Fibroblasts mediate endothelium response to angiogenic cues in a newly developed 3D stroma engineered model.

Three-dimensional stroma engineered models would enable fundamental and applicative studies of human tissues interaction and remodeling in both physiological and pathological conditions. In this work, we propose a 3D vascularized stroma model to be used as in vitro platform for drug testing. A pullulan/dextran-based porous scaffold containing pre-patterned microchannels of 100 µm diameter is used for co-culturing of fibroblasts within the matrix pores and endothelial cells to form the lumen. Optical clearing of the constructs by hyperhydration allows for in-depth imaging of the model up to 1 mm by lightsheet and confocal microscopy. Our 3D vascularized stroma model allows for higher viability, metabolism and cytokines expression compared to a monocultured vascular model. Stroma-endothelium cross-talk is then investigated by exposing the system to pro and anti-angiogenic molecules. The results highlight the protective role played by fibroblasts on the vasculature, as demonstrated by decreased cytotoxicity, restoration of nitric oxide levels upon challenge, and sustained expression of endothelial markers CD31, vWF and VEGF. Our tissue model provides a 3D engineered platform for in vitro studies of stroma remodeling in angiogenesis-driven events, known to be a leading mechanism in diseased conditions, such as metastatic cancers, retinopathies and ischemia, and to investigate related potential therapies.

Engineered human liver based on pullulan-dextran hydrogel promotes mice survival after liver failure.

Liver tissue engineering approaches aim to support drug testing, assistance devices, or transplantation. However, their suitability for clinical application remains unsatisfactory. Herein, we demonstrate the beneficial and biocompatible use of porous pullulan-dextran hydrogel for the self-assembly of hepatocytes and biliary-like cells into functional 3D microtissues. Using HepaRG cells, we obtained 21 days maintenance of engineered liver polarity, functional detoxification and excretion systems, as well as glycogen storage in hydrogel. Implantation on two liver lobes in mice of hydrogels containing 3800 HepaRG 3D structures of 100 â€‹µm in diameter, indicated successful engraftment and no signs of liver toxicity after one month. Finally, after acetaminophen-induced liver failure, when mice were transplanted with engineered livers on left lobe and peritoneal cavity, the survival rate at 7 days significantly increased by 31.8% compared with mice without cell therapy. These findings support the clinical potential of pullulan-dextran hydrogel for liver failure management.

Spatial-Controlled Coating of Pro-Angiogenic Proteins on 3D Porous Hydrogels Guides Endothelial Cell Behavior.

In tissue engineering, the composition and the structural arrangement of molecular components within the extracellular matrix (ECM) determine the physical and biochemical features of a scaffold, which consequently modulate cell behavior and function. The microenvironment of the ECM plays a fundamental role in regulating angiogenesis. Numerous strategies in tissue engineering have attempted to control the spatial cues mimicking in vivo angiogenesis by using simplified systems. The aim of this study was to develop 3D porous crosslinked hydrogels with different spatial presentation of pro-angiogenic molecules to guide endothelial cell (EC) behavior. Hydrogels with pores and preformed microchannels were made with pharmaceutical-grade pullulan and dextran and functionalized with novel pro-angiogenic protein polymers (Caf1-YIGSR and Caf1-VEGF). Hydrogel functionalization was achieved by electrostatic interactions via incorporation of diethylaminoethyl (DEAE)-dextran. Spatial-controlled coating of hydrogels was realized through a combination of freeze-drying and physical absorption with Caf1 molecules. Cells in functionalized scaffolds survived, adhered, and proliferated over seven days. When incorporated alone, Caf1-YIGSR mainly induced cell adhesion and proliferation, whereas Caf1-VEGF promoted cell migration and sprouting. Most importantly, directed cell migration required the presence of both proteins in the microchannel and in the pores, highlighting the need for an adhesive substrate provided by Caf1-YIGSR for Caf1-VEGF to be effective. This study demonstrates the ability to guide EC behavior through spatial control of pro-angiogenic cues for the study of pro-angiogenic signals in 3D and to develop pro-angiogenic implantable materials.

In Vitro Strategies to Vascularize 3D Physiologically Relevant Models.

