SARS-CoV-2 particles promote airway epithelial differentiation and ciliation.

Introduction: The Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), which caused the coronavirus disease 2019 (COVID-19) pandemic, enters the human body via the epithelial cells of the airway tract. To trap and eject pathogens, the airway epithelium is composed of ciliated and secretory cells that produce mucus which is expelled through a process called mucociliary clearance. Methods: This study examines the early stages of contact between SARS-CoV-2 particles and the respiratory epithelium, utilizing 3D airway tri-culture models exposed to ultraviolet light-irradiated virus particles. These cultures are composed of human endothelial cells and human tracheal mesenchymal cells in a fibrin hydrogel matrix covered by mucociliated human tracheal epithelial cells. Results: We found that SARS-CoV-2 particles trigger a significant increase in ciliation on the epithelial surface instructed through a differentiation of club cells and basal stem cells. The contact with SARS-CoV-2 particles also provoked a loss of cell-cell tight junctions and impaired the barrier integrity. Further immunofluorescence analyses revealed an increase in FOXJ1 expression and PAK1/2 phosphorylation associated with particle-induced ciliation. Discussion: An understanding of epithelial responses to virus particles may be instrumental to prevent or treat respiratory infectious diseases such as COVID-19.

Dual Labeling of Primary Cells with Fluorescent Gadolinium Oxide Nanoparticles.

The interest in mesenchymal stromal cells as a therapy option is increasing rapidly. To improve their implementation, location, and distribution, the properties of these must be investigated. Therefore, cells can be labeled with nanoparticles as a dual contrast agent for fluorescence and magnetic resonance imaging (MRI). In this study, a more efficient protocol for an easy synthesis of rose bengal-dextran-coated gadolinium oxide (Gd2O3-dex-RB) nanoparticles within only 4 h was established. Nanoparticles were characterized by zeta potential measurements, photometric measurements, fluorescence and transmission electron microscopy, and MRI. In vitro cell experiments with SK-MEL-28 and primary adipose-derived mesenchymal stromal cells (ASC), nanoparticle internalization, fluorescence and MRI properties, and cell proliferation were performed. The synthesis of Gd2O3-dex-RB nanoparticles was successful, and they were proven to show adequate signaling in fluorescence microscopy and MRI. Nanoparticles were internalized into SK-MEL-28 and ASC via endocytosis. Labeled cells showed sufficient fluorescence and MRI signal. Labeling concentrations of up to 4 mM and 8 mM for ASC and SK-MEL-28, respectively, did not interfere with cell viability and proliferation. Gd2O3-dex-RB nanoparticles are a feasible contrast agent to track cells via fluorescence microscopy and MRI. Fluorescence microscopy is a suitable method to track cells in in vitro experiments with smaller samples.

Transformative Materials to Create 3D Functional Human Tissue Models In Vitro in a Reproducible Manner.

Recreating human tissues and organs in the petri dish to establish models as tools in biomedical sciences has gained momentum. These models can provide insight into mechanisms of human physiology, disease onset, and progression, and improve drug target validation, as well as the development of new medical therapeutics. Transformative materials play an important role in this evolution, as they can be programmed to direct cell behavior and fate by controlling the activity of bioactive molecules and material properties. Using nature as an inspiration, scientists are creating materials that incorporate specific biological processes observed during human organogenesis and tissue regeneration. This article presents the reader with state-of-the-art developments in the field of in vitro tissue engineering and the challenges related to the design, production, and translation of these transformative materials. Advances regarding (stem) cell sources, expansion, and differentiation, and how novel responsive materials, automated and large-scale fabrication processes, culture conditions, in situ monitoring systems, and computer simulations are required to create functional human tissue models that are relevant and efficient for drug discovery, are described. This paper illustrates how these different technologies need to converge to generate in vitro life-like human tissue models that provide a platform to answer health-based scientific questions.

Influence of Aerosolization on Endothelial Cells for Efficient Cell Deposition in Biohybrid and Regenerative Applications.

