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- Abteilung für Funktionswerkstoffe der Medizin und der Zahnheilkunde (10) (entfernen)
Despite advances in cartilage repair strategies, treatment of focal chondral lesions remains an important challenge to prevent osteoarthritis. Articular cartilage is organized into several layers and lack of zonal organization of current grafts is held responsible for insufficient biomechanical and biochemical quality of repair-tissue. The aim was to develop a zonal approach for cartilage regeneration to determine whether the outcome can be improved compared to a non-zonal strategy. Hydrogel-filled polycaprolactone (PCL)-constructs with a chondrocyte-seeded upper-layer deemed to induce hyaline cartilage and a mesenchymal stromal cell (MSC)-containing bottom-layer deemed to induce calcified cartilage were compared to chondrocyte-based non-zonal grafts in a minipig model. Grafts showed comparable hardness at implantation and did not cause visible signs of inflammation. After 6 months, X-ray microtomography (µCT)-analysis revealed significant bone-loss in both treatment groups compared to empty controls. PCL-enforcement and some hydrogel-remnants were retained in all defects, but most implants were pressed into the subchondral bone. Despite important heterogeneities, both treatments reached a significantly lower modified O’Driscoll-score compared to empty controls. Thus, PCL may have induced bone-erosion during joint loading and misplacement of grafts in vivo precluding adequate permanent orientation of zones compared to surrounding native cartilage.
In the treatment of bone non-unions, an alternative to bone autografts is the use of bone morphogenetic proteins (BMPs), e.g., BMP–2, BMP–7, with powerful osteoinductive and osteogenic properties. In clinical settings, these osteogenic factors are applied using absorbable collagen sponges for local controlled delivery. Major side effects of this strategy are derived from the supraphysiological doses of BMPs needed, which may induce ectopic bone formation, chronic inflammation, and excessive bone resorption. In order to increase the efficiency of the delivered BMPs, we designed cryostructured collagen scaffolds functionalized with hydroxyapatite, mimicking the structure of cortical bone (aligned porosity, anisotropic) or trabecular bone (random distributed porosity, isotropic). We hypothesize that an anisotropic structure would enhance the osteoconductive properties of the scaffolds by increasing the regenerative performance of the provided rhBMP–2. In vitro, both scaffolds presented similar mechanical properties, rhBMP–2 retention and delivery capacity, as well as scaffold degradation time. In vivo, anisotropic scaffolds demonstrated better bone regeneration capabilities in a rat femoral critical-size defect model by increasing the defect bridging. In conclusion, anisotropic cryostructured collagen scaffolds improve bone regeneration by increasing the efficiency of rhBMP–2 mediated bone healing.
Fabrication of microchannels using 3D printing of sugars as fugitive material is explored in different fields, including microfluidics. However, establishing reproducible methods for the controlled production of sugar structures with sub-100 μm dimensions remains a challenge.
This study pioneers the processing of sugars by melt electrowriting (MEW) enabling the fabrication of structures with so far unprecedented resolution from Isomalt. Based on a systematic variation of process parameters, fibers with diameters down to 20 μm can be fabricated. The flexibility in the adjustment of fiber diameter by on-demand alteration of MEW parameters enables generating constructs with perfusable channels within polydimethylsiloxane molds. These channels have a diameter that can be adjusted from 30 to 200 μm in a single design.
Taken together, the experiments show that MEW strongly benefits from the thermal and physical stability of Isomalt, providing a robust platform for the fabrication of small-diameter embedded microchannel systems.
Supplement-free induction of cellular differentiation and polarization solely through the topography of materials is an auspicious strategy but has so far significantly lagged behind the efficiency and intensity of media-supplementation-based protocols. Consistent with the idea that 3D structural motifs in the extracellular matrix possess immunomodulatory capacity as part of the natural healing process, it is found in this study that human-monocyte-derived macrophages show a strong M2a-like prohealing polarization when cultured on type I rat-tail collagen fibers but not on collagen I films. Therefore, it is hypothesized that highly aligned nanofibrils also of synthetic polymers, if packed into larger bundles in 3D topographical biomimetic similarity to native collagen I, would induce a localized macrophage polarization. For the automated fabrication of such bundles in a 3D printing manner, the strategy of “melt electrofibrillation” is pioneered by the integration of flow-directed polymer phase separation into melt electrowriting and subsequent selective dissolution of the matrix polymer postprocessing. This process yields nanofiber bundles with a remarkable structural similarity to native collagen I fibers, particularly for medical-grade poly(ε-caprolactone). These biomimetic fibrillar structures indeed induce a pronounced elongation of human-monocyte-derived macrophages and unprecedentedly trigger their M2-like polarization similar in efficacy as interleukin-4 treatment.
Biofabrication aims to fabricate biologically functional products through bioprinting or bioassembly (Groll et al 2016 Biofabrication 8 013001). In biofabrication processes, cells are positioned at defined coordinates in three-dimensional space using automated and computer controlled techniques (Moroni et al 2018 Trends Biotechnol. 36 384–402), usually with the aid of biomaterials that are either (i) directly processed with the cells as suspensions/dispersions, (ii) deposited simultaneously in a separate printing process, or (iii) used as a transient support material. Materials that are suited for biofabrication are often referred to as bioinks and have become an important area of research within the field. In view of this special issue on bioinks, we aim herein to briefly summarize the historic evolution of this term within the field of biofabrication. Furthermore, we propose a simple but general definition of bioinks, and clarify its distinction from biomaterial inks.
