Complex and patient-specific scaffolds and tissue engineering constructs by extrusion-based 3D (bio) printing

Extrusion-based additive manufacturing (“3D plotting”) is a very versatile technology as in principle any pasty material can be utilised. In addition, the printers used for 3D plotting are less complicated and therefore also cheaper in comparison to e.g. laser-based systems. In our lab several suitable biomaterials – biopolymer hydrogels, composites but also a pasty calcium phosphate bone cement (CPC) – have been developed for 3D plotting of scaffolds as well as for biofabrication purposes. Of special interest are alginate/methylcellulose blends with or without laponite (a synthetic clay) as additional component which both allow bioprinting of macroscopic but still open-porous, cell-laden constructs. Beside bioprinting of mammalian cells (human MSC) we could demonstrate successful utilisation of microalgae and plant cells. By using a multi-channel 3D plotting device (BioScaffolder 3.1 from Gesim, Germany) we could combine two different materials in an alternating fashion within one construct. This also works for the combination of cell-laden biopolymer hydrogel blends and the self-setting calcium phosphate bone cement which provides mechanical stiffness. Another option for combining two materials is extrusion through a double nozzle system, leading to strands with core/shell morphology (Fig. 1). Especially if stiff, highly concentrated alginate-based hydrogels or CPC are used as shell material mechanically robust and open-porous constructs with tailored properties can be manufactured. By loading shell and core part with different drugs or growth factors dual release systems with adjustable release properties can be realised. Finally, also living cells can be suspended in the soft biopolymer hydrogels, acting as core material in core/shell bioprinting, leading to stable tissue engineering constructs. Please click Additional Files below to see the full abstract

Kinetisches Modell für die Prozessanalyse von Displacement-Assays mit mono- und bivalenten Antikörpern