The endothelialization of gas exchange membranes can increase the hemocompatibility of extracorporeal membrane oxygenators and thus become a long-term lung replacement option. Cell seeding on large or uneven surfaces of oxygenator membranes is challenging, with cell aerosolization being a possible solution. In this study, we evaluated the endothelial cell aerosolization for biohybrid lung application. A Vivostat® system was used for the aerosolization of human umbilical vein endothelial cells with non-sprayed cells serving as a control. The general suitability was evaluated using various flow velocities, substrate distances and cell concentrations. Cells were analyzed for survival, apoptosis and necrosis levels. In addition, aerosolized and non-sprayed cells were cultured either static or under flow conditions in a dynamic microfluidic model. Evaluation included immunocytochemistry and gene expression via quantitative PCR. Cell survival for all tested parameters was higher than 90%. No increase in apoptosis and necrosis levels was seen 24 h after aerosolization. Spraying did not influence the ability of the endothelial cells to form a confluent cell layer and withstand shear stresses in a dynamic microfluidic model. Immunocytochemistry revealed typical expression of CD31 and von Willebrand factor with cobble-stone cell morphology. No change in shear stress-induced factors after aerosolization was reported by quantitative PCR analysis. With this study, we have shown the feasibility of endothelial cell aerosolization with no significant changes in cell behavior. Thus, this technique could be used for efficient the endothelialization of gas exchange membranes in biohybrid lung applications.

Effervescent Atomizer as Novel Cell Spray Technology to Decrease the Gas-to-Liquid Ratio.

Cell spraying has become a feasible application method for cell therapy and tissue engineering approaches. Different devices have been used with varying success. Often, twin-fluid atomizers are used, which require a high gas velocity for optimal aerosolization characteristics. To decrease the amount and velocity of required air, a custom-made atomizer was designed based on the effervescent principle. Different designs were evaluated regarding spray characteristics and their influence on human adipose-derived mesenchymal stromal cells. The arithmetic mean diameters of the droplets were 15.4−33.5 µm with decreasing diameters for increasing gas-to-liquid ratios. The survival rate was >90% of the control for the lowest gas-to-liquid ratio. For higher ratios, cell survival decreased to approximately 50%. Further experiments were performed with the design, which had shown the highest survival rates. After seven days, no significant differences in metabolic activity were observed. The apoptosis rates were not influenced by aerosolization, while high gas-to-liquid ratios caused increased necrosis levels. Tri-lineage differentiation potential into adipocytes, chondrocytes, and osteoblasts was not negatively influenced by aerosolization. Thus, the effervescent aerosolization principle was proven suitable for cell applications requiring reduced amounts of supplied air. This is the first time an effervescent atomizer was used for cell processing.

Bone Marrow Derived Mesenchymal Stromal Cells Promote Vascularization and Ciliation in Airway Mucosa Tri-Culture Models in Vitro.

Patients suffering from irresectable tracheal stenosis often face limited treatment options associated with low quality of life. To date, an optimal tracheal replacement strategy does not exist. A tissue-engineered tracheal substitute promises to overcome limitations such as implant vascularization, functional mucociliary clearance and mechanical stability. In order to advance a tracheal mucosa model recently developed by our group, we examined different supporting cell types in fibrin-based tri-culture with primary human umbilical vein endothelial cells (HUVEC) and primary human respiratory epithelial cells (HRE). Bone marrow-derived mesenchymal stromal cells (BM-MSC), adipose-derived mesenchymal stromal cells (ASC) and human nasal fibroblasts (HNF) were compared regarding their ability to promote mucociliary differentiation and vascularization in vitro. Three-dimensional co-cultures of the supporting cell types with either HRE or HUVEC were used as controls. Mucociliary differentiation and formation of vascular-like structures were analyzed by scanning electron microscopy (SEM), periodic acid Schiff's reaction (PAS reaction), two-photon laser scanning microscopy (TPLSM) and immunohistochemistry. Cytokine levels of vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), interleukin-6 (IL6), interleukin-8 (IL8), angiopoietin 1, angiopoietin 2, fibroblast growth factor basic (FGF-b) and placenta growth factor (PIGF) in media supernatant were investigated using LEGENDplex™ bead-based immunoassay. Epithelial morphology of tri-cultures with BM-MSC most closely resembled native respiratory epithelium with respect to ciliation, mucus production as well as expression and localization of epithelial cell markers pan-cytokeratin, claudin-1, α-tubulin and mucin5AC. This was followed by tri-cultures with HNF, while ASC-supported tri-cultures lacked mucociliary differentiation. For all supporting cell types, a reduced ciliation was observed in tri-cultures compared to the corresponding co-cultures. Although formation of vascular-like structures was confirmed in all cultures, vascular networks in BM-MSC-tri-cultures were found to be more branched and extended. Concentrations of pro-angiogenic and inflammatory cytokines, in particular VEGF and angiopoietin 2, revealed to be reduced in tri-cultures compared to co-cultures. With these results, our study provides an important step towards a vascularized and ciliated tissue-engineered tracheal replacement. Additionally, our tri-culture model may in the future contribute to an improved understanding of cell-cell interactions in diseases associated with impaired mucosal function.