Vascularization of 3D models represents a major challenge of tissue engineering and a key prerequisite for their clinical and industrial application. The use of prevascularized models built from dedicated materials could solve some of the actual limitations, such as suboptimal integration of the bioconstructs within the host tissue, and would provide more in vivo-like perfusable tissue and organ-specific platforms. In the last decade, the fabrication of vascularized physiologically relevant 3D constructs has been attempted by numerous tissue engineering strategies, which are classified here in microfluidic technology, 3D coculture models, namely, spheroids and organoids, and biofabrication. In this review, the recent advancements in prevascularization techniques and the increasing use of natural and synthetic materials to build physiological organ-specific models are discussed. Current drawbacks of each technology, future perspectives, and translation of vascularized tissue constructs toward clinics, pharmaceutical field, and industry are also presented. By combining complementary strategies, these models are envisioned to be successfully used for regenerative medicine and drug development in a near future.

Overcoming the Design Challenge in 3D Biomimetic Hybrid Scaffolds for Bone and Osteochondral Regeneration by Factorial Design.

Scaffolds for bone regeneration have been engineered by a plethora of manufacturing technologies and biomaterials. However, the performance of these systems is often limited by lack of robustness in the process design, that hampers their scalability to clinical application. In the present study, Design of Experiment (DoE) was used as statistical tool to design the biofabrication of hybrid hydroxyapatite (HA)/collagen scaffolds for bone regeneration and optimize their integration in a multilayer osteochondral device. The scaffolds were synthesized via a multi-step bioinspired process consisting in HA nano-crystals nucleation on the collagen self-assembling fibers and ribose glycation was used as collagen cross-linking method to modulate the mechanical and physical properties. The process design was performed by selecting hydrogel concentration, HA/collagen ratio and cross-linker content as key variables and the fabrication was carried out basing on a full factorial design. Scaffold performances were tested by evaluating porosity, swelling ratio, degradation rate and mechanical behavior as model output responses while physicochemical properties of the constructs were evaluated by TGA, ICP, FT-IR spectroscopy, and XRD analysis. Physicochemical characterizations confirmed the nucleation of a biomimetic inorganic phase and the interaction of the HA and collagenic components. The DoE model revealed a significant interaction between HA content and collagen cross-linking in determining porosity, swelling and mechanical properties of the scaffolds. The combined effect of hydrogel concentration and mineral phase played a key role on porosity and swelling while degradation resulted to be mainly affected by the HA loading and ribose content. The model was then used to determine the suitable input parameters for the synthesis of multi-layer scaffolds with graded mineralization rate, that can be used to mimic the whole cartilage-bone interface. This work proved that experimental design applied to complex biofabrication processes represents an effective and reliable way to design hybrid constructs with standardized and tunable properties for osteochondral tissue engineering.

Rapid Fabrication of Membrane-Integrated Thermoplastic Elastomer Microfluidic Devices.

Leveraging the advantageous material properties of recently developed soft thermoplastic elastomer materials, this work presents the facile and rapid fabrication of composite membrane-integrated microfluidic devices consisting of FlexdymTM polymer and commercially available porous polycarbonate membranes. The three-layer devices can be fabricated in under 2.5 h, consisting of a 2-min hot embossing cycle, conformal contact between device layers and a low-temperature baking step. The strength of the FlexdymTM-polycarbonate seal was characterized using a specialized microfluidic delamination device and an automated pressure controller configuration, offering a standardized and high-throughput method of microfluidic burst testing. Given a minimum bonding distance of 200 µm, the materials showed bonding that reliably withstood pressures of 500 mbar and above, which is sufficient for most microfluidic cell culture applications. Bonding was also stable when subjected to long term pressurization (10 h) and repeated use (10,000 pressure cycles). Cell culture trials confirmed good cell adhesion and sustained culture of human dermal fibroblasts on a polycarbonate membrane inside the device channels over the course of one week. In comparison to existing porous membrane-based microfluidic platforms of this configuration, most often made of polydimethylsiloxane (PDMS), these devices offer a streamlined fabrication methodology with materials having favourable properties for cell culture applications and the potential for implementation in barrier model organ-on-chips.