Molecular and functional analysis of small molecule binding to protein can provoke insights into cellular signaling and regulatory systems as well as facilitate pharmaceutical drug discovery. In label free small molecule detection the displacement assay format can be applied. This assay format comprises the displacement of receptor molecules bond to immobilized ligand by a competition reaction with ligand in solution. This is beneficial because displacement of high molecular receptors is detected compared to low molecular ligand as in classical binding analysis therefore potentially lowering the method detection limit. It was hypothesized that with choosing appropriate measuring methods and theoretical modeling reaction rate constants can be determined separately in every kinetic stage of the assay format. Herein elucidating the dominant valence of antibody antigen binding in the established assay was of great importance. Using the Influenza Hemagglutinin (HA) peptid binding to mono or bivalent Anti-Hemagglutinin peptide antibody displacement assay formats could be established. The exact time resolved analysis of binding and dissolution of ligand HA and Anti-Hemagglutinin peptide antibody was achieved with surface plasmon resonance (SPR) spectroscopy. Mathematical models could be developed from kinetic equations of ligand binding to mono or bivalent antibody. With this, an accurate simulation of the SPR results was reached. The simulation plot had to be exactly adjusted to the SPR results to determine all kinetic rate constants defining ligand and receptor binding kinetics. Large variations in receptor concentration gave almost identical rate constants in binding; this proves the quality of SPR measurements and demonstrates consistence between measurement, simulation, and binding model. Maximum decline of SPR response could be used to determine ligand concentrations in analyte. Displacement dependence from antigen concentration was found to be exponential and was explained by rebinding. Kinetic data and models could be transferred for the simulation of almost stationary displacement assay formats realized with impedance and fluorescence spectroscopy. With the obtained results it was possible to detect the displacement of the bacterial signaling autoinducer AI-2 by a displacement assay format using periplasmic binding protein LuxP as receptor. Concluding it can be said that the hypothesis could be proved and the obtained results can facilitate the use of displacement assay formats in biosensing. Displacement assay formats should be especially interesting in small molecule detection and in compact integrated mass sensitive sensor designs suitable as mobile sensors in outdoor screening.:Zusammenfassung i Summery iii Inhaltsverzeichnis v 1. Problemstellung 1 2. Kinetisches Modell für Displacement-Assays mit monovalenten Antikörpern 5 2.1 Kinetisches Modell 6 2.1.1 Grundgleichungen 6 2.1.2 Analytische Näherungslösung 8 2.1.3 Numerische Lösung 10 2.2 Vergleich mit experimentellen Beispielen 18 2.2.1 Surface-Plasmonen-Resonanzspektroskopie eines Hämagglutinin- Peptid/Hämagglutinin-Antikörper-Displacement-Assays 18 2.2.2 Fluoreszenzspektroskopie eines Hämagglutinin-Peptid/Hämagglutinin-Antikörper-Displacement-Assays 36 2.2.3 Analyse eines LuxP/AI2-Displacementassays mittels SPR-Spektroskopie 52 3. Kinetisches Modell für Displacement-Assays mit bivalenten Antikörpern 70 3.1 Kinetisches Modell 70 3.1.1 Grundgleichungen 70 3.1.2 Numerische Lösung 72 3.2 Vergleich mit experimentellen Beispielen 80 3.2.1 Surface-Plasmonen-Resonanzspektroskopie eines Hämagglutinin- Peptid/Hämagglutinin-Antikörper-Displacement-Assays 80 3.2.2 Impedanzspektroskopie eines Hämagglutinin-Peptid/Hämagglutinin-Antikörper-Displacement-Assays 88 4. Konklusionen und Ausblick 92 5. Anhänge 98 A1: Oberflächenplasmonenresonanzspektroskopie 98 A2: Aufbau der Messschichten und Messreihen eines Hämagglutinin-Peptid/Hämagglutinin-Antikörper-Displacement-Assays 105 A3: Schichtaufbau zur Analyse eines LuxP/AI2-Displacementassays mittels SPR-Spektroskopie 119 A4: Aufbau zur Fluoreszenzspektroskopie eines Hämagglutinin-Peptid/Hämagglutinin-Antikörper-Displacement-Assays 125 A5: Impedanzspektroskopie 128 A6: Transferratenkonstanten 139 A7: Abkürzungsverzeichnis 141 6. Literatur 146 7. Danksagung 155 8. Selbständigkeitserklärung 157Die Analyse des Bindungsverhaltens niedermolekularer Liganden an Proteine ist für die Aufklärung von biologischen Regulationssystemen oder bei der Suche neuer medizinischer Wirkstoffe von Wichtigkeit. Ein markierungsfreies Detektions¬prinzip zur Erfassung niedermolekularer Liganden ist die Displacement- oder Replacement-Methode. Bei dieser tritt die Bindung des