Establishment of a Pre-vascularized 3D Lung Cancer Model in Fibrin Gel-Influence of Hypoxia and Cancer-Specific Therapeutics.

Lung cancer is the most frequently diagnosed cancer worldwide and the one that causes the highest mortality. In order to understand the disease and to develop new treatments, in vitro human lung cancer model systems which imitate the physiological conditions is of high significance. In this study, a human 3D lung cancer model was established that features the organization of a tumor with focus on tumor angiogenesis. Vascular networks were formed by co-culture of human umbilical vein endothelial cells and adipose tissue-derived mesenchymal stem cells (ASC) for 14 days in fibrin. A part of the pre-vascularized fibrin gel was replaced by fibrin gel containing lung cancer cells (A549) to form tri-cultures. This 3D cancer model system was cultured under different culture conditions and its behaviour after treatment with different concentrations of tumor-specific therapeutics was evaluated. The evaluation was performed by measurement of metabolic activity, viability, quantification of two-photon laser scanning microscopy and measurement of the proangiogenic factor vascular endothelial growth factor in the supernatant. Hypoxic conditions promoted vascularization compared to normoxic cultured controls in co- and tri-cultures as shown by significantly increased vascular structures, longer structures with a higher area and volume, and secretion of vascular endothelial growth factor. Cancer cells also promoted vascularization. Treatment with 50 µM gefitinib or 50 nM paclitaxel decreased the vascularization significantly. VEGF secretion was only reduced after treatment with gefitinib, while in contrast secretion remained constant during medication with paclitaxel. The findings suggest that the herein described 3D lung cancer model provides a novel platform to investigate the angiogenic potential of cancer cells and its responses to therapeutics. Thus, it can serve as a promising approach for the development and patient-specific pre-selection of anticancer treatment.

FRESH bioprinting technology for tissue engineering - the influence of printing process and bioink composition on cell behavior and vascularization.

The rapid and tailored biofabrication of natural materials is of high interest for the field of tissue engineering and regenerative medicine. Scaffolds require both high biocompatibility and tissue-dependent mechanical strength to function as basis for tissue-engineered implants. Thus, natural hydrogels such as fibrin are promising but their rapid biofabrication remains challenging. Printing of low viscosity and slow polymerizing solutions with good spatial resolution can be achieved by freeform reversible embedding of suspended hydrogels (FRESH) bioprinting of cell-laden natural hydrogels. In this study, fibrin and hyaluronic acid were used as single components as well as blended ink mixtures for the FRESH bioprinting. Rheometry revealed that single materials were less viscous than the blended bioink showing higher values for viscosity over a shear rate of 10-1000 s-1. While fibrin showed viscosities between 0.1624 and 0.0017 Pa·s, the blended ink containing fibrin and hyaluronic acid were found to be in a range of 0.1-1 Pa·s. In 3D vascularization assays, formation of vascular structures within the printed constructs was investigated indicating that the printing process did not harm cells and allowed formation of vasculature comparable to moulded control samples. Best values for vascularization were achieved in bioinks consisting of 1.0% fibrin-0.5% hyaluronic acid. The vascular structure area and length were three times higher compared to other tested bioinks, and structure volume as well as number of branches revealed almost four times higher values. In this study, we combined the benefits of the FRESH printing technique with in vitro vascularization, showing that it is possible to achieve a mechanically stable small-scale hydrogel construct incorporating vascular network formation.

Endoscopic atomization of mesenchymal stromal cells: in vitro study for local cell therapy of the lungs.

