at least one artificially introduced crosslinking group. At least one artificially introduced non-crosslinking functional group is e.g. halogen, alkyl-, heteroarylalkyl- group. Producing a two- or three-dimensional structure on a substrate, comprises: (a) applying at least a gelatin derivative present in a liquid medium to the substrate and (b) fixing the gelatin derivative applied in step (a) by intermolecular cross-linking the gelatin derivative. The gelatin derivative comprises at least one artificially introduced non-crosslinking functional group and at least one artificially introduced crosslinking group. At least one artificially introduced non-crosslinking functional group is halogen, alkyl-, haloalkyl-, heteroalkyl-, cycloalkyl-, heterocycloalkyl-, aryl-, heteroaryl-, arylalkyl-, heteroarylalkyl-, arylheteroalkyl-, alkoxy-, alkoxyalkyl-, alkoxyaryl-group (all preferred), aldehyde- or acyl group. At least one artificially introduced crosslinking group comprises maleimide group and carbon-carbon double bond, or azide group, carbon-carbon double bond, carbon-carbon triple bond, aldehyde, ketone, imine group, thioketone thiol group, carbon-nitrogen triple bond and diene group (preffered). Independent claims are also included for: (1) a gelatin derivative comprising at least one artificially introduced non-crosslinking functional group and at least one artificially introduced crosslinking group; and (2) a two- or three-dimensional structure produced by the above mentioned method

Methacrylated gelatin and mature adipocytes are promising components for adipose tissue engineering

In vitro engineering of autologous fatty tissue constructs is still a major challenge for the treatment of congenital deformities, tumor resections or high-graded burns. In this study, we evaluated the suitability of photo-crosslinkable methacrylated gelatin (GM) and mature adipocytes as components for the composition of three-dimensional fatty tissue constructs. Cytocompatibility evaluations of the GM and the photoinitiator Lithium phenyl-2,4,6 trimethylbenzoylphosphinate (LAP) showed no cytotoxicity in the relevant range of concentrations. Matrix stiffness of cell-laden hydrogels was adjusted to native fatty tissue by tuning the degree of crosslinking and was shown to be comparable to that of native fatty tissue. Mature adipocytes were then cultured for 14 days within the GM resulting in a fatty tissue construct loaded with viable cells expressing cell markers perilipin A and laminin. This work demonstrates that mature adipocytes are a highly valuable cell source for the composition of fatty tissue equivalents in vitro. Photo-crosslinkable methacrylated gelatin is an excellent tissue scaffold and a promising bioink for new printing techniques due to its biocompatibility and tunable properties

Bone matrix production in hydroxyapatite-modified hydrogels suitable for bone bioprinting

Though bioprinting is a forward-looking approach in bone tissue engineering, the development of bioinks which are on the one hand processable with the chosen printing technique, and on the other hand possess the relevant mechanical as well as osteoconductive features remains a challenge. In the present study, polymer solutions based on methacrylated gelatin and methacrylated hyaluronic acid modified with hydroxyapatite (HAp) particles (5 wt%) were prepared. Encapsulation of primary human adipose derived stem cells in the HAp-containing gels and culture for 28 d resulted in a storage moduli significantly increased to 126% ± 9.6% compared to the value on day 1 by the sole influence of the HAp. Additional use of osteogenic media components resulted in an increase of storage module up to 199% ± 27.8%. Similarly, the loss moduli was increased to 370% ± 122.1% under the influence of osteogenic media components and HAp. Those changes in rheological material characteristics indicate a distinct change in elastic and viscous hydrogel properties, and are attributed to extensive matrix production in the hydrogels by the encapsulated cells, what could also be proven by staining of bone matrix components like collagen I, fibronectin, alkaline phosphatase and osteopontin. When using the cell-laden polymer solutions as bioinks to build up relevant geometries, the ink showed excellent printability and the printed grid structure's integrity remained intact over a culture time of 28 d. Again, an intense matrix formation as well as upregulation of osteogenic markers by the encapsulated cells could be shown. In conclusion, we demonstrated that our HAp-containing bioinks and hydrogels on basis of methacrylated gelatin and hyaluronic acid are on the one hand highly suitable for the build up of relevant three-dimensional geometries with microextrusion bioprinting, and on the other hand exhibit a significant positive effect on bone matrix development and remodeling in the hydrogels, as indicated by rheological measurements and staining of bone components. This makes the developed composite hydrogels an excellent material for bone bioprinting approaches

Characterization of bone matrix development in gelatin-based hydrogels

For the fabrication of bone tissue equivalents via tissue engineering and bioprinting approaches, one crucial element is the used biomaterial which on the one hand should support and enhance the function of the used cells, and on the other hand guarantee processability with the chosen manufacturing technique. The use of gelatin-based hydrogels for cell encapsulation provides the positive properties of the base material collagen, and offers the possibility of adjusting composition as well as characteristics like viscosity and gelling behavior [1,2]. Further modification of the material with components like hydroxyapatite (HAp) was shown to have a positive impact on processability with bioprinting approaches and properties of the material as well as its bioactivity [3,4,5]. In the current study, bioinks based on methacrylated gelatin (GM) and modified with HAp particles were used and analyzed for said parameters. The viscosity of the bioinks was measured, as well as water uptake by the hydrogels and the gels’ mechanical properties. Human mesenchymal stem cells from adipose tissue (hASCs) of three donors were encapsulated in differently modified GM hydrogels, and matrix development during culture for 28 days in standard and osteogenic media was examined. To prove the development of a bone matrix, the mechanical properties of the hydrogels were analyzed, and the hydrogels were stained for bone matrix markers like collagen type I and osteocalcin. Further analysis of matrix composition was conducted via Raman spectroscopy. The modification with HAp led to improved properties of the bioinks as well as the resulting hydrogels, as being shown by increased ink viscosity and mechanical properties of the hydrogels. The degree of water uptake, however, was not influenced. The staining of the hASC-laden hydrogels cultured under osteogenic conditions showed an increased deposition of bone components like collagen and non-collagenous proteins like osteocalcin in the modified hydrogels compared to the control GM gels after 28 days of culture. Surprisingly, this increase was also seen in the HAp-containing gels cultured under standard conditions (Fig. 1A), what illustrated the significant positive impact of the added HAp onto osteogenesis. Similarly, the spectroscopic analysis of said hydrogels on day 1 and day 28 of culture revealed peaks resulting from typical bone components like the collagen-related amino-acid proline (853/869/1067 cm-1) or carbonate which is found in the biological mineral phase of bone (1074 cm-1) (Fig. 1B). The developing bone matrix could also be detected by a rising storage module of the gels, resulting in an increase between 25% and 100% compared to day 1 (Fig. 1C) and representing the mechanical strength of the newly formed matrix. In the current study, we could show the suitability of a GM-based material for the use in bone tissue engineering. The properties of the analyzed inks and resulting hydrogels could be modified by the addition of HAp, and the osteoinductive impact of the added mineral phase leading to a significant increase in osteogenesis in the hydrogels was shown. We therefore anticipate that the material will be highly suitable as a bioink for the bioprinting of bone tissue equivalents