Rezeptors an den immobilisierten Liganden mit der Bindung an freien Liganden in Konkurrenz, sodass anstelle der niedermolekularen Liganden die hochmolekularen Rezeptoren detektiert werden können. In dieser Arbeit wurde von der Hypothese ausgegangen, dass durch die Auswahl geeigneter Messverfahren und der zugeordneten Modellierung die einzelnen kinetischen Stadien des Displacements separat zur Bestimmung der kinetischen Konstanten der Displacementprozesse genutzt werden können. Dabei sollte unter anderem auch eine Aussage über die dominierende Valenz der Antigen-Antikörper-Bindung erreicht werden. Hierzu wurden auf der Basis des Modellsystems Hämagglutinin-Peptid/ Hämagglutinin-Antikörper Displacement-Assays mit mono- und bivalenten Anti-körpern entwickelt, anhand derer eine genaue zeitaufgelöste Analyse des Bindungs- und Ablösungsverhaltens vom Liganden HA an den Anti-HA-Antikörper (Rezeptor) mittels Oberflächenplasmonenresonanz(SPR)-Spektroskopie erzielt wurde. Ausgehend von den Reaktionsgleichungen zwischen Liganden und mono- und bivalenten Rezeptoren wurden mathematische Modelle entwickelt, die eine exakte Simulation der SPR-Ergebnisse ermöglichten. Durch genaues Anpassen der Simulationsplots an die Messplots konnten alle Ratenkonstanten, die die Kinetik der Reaktionen zwischen Liganden, Rezeptoren und ihren Komplexen bestimmen, ermittelt werden. Da auch für eine große Variation der Rezeptorkonzentrationen in der Analytlösung nahezu identische Werte für die Ratenkonstanten erhalten wurden, ergeben Messungen und Simulationen ein konsistentes Bild der Anbindungskinetik und bestätigen die Qualität der Messungen. Aus Messungen des maximalen Responsabfalles kann die Konzentration der freien Antigene beim Displacement ermittelt werden. Man findet eine exponentielle Abhängigkeit des Displacements von der Konzentration der freien Antigene, die sich durch den sogenannten „Rebindingeffekt“ erklären lässt. Die gewonnenen kinetischen Daten und entwickelten Modellierungsverfahren konnten zur Simulation quasistationärer Detektionsverfahren, die mit Fluoreszenz- und Impedanzspektroskopie durchgeführt wurden, erfolgreich angewandt werden. Die erzielten Erkenntnisse konnten auf ein wissenschaftlich herausforderndes biologisches System (LuxP/AI2) angewandt werden, bei dem das niedermolekulare Signalmolekül AI2 über ein Displacementassay detektiert wurde. Dieses System ermöglicht einen Einblick in die Intra- und Interspezieskommunikation bei Bakterien. Insgesamt zeigt sich, dass die hier formulierte Hypothese als bewiesen angesehen werden kann. Die in dieser Arbeit gewonnenen Erkenntnisse eröffnen verschiedene Einsätze der Displacementmethode in der Biosensorik. Insbesondere lassen sich damit kleine Moleküle markierungsfrei quantitativ bestimmen, ohne hoch präzise Analysengeräte einsetzen zu müssen. Damit ergibt sich die Möglichkeit, sehr kompakte integrierte massensensitive Sensoren, die nicht die Empfindlichkeit hochempfindlicher SPR-Spektrometer erreichen, zur Detektion kleiner Moleküle einzusetzen. Dies ist besonders für mobile Anwendungen von Interesse.:Zusammenfassung i Summery iii Inhaltsverzeichnis v 1. Problemstellung 1 2. Kinetisches Modell für Displacement-Assays mit monovalenten Antikörpern 5 2.1 Kinetisches Modell 6 2.1.1 Grundgleichungen 6 2.1.2 Analytische Näherungslösung 8 2.1.3 Numerische Lösung 10 2.2 Vergleich mit experimentellen Beispielen 18 2.2.1 Surface-Plasmonen-Resonanzspektroskopie eines Hämagglutinin- Peptid/Hämagglutinin-Antikörper-Displacement-Assays 18 2.2.2 Fluoreszenzspektroskopie eines Hämagglutinin-Peptid/Hämagglutinin-Antikörper-Displacement-Assays 36 2.2.3 Analyse eines LuxP/AI2-Displacementassays mittels SPR-Spektroskopie 52 3. Kinetisches Modell für Displacement-Assays mit bivalenten Antikörpern 70 3.1 Kinetisches Modell 70 3.1.1 Grundgleichungen 70 3.1.2 Numerische Lösung 72 3.2 Vergleich mit experimentellen Beispielen 80 3.2.1 Surface-Plasmonen-Resonanzspektroskopie eines Hämagglutinin- Peptid/Hämagglutinin-Antikörper-Displacement-Assays 80 3.2.2 Impedanzspektroskopie eines Hämagglutinin-Peptid/Hämagglutinin-Antikörper-Displacement-Assays 88 4. Konklusionen und Ausblick 92 5. Anhänge 98 A1: Oberflächenplasmonenresonanzspektroskopie 98 A2: Aufbau der Messschichten und Messreihen eines Hämagglutinin-Peptid/Hämagglutinin-Antikörper-Displacement-Assays 105 A3: Schichtaufbau zur Analyse eines LuxP/AI2-Displacementassays mittels SPR-Spektroskopie 119 A4: Aufbau zur Fluoreszenzspektroskopie eines Hämagglutinin-Peptid/Hämagglutinin-Antikörper-Displacement-Assays 125 A5: Impedanzspektroskopie 128 A6: Transferratenkonstanten 139 A7: Abkürzungsverzeichnis 141 6. Literatur 146 7. Danksagung 155 8. Selbständigkeitserklärung 15