BACKGROUND AIMS: Cell-based therapies of pulmonary diseases with mesenchymal stromal cells (MSCs) are increasingly under experimental investigation. In most of these, MSCs are administered intravenously or by direct intratracheal instillation. A parallel approach is to administer the cells into the lung by endoscopic atomization (spraying). In a previous study, the authors developed a flexible endoscopic atomization device that allows administration of respiratory epithelial cells in the lungs with high survival. METHODS: In this study, the authors evaluated the feasibility of spraying MSCs with two different endoscopic atomization devices (air and pressure atomization). Following atomization, cell viability was evaluated with live/dead staining. Subsequent effects on cytotoxicity, trilineage differentiation and expression of MSC-specific markers as well as on MSC metabolic activity and morphology were analyzed for up to 7 days. RESULTS: MSC viability immediately after spraying and subsequent metabolic activity for 7 days was not influenced by either of the devices. Slightly higher cytotoxicity rates could be observed for pressure-atomized compared with control and air-atomized MSCs over 7 days. Flow cytometry revealed no changes in characteristic MSC cell surface marker expression, and morphology remained unchanged. Standard differentiation into osteocytes, chondrocytes and adipocytes was inducible after atomization. CONCLUSIONS: In the literature, a minimal survival of 50% was previously defined as the cutoff value for successful cell atomization. This is easily met with both of the authors' devices, with more than 90% survival. Thus, there is a potential role for atomization in intrapulmonary MSC-based cell therapies, as it is a feasible and easily utilizable approach based on clinically available equipment.

Extracellular Vesicles-Loaded Fibrin Gel Supports Rapid Neovascularization for Dental Pulp Regeneration.

Rapid vascularization is required for the regeneration of dental pulp due to the spatially restricted tooth environment. Extracellular vesicles (EVs) released from mesenchymal stromal cells show potent proangiogenic effects. Since EVs suffer from rapid clearance and low accumulation in target tissues, an injectable delivery system capable of maintaining a therapeutic dose of EVs over a longer period would be desirable. We fabricated an EV-fibrin gel composite as an in situ forming delivery system. EVs were isolated from dental pulp stem cells (DPSCs). Their effects on cell proliferation and migration were monitored in monolayers and hydrogels. Thereafter, endothelial cells and DPSCs were co-cultured in EV-fibrin gels and angiogenesis as well as collagen deposition were analyzed by two-photon laser microscopy. Our results showed that EVs enhanced cell growth and migration in 2D and 3D cultures. EV-fibrin gels facilitated vascular-like structure formation in less than seven days by increasing the release of VEGF. The EV-fibrin gel promoted the deposition of collagen I, III, and IV, and readily induced apoptosis during the initial stage of angiogenesis. In conclusion, we confirmed that EVs from DPSCs can promote angiogenesis in an injectable hydrogel in vitro, offering a novel and minimally invasive strategy for regenerative endodontic therapy.

EndOxy: Mid-term stability and shear stress resistance of endothelial cells on PDMS gas exchange membranes.

Endothelialized oxygenator devices (EndOxy) with a physiological, nonthrombogenic, and anti-inflammatory surface offer the potential to overcome current shortcomings of conventional extracorporeal membrane oxygenation such as complications like thromboembolism and bleeding that deteriorate adequate long-term hemocompatibility. The approach of endothelialization of gas exchange membranes, and thus the formation of a nonthrombogenic and anti-inflammatory surface, is promising. In this study, we investigated the mid-term shear stress resistance as well as gas transfer rates and cell densities of endothelial cells seeded on RGD-conjugated polydimethylsiloxane (RGD-PDMS) gas exchange membranes under dynamic conditions. Human umbilical vein endothelial cells were seeded on RGD-PDMS and exposed to defined shear stresses in a microfluidic bioreactor. Endothelial cell morphology was assessed by bright field microscopy and immunocytochemistry. Furthermore, gas transfer measurement of blank, RGD-conjugated, and endothelialized PDMS oxygenator membranes was performed. RGD-PDMS gas exchange membranes proved suitable for the dynamic culture of endothelial cells for up to 21 days at a wall shear stress of 2.9 dyn/cm2 . Furthermore, the cells resisted increased wall shear stresses up to 8.6 dyn/cm2 after a previous dynamic preculture of each one hour at 2.9 dyn/cm2 and 5.7 dyn/cm2 . Also, after a longer dynamic preculture of three days at 2.9 dyn/cm2 and one hour at 5.7 dyn/cm2 , increased wall shear stresses of 8.6 dyn/cm2 were tolerated by the cells and cell integrity could be remained. Gas transfer (GT) tests revealed that neither RGD conjugation nor endothelialization of RGD-PDMS significantly decrease the gas transfer rates of the membranes during short-term trials. Gas transfer rates are stable for at least 72 hours of dynamic cultivation of endothelial cells. Immunocytochemistry showed that the cell layer stained positive for typical endothelial cell markers CD31 and von Willebrand factor (VWF) after all trials. Cell density of EC on RGD-PDMS increased between 3 and 21 days of dynamic culture. In this study, we show the suitability of RGD-PDMS membranes for flow resistant endothelialization of gas-permeable membranes, demonstrating the feasibility of this approach for a biohybrid lung.