Macrophages are key players of the innate immune system that can roughly be divided into the pro-inflammatory M1 type and the anti-inflammatory, pro-healing M2 type. While a transient initial pro-inflammatory state is helpful, a prolonged inflammation deteriorates a proper healing and subsequent regeneration. One promising strategy to drive macrophage polarization by biomaterials is precise control over biomaterial geometry. For regenerative approaches, it is of particular interest to identify geometrical parameters that direct human macrophage polarization. For this purpose, we advanced melt electrowriting (MEW) towards the fabrication of fibrous scaffolds with box-shaped pores and precise inter-fiber spacing from 100 μm down to only 40 μm. These scaffolds facilitate primary human macrophage elongation accompanied by differentiation towards the M2 type, which was most pronounced for the smallest pore size of 40 μm. These new findings can be important in helping to design new biomaterials with an enhanced positive impact on tissue regeneration.
Additive manufacturing of scaffolds with sub-micron filaments via melt electrospinning writing
(2015)
The aim of this study was to explore the lower resolution limits of an electrohydrodynamic process combined with direct writing technology of polymer melts. Termed melt electrospinning writing, filaments are deposited layer-by-layer to produce discrete three-dimensional scaffolds for in vitro research. Through optimization of the parameters (flow rate, spinneret diameter, voltage, collector distance) for poly-ϵ-caprolactone, we could direct-write coherent scaffolds with ultrafine filaments, the smallest being 817 ± 165 nm. These low diameter filaments were deposited to form box-structures with a periodicity of 100.6 ± 5.1 μm and a height of 80 μm (50 stacked filaments; 100 overlap at intersections). We also observed oriented crystalline regions within such ultrafine filaments after annealing at 55 °C. The scaffolds were printed upon NCO-sP(EO-stat-PO)-coated glass slide surfaces and withstood frequent liquid exchanges with negligible scaffold detachment for at least 10 days in vitro.
The development and formulation of printable inks for extrusion-based 3D bioprinting has been a major challenge in the field of biofabrication. Inks, often polymer solutions with the addition of crosslinking to form hydrogels, must not only display adequate mechanical properties for the chosen application but also show high biocompatibility as well as printability. Here we describe a reproducible two-step method for the assessment of the printability of inks for bioprinting, focussing firstly on screening ink formulations to assess fibre formation and the ability to form 3D constructs before presenting a method for the rheological evaluation of inks to characterise the yield point, shear thinning and recovery behaviour. In conjunction, a mathematical model was formulated to provide a theoretical understanding of the pressure-driven, shear thinning extrusion of inks through needles in a bioprinter. The assessment methods were trialled with a commercially available crème, poloxamer 407, alginate-based inks and an alginate-gelatine composite material. Yield stress was investigated by applying a stress ramp to a number of inks, which demonstrated the necessity of high yield for printable materials. The shear thinning behaviour of the inks was then characterised by quantifying the degree of shear thinning and using the mathematical model to predict the window of printer operating parameters in which the materials could be printed. Furthermore, the model predicted high shear conditions and high residence times for cells at the walls of the needle and effects on cytocompatibility at different printing conditions. Finally, the ability of the materials to recover to their original viscosity after extrusion was examined using rotational recovery rheological measurements. Taken together, these assessment techniques revealed significant insights into the requirements for printable inks and shear conditions present during the extrusion process and allow the rapid and reproducible characterisation of a wide variety of inks for bioprinting.
Identification of articular cartilage progenitor cells (ACPCs) has opened up new opportunities for cartilage repair. These cells may be used as alternatives for or in combination with mesenchymal stromal cells (MSCs) in cartilage engineering. However, their potential needs to be further investigated, since only a few studies have compared ACPCs and MSCs when cultured in hydrogels. Therefore, in this study, we compared chondrogenic differentiation of equine ACPCs and MSCs in agarose constructs as monocultures and as zonally layered co-cultures under both normoxic and hypoxic conditions. ACPCs and MSCs exhibited distinctly differential production of the cartilaginous extracellular matrix (ECM). For ACPC constructs, markedly higher glycosaminoglycan (GAG) contents were determined by histological and quantitative biochemical evaluation, both in normoxia and hypoxia. Differential GAG production was also reflected in layered co-culture constructs. For both cell types, similar staining for type II collagen was detected. However, distinctly weaker staining for undesired type I collagen was observed in the ACPC constructs. For ACPCs, only very low alkaline phosphatase (ALP) activity, a marker of terminal differentiation, was determined, in stark contrast to what was found for MSCs. This study underscores the potential of ACPCs as a promising cell source for cartilage engineering.
Intercellular adhesion plays a major role in tissue development and homeostasis. Yet, technologies to measure mature cell-cell contacts are not available. We introduce a methodology based on fluidic probe force microscopy to assess cell-cell adhesion forces after formation of mature intercellular contacts in cell monolayers. With this method we quantify that L929 fibroblasts exhibit negligible cell-cell adhesion in monolayers whereas human endothelial cells from the umbilical artery (HUAECs) exert strong intercellular adhesion forces per cell. We use a new in vitro model based on the overexpression of Muscle Segment Homeobox 1 (MSX1) to induce Endothelial-to-Mesenchymal Transition (EndMT), a process involved in cardiovascular development and disease. We reveal how intercellular adhesion forces in monolayer decrease significantly at an early stage of EndMT and we show that cells undergo stiffening and flattening at this stage. This new biomechanical insight complements and expands the established standard biomolecular analyses. Our study thus introduces a novel tool for the assessment of mature intercellular adhesion forces in a physiological setting that will be of relevance to biological processes in developmental biology, tissue regeneration and diseases like cancer and fibrosis.