Bioactive SrO-SiO2 glass with well-ordered mesopores: Characterization, physiochemistry and biological properties

For a biomaterial to be considered suitable for bone repair it should ideally be both bioactive and have a capacity for controllable drug delivery; as such, mesoporous SiO2 glass has been proposed as a new class of bone regeneration material by virtue of its high drug-loading ability and generally good biocompatibility. It does, however, have less than optimum bioactivity and controllable drug delivery properties. In this study, we incorporated strontium (Sr) into mesoporous SiO2 in an effort to develop a bioactive mesoporous SrO–SiO2 (Sr–Si) glass with the capacity to deliver Sr2+ ions, as well as a drug, at a controlled rate, thereby producing a material better suited for bone repair. The effects of Sr2+ on the structure, physiochemistry, drug delivery and biological properties of mesoporous Sr–Si glass were investigated. The prepared mesoporous Sr–Si glass was found to have an excellent release profile of bioactive Sr2+ ions and dexamethasone, and the incorporation of Sr2+ improved structural properties, such as mesopore size, pore volume and specific surface area, as well as rate of dissolution and protein adsorption. The mesoporous Sr–Si glass had no cytotoxic effects and its release of Sr2+ and SiO44− ions enhanced alkaline phosphatase activity – a marker of osteogenic cell differentiation – in human bone mesenchymal stem cells. Mesoporous Sr–Si glasses can be prepared to porous scaffolds which show a more sustained drug release. This study suggests that incorporating Sr2+ into mesoporous SiO2 glass produces a material with a more optimal drug delivery profile coupled with improved bioactivity, making it an excellent material for bone repair applications. Keywords: Mesoporous Sr–Si glass; Drug delivery; Bioactivity; Bone repair; Scaffold

Swelling and mechanical properties of alginate hydrogels with respect to promotion of neural growth

Soft alginate hydrogels support robust neurite outgrowth, but their rapid disintegration in solutions of high ionic strength restricts them from long-term in vivo applications. Aiming to enhance the mechanical stability of soft alginate hydrogels, we investigated how changes in pH and ionic strength during gelation influence the swelling, stiffness, and disintegration of a three-dimensional (3D) alginate matrix and its ability to support neurite outgrowth. Hydrogels were generated from dry alginate layers through ionic crosslinks with Ca(2+) (<=10 mM) in solutions of low or high ionic strength and at pH 5.5 or 7.4. High- and low-viscosity alginates with different molecular compositions demonstrated pH and ionic strength-independent increases in hydrogel volume with decreases in Ca(2+) concentrations from 10 to 2 mM. Only soft hydrogels that were synthesized in the presence of 150 mM of NaCl (Ca-alginateNaCl) displayed long-term volume stability in buffered physiological saline, whereas analogous hydrogels generated in NaCl-free conditions (Ca-alginate) collapsed. The stiffnesses of Ca-alginateNaCl hydrogels elevated from 0.01 to 19 kPa as the Ca(2+)-concentration was raised from 2 to 10 mM; however, only Ca-alginateNaCl hydrogels with an elastic modulus <=1.5 kPa that were generated with <=4 mM of Ca(2+) supported robust neurite outgrowth in primary neuronal cultures. In conclusion, soft Ca-alginateNaCl hydrogels combine mechanical stability in solutions of high ionic strength with the ability to support neural growth and could be useful as 3D implants for neural regeneration in vivo

Three-dimensional bioprinting of volumetric tissues and organs

Three-dimensional (3D) bioprinting has become a fast-developing research field in the last few years. Many different technical solutions are available, with extrusion-based printing being the most promising and versatile method. In addition, a variety of biomaterials are already available for 3D printing of live cells. The real challenge, however, remains bioprinting of macroscopic, volumetric constructs of well-defined structures since hydrogels used for cell-embedding must consist of rather soft materials. This article describes recent developments to overcome these limitations that prevent clinical applications of bioprinted human tissues. New approaches include technical solutions such as in situ cross-linking or gelation processes that now can be performed during the bioprinting process, modified bioinks that combine suitable viscosity and cytocompatible gelation mechanisms, and utilization of additional materials to provide mechanical strength to the cell-laden constructs