Choosing the Right Differentiation Medium to Develop Mucociliary Phenotype of Primary Nasal Epithelial Cells In Vitro.

In vitro differentiation of airway epithelium is of interest for respiratory tissue engineering and studying airway diseases. Both applications benefit from the use of primary cells to maintain a mucociliated phenotype and thus physiological functionality. Complex differentiation procedures often lack standardization and reproducibility. To alleviate these shortfalls, we compared differentiation behavior of human nasal epithelial cells in four differentiation media. Cells were differentiated at the air-liquid interface (ALI) on collagen-coated inserts. Mucociliary differentiation status after five weeks was analyzed by electron microscopy, histology and immunohistochemistry. The amount of ciliation was estimated and growth factor concentrations were evaluated using ELISA. We found that retinoic-acid-supplemented mixture of DMEM and Airway Epithelial Cell Growth Medium gave most promising results to obtain ciliated and mucus producing nasal epithelium in vitro. We discovered the balance between retinoic acid (RA), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF) and fibroblast growth factor ß (FGF-ß) to be relevant for differentiation. We could show that low VEGF, EGF and FGF-ß concentrations in medium correspond to absent ciliation in specific donors. Therefore, our results may in future facilitate donor selection and non-invasive monitoring of ALI cultures and by this contribute to improved standardization of epithelial in vitro culture.

Influence of Different Cell Types and Sources on Pre-Vascularisation in Fibrin and Agarose-Collagen Gels.

Vascularisation is essential for the development of tailored, tissue-engineered organs and tissues due to diffusion limits of nutrients and the lack of the necessary connection to the cardiovascular system. To pre-vascularize, endothelial cells and supporting cells can be embedded in the scaffold to foster an adequate nutrient and oxygen supply after transplantation. This technique is applied for tissue engineering of various tissues, but there have been few studies on the use of different cell types or cells sources. We compare the effect of supporting cells from different sources on vascularisation. Fibrin gels and agarose-collagen hydrogels were used as scaffolds. The supporting cells were primary human dermal fibroblasts (HDFs), human nasal fibroblasts (HNFs), human mesenchymal stem cells from umbilical cord's Wharton's jelly (WJ MSCs), adipose-derived MSCs (AD MSCs) and femoral bone marrow-derived MSCs (BM MSCs). The tissue constructs were incubated for 14 days and analyzed by two-photon laser scanning microscopy. Vascularisation was supported by all cell types, forming branched networks of tubular vascular structures in both hydrogels. In general, fibrin gels present a higher angiogenic promoting environment compared to agarose-collagen hydrogels and fibroblasts show a high angiogenic potential in co-culture with endothelial cells. In agarose-collagen hydrogels, vascular structures supported by AD MSCs were comparable to our HDF control in terms of volume, area and length. BM MSCs formed a homogeneous network of smaller structures in both hydrogels. This study provides data toward understanding the pre-vascularisation properties of different supporting cell types and sources for tissue engineering of different organs and tissues.

EndOxy: Dynamic Long-Term Evaluation of Endothelialized Gas Exchange Membranes for a Biohybrid Lung.

In the concept of a biohybrid lung, endothelial cells seeded on gas exchange membranes form a non-thrombogenic an anti-inflammatory surface to overcome the lacking hemocompatibility of today's oxygenators during extracorporeal membrane oxygenation. To evaluate this concept, the long-term stability and gas exchange performance of endothelialized RGD-conjugated polydimethylsiloxane (RGD-PDMS) membranes was evaluated. Human umbilical vein endothelial cells (ECs) were cultured on RGD-PDMS in a model system under physiological wall shear stress (WSS) of 0.5 Pa for up to 33 days. Gas exchange performance was tested with three biological replicates under elevated WSS of 2.5 Pa using porcine blood adjusted to venous values following ISO 7199 and blood gas analysis. EC morphology was assessed by immunocytochemistry (n = 3). RGD-PDMS promoted endothelialization and stability of endothelialized membranes was shown for at least 33 days and for a maximal WSS of 2.5 Pa. Short-term exposure to porcine blood did not affect EC integrity. The gas transfer tests provided evidence for the oxygenation and decarboxylation of the blood across endothelialized membranes with a decrease of transfer rates over time that needs to be addressed in further studies with larger sample sizes. Our results demonstrate the general suitability of RGD-PDMS for biohybrid lung applications, which might enable long-term support of patients with chronic lung failure in the future.

Comparison of Covered Laser-cut and Braided Respiratory Stents: From Bench to Pre-Clinical Testing.

Lung cancer patients often suffer from severe airway stenosis, the symptoms of which can be relieved by the implantation of stents. Different respiratory stents are commercially available, but the impact of their mechanical performance on tissue responses is not well understood. Two novel laser-cut and hand-braided nitinol stents, partially covered with polycarbonate urethane, were bench tested and implanted in Rhön sheep for 6 weeks. Bench testing highlighted differences in mechanical behavior: the laser-cut stent showed little foreshortening when crimped to a target diameter of 7.5 mm, whereas the braided stent elongated by more than 50%. Testing also revealed that the laser-cut stent generally exerted higher radial resistive and chronic outward forces than the braided stent, but the latter produced significantly higher radial resistive forces at diameters below 9 mm. No migration was observed for either stent type in vivo. In terms of granulation, most stents exerted a low to medium tissue response with only minimal formation of granulation tissue. We have developed a mechanical and in vivo framework to compare the behavior of different stent designs in a large animal model, providing data, which may be employed to improve current stent designs and to achieve better treatment options for lung cancer patients.

Evaluating the interaction of a tracheobronchial stent in an ovine in-vivo model.

Tracheobronchial stents are used to restore patency to stenosed airways. However, these devices are associated with many complications such as stent migration, granulation tissue formation, mucous plugging and stent strut fracture. Of these, granulation tissue formation is the complication that most frequently requires costly secondary interventions. In this study a biomechanical lung modelling framework recently developed by the authors to capture the lung in-vivo stress state under physiological loading is employed in conjunction with ovine pre-clinical stenting results and device experimental data to evaluate the effect of stent interaction on granulation tissue formation. Stenting is simulated using a validated model of a prototype covered laser-cut tracheobronchial stent in a semi-specific biomechanical lung model, and physiological loading is performed. Two computational methods are then used to predict possible granulation tissue formation: the standard method which utilises the increase in maximum principal stress change, and a newly proposed method which compares the change in contact pressure over a respiratory cycle. These computational predictions of granulation tissue formation are then compared to pre-clinical stenting observations after a 6-week implantation period. Experimental results of the pre-clinical stent implantation showed signs of granulation tissue formation both proximally and distally, with a greater proximal reaction. The standard method failed to show a correlation with the experimental results. However, the contact change method showed an apparent correlation with granulation tissue formation. These results suggest that this new method could be used as a tool to improve future device designs.

Towards a Biohybrid Lung: Endothelial Cells Promote Oxygen Transfer through Gas Permeable Membranes.

In patients with respiratory failure, extracorporeal lung support can ensure the vital gas exchange via gas permeable membranes but its application is restricted by limited long-term stability and hemocompatibility of the gas permeable membranes, which are in contact with the blood. Endothelial cells lining these membranes promise physiological hemocompatibility and should enable prolonged application. However, the endothelial cells increase the diffusion barrier of the blood-gas interface and thus affect gas transfer. In this study, we evaluated how the endothelial cells affect the gas exchange to optimize performance while maintaining an integral cell layer. Human umbilical vein endothelial cells were seeded on gas permeable cell culture membranes and cultivated in a custom-made bioreactor. Oxygen transfer rates of blank and endothelialized membranes in endothelial culture medium were determined. Cell morphology was assessed by microscopy and immunohistochemistry. Both setups provided oxygenation of the test fluid featuring small standard deviations of the measurements. Throughout the measuring range, the endothelial cells seem to promote gas transfer to a certain extent exceeding the blank membranes gas transfer performance by up to 120%. Although the underlying principles hereof still need to be clarified, the results represent a significant step towards the development of a biohybrid lung.