Hollow fibres integrated in a microfluidic cell culture system

Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG geförderten) Allianz- bzw. Nationallizenz frei zugänglich.This publication is with permission of the rights owner freely accessible due to an Alliance licence and a national licence (funded by the DFG, German Research Foundation) respectively.For in vitro drug screening a modified perfusion micro-bioreactor system with integrated hollow fibres will be demonstrated. This biocompatible system consists of an integrated closed flow circuit that includes reservoirs and pneumatic micro pumps. Additional optical online-monitoring devices allow the observation during the cell cultivation. The embedded hollow fibre system which acts as cell carrier consists of a biodegradable biopolymer. One option to fabricate such 3D structures is the technology of Organ Printing which is realised by an adapted rapid prototyping system entitled 3D Scaffold Printer. With this device specimens consisting of tubes with a diameter smaller than 2 mm can be prepared. It should be possible to cultivate the hollow fibres inside and outside with different kinds of cells and therefore generate models of complex tissues

Podoplanin immunopositive lymphatic vessels at the implant interface in a rat model of osteoporotic fractures

Insertion of bone substitution materials accelerates healing of osteoporotic fractures. Biodegradable materials are preferred for application in osteoporotic patients to avoid a second surgery for implant replacement. Degraded implant fragments are often absorbed by macrophages that are removed from the fracture side via passage through veins or lymphatic vessels. We investigated if lymphatic vessels occur in osteoporotic bone defects and whether they are regulated by the use of different materials. To address this issue osteoporosis was induced in rats using the classical method of bilateral ovariectomy and additional calcium and vitamin deficient diet. In addition, wedge-shaped defects of 3, 4, or 5 mm were generated in the distal metaphyseal area of femur via osteotomy. The 4 mm defects were subsequently used for implantation studies where bone substitution materials of calcium phosphate cement, composites of collagen and silica, and iron foams with interconnecting pores were inserted. Different materials were partly additionally functionalized by strontium or bisphosphonate whose positive effects in osteoporosis treatment are well known. The lymphatic vessels were identified by immunohistochemistry using an antibody against podoplanin. Podoplanin immunopositive lymphatic vessels were detected in the granulation tissue filling the fracture gap, surrounding the implant and growing into the iron foam through its interconnected pores. Significant more lymphatic capillaries were counted at the implant interface of composite, strontium and bisphosphonate functionalized iron foam. A significant increase was also observed in the number of lymphatics situated in the pores of strontium coated iron foam. In conclusion, our results indicate the occurrence of lymphatic vessels in osteoporotic bone. Our results show that lymphatic vessels are localized at the implant interface and in the fracture gap where they might be involved in the removal of lymphocytes, macrophages, debris and the implants degradation products. Therefore the lymphatic vessels are involved in implant integration and fracture healing

3D Extrusion Printing of Biphasic Anthropomorphic Brain Phantoms Mimicking MR Relaxation Times Based on Alginate-Agarose-Carrageenan Blends

The availability of adapted phantoms mimicking different body parts is fundamental to establishing the stability and reliability of magnetic resonance imaging (MRI) methods. The primary purpose of such phantoms is the mimicking of physiologically relevant, contrast-creating relaxation times T1 and T2. For the head, frequently examined by MRI, an anthropomorphic design of brain phantoms would imply the discrimination of gray matter and white matter (WM) within defined, spatially distributed compartments. Multichannel extrusion printing allows the layer-by layer fabrication of multiple pastelike materials in a spatially defined manner with a predefined shape. In this study, the advantages of this method are used to fabricate biphasic brain phantoms mimicking MR relaxation times and anthropomorphic geometry. The printable ink was based on purely naturally derived polymers: alginate as a calcium-cross-linkable gelling agent, agarose, iota- carrageenan, and GdCl3 in different concentrations (0-280 mu mol kg-1) as the paramagnetic component. The suggested inks (e.g., 3Alg-1Agar-6Car) fulfilled the requirements of viscoelastic behavior and printability of large constructs (&gt;150 mL). The microstructure and distribution of GdCl3 were assessed by scanning electron microscopy (SEM) with energy-dispersive X-ray spectroscopy (EDX). In closely monitored steps of technological development and characterization, from monophasic and biphasic samples as printable inks and cross-linked gels, we describe the construction of large-scale phantom models whose relaxation times were characterized and checked for stability over time