Three-Dimensional Printing and Angiogenesis: Tailored Agarose-Type I Collagen Blends Comprise Three-Dimensional Printability and Angiogenesis Potential for Tissue-Engineered Substitutes.

Three-dimensional (3D) bioprinting is a promising technology for manufacturing cell-laden tissue-engineered constructs. Larger tissue substitutes, however, require a vascularized network to ensure nutrition supply. Therefore, tailored bioinks combining 3D printability and cell-induced vascularization are needed. We hypothesize that tailored hydrogel blends made of agarose-type I collagen and agarose-fibrinogen are 3D printable and will allow the formation of capillary-like structures by human umbilical vein endothelial cells and human dermal fibroblasts. Samples were casted, incubated for 14 days, and analyzed by immunohistology and two-photon laser scanning microscopy. The 3D printability of the hydrogel blends was examined using a drop-on-demand printing system. The rheological behavior was also investigated. Substantial capillary network formation was observed in agarose-type I collagen hydrogel blends with concentrations of 0.2% or 0.5% collagen and 0.5% agarose. Furthermore, storage moduli of agarose-collagen blends were significantly increased compared to those of the corresponding single components (448 Pa for 0.5% agarose, 148 Pa for 0.5% collagen, and 1551 Pa for 0.5% agarose-0.5% collagen). Neither the addition of collagen nor fibrinogen significantly impaired the printing resolution. In conclusion, we present a tailored hydrogel blend that can be printed in 3D and in parallel exhibits cell-induced vascularization capability.

An ovine in vivo framework for tracheobronchial stent analysis.

Tracheobronchial stents are most commonly used to restore patency to airways stenosed by tumour growth. Currently all tracheobronchial stents are associated with complications such as stent migration, granulation tissue formation, mucous plugging and stent strut fracture. The present work develops a computational framework to evaluate tracheobronchial stent designs in vivo. Pressurised computed tomography is used to create a biomechanical lung model which takes into account the in vivo stress state, global lung deformation and local loading from pressure variation. Stent interaction with the airway is then evaluated for a number of loading conditions including normal breathing, coughing and ventilation. Results of the analysis indicate that three of the major complications associated with tracheobronchial stents can potentially be analysed with this framework, which can be readily applied to the human case. Airway deformation caused by lung motion is shown to have a significant effect on stent mechanical performance, including implications for stent migration, granulation formation and stent fracture.

Gefitinib/gefitinib microspheres loaded polyurethane constructs as drug-eluting stent coating.

One of the complications of bronchotracheal cancer is obstruction of the upper airways. Local tumor resection in combination with an airway stent can suppress intraluminal tumor (re)growth. We have investigated a novel drug-eluting stent coating for local release of the anticancer drug gefitinib. A polyurethane (PU) sandwich construct was prepared by a spray coating method in which gefitinib was embedded between a PU support layer of 200µm and a PU top layer of 50-200µm. Gefitinib was either embedded in the construct as small crystals or as gefitinib-loaded poly(lactic-co-glycolic acid) (PLGA) microspheres (MSP). The drug was incorporated in the PU constructs with high recovery (83-93%), and the spray coating procedure did not affect the morphologies of the embedded microspheres as demonstrated by scanning electron microscopy (SEM), confocal laser scanning microscopy and fluorescence microscopy analysis. PU constructs loaded with gefitinib crystals released the drug for 7-21days and showed diffusion based release kinetics. Importantly, directional release of the drug towards the top layer, which is supposed to face the tumor mass, was controlled by the thicknesses of the PU top layer. PU constructs loaded with gefitinib microspheres released the drug in a sustained manner for >6months indicating that drug release from the microspheres became the rate limiting step. In conclusion, the sandwich structure of drug-loaded PLGA microspheres in PU coating is a promising coating for airway stents that release anticancer drugs locally for a prolonged time.