Multivascular networks and functional intravascular topologies within biocompatible hydrogels

The morphologies of the circulatory and pulmonary systems are entangled physically and evolutionarily (1). In air – breathing vertebrates, these conserved and delineated vessel topologies interact to allow oxygen – dependent breathing of the entire organism (2–4). To construct and interrogate soft hydrogels containing such biomimetic and multivascular architectures, we used stereolithography (Figure S1) (5), commonly used to efficiently convert photoactive liquid resins into structured plastic parts by localized photopolymerization (6, 7). Compared to 3D extrusion printing, which deposits voxels in series (8–12), photocrosslinking can be highly parallelized via image projection to address simultaneously and independently millions of voxels in time steps. In stereolithography, xy the resolution is determined by the path of light, whereas z the resolution is dictated by light-reducing additives that absorb excess light and limit the polymerization to the desired layer thickness, thereby improving pattern fidelity. In the absence of appropriate photoabsorbent additives, the type of 3D pattern obtained by the soft hydrogel was limited in the types of patterns that can be generated (13–16) or required complex, expensive and low-throughput microscopy to improve z resolution via the multiphoton effect (17–19). However, light-blocking chemicals commonly used for photoresist structuring or for the manufacture of plastic parts, such as Sudan I, are not suitable for bioproduction because of their known genotoxic and carcinogenic characteristics (20). Therefore, we hypothesized that the identification of non-toxic light blockers for projection stereolithography could be a major advance for the architectural richness available for the design and production of widely used biocompatible hydrogels.

Here, we establish that synthetic and natural food dyes, widely used in the food industry, can be applied as powerful biocompatible photoabsorbents to enable the stereolithographic production of hydrogels containing complex and functional vascular architectures. We have identified candidate photosorbents among food additives whose absorbance spectra include visible light wavelengths that can be used for biocompatible photopolymerization. We initially sought to generate monolithic hydrogels, mainly composed of water and poly (ethylene glycol) diacrylate. [PEGDA, 6 kDa, 20 weight % (wt %)], with a 1 mm cylindrical channel oriented perpendicular to the projection axis of the light. Even the making of this trivial design can not be easily accomplished because of the diluted nature of such aqueous formulations, in which the small mass fraction of crosslinkable groups and the longer required polymerization times result in inadvertent polymerization and solidification in the narrow voids designed to be a hollow perfusable vascular system (Figs S2 to S4).

We determined that aqueous solutions of pre-hydrogel containing tartrazine (FD & C Yellow 5, E102 yellow food coloring), curcumin (turmeric) or anthocyanin (blueberry) can each produce hydrogels with a patented container (Figs S2 to S5). In addition to these organic molecules, inorganic gold nanoparticles (50 nm), widely recognized for their biocompatibility and light attenuation properties (21), also serve as an effective photoabsorbent additive for generating perfusable hydrogels (Fig. S4).

To understand how these photoabsorbants affect the gel kinetics of photopolymerizable hydrogels, we performed photorheological characterization with short-term light exposures, indicating that these additives cause a dose-dependent delay in the induction of photocrosslinking (Figs S2D and S4E). The saturating light exposures that extend beyond the endpoint of the reaction demonstrate that the appropriate additives do not interfere with the reaction because the hydrogels have finally reached an equivalent storage modulus regardless of the concentration of additive (Figs S2D and S4E). We selected tartrazine as a photosorbent for further studies. In addition to its low toxicity in humans and its wide utility in the food industry (22), we observed that this hydrophilic dye is easily removed by washing the generated hydrogels (70% elute within 3 hours for small gels), giving almost transparent constructions suitable for imaging (Figure S2E). Part of the tartrazine can also be degraded during the polymerization, because it is known that tartrazine is sensitive to free radicals (23). Submerging gels in water or saline to remove soluble tartrazine also rinses the vascular topology and removes the pre-hydrogel solution that has not reacted. Unlike tartrazine, curcumin is lipophilic and does not wash in aqueous solution; anthocyanins have maximum absorption away from our expected 405 nm light source, which requires high concentrations for proper power; and the gold nanoparticles are physically trapped and make transmission or fluorescence microscopy impracticable (Fig. S4E).

We assessed whether this knowledge of materials could similarly confer new architectural freedoms on more advanced photoactive materials. The photoabsorbent additives are necessary and sufficient to allow the construction of vessels during thiolene staged growth photopolymerization (24) hydrogels and in a continuous liquid interface production (6) Workflow for the generation of hydrogels (Figure S5). We observed a strong stratification between the adjacent fabricated layers and a fast patterned hydrogel response to mechanical deformations (Figure S6). This easy generation of soft hydrogels with patented cylindrical vessels oriented orthogonally to the light projection axis suggests considerable design flexibility for the generation of complex vascular topologies, and the optical clarity of the resulting hydrogels involves Appropriate imaging methodologies for the characterization and validation of fluid flows.

We then studied the ability to form hydrogels containing functional intravascular topologies. We first explored chaotic mixers: intravascular topologies that homogenize fluids as a result of interactions between fluid flow and vessel geometry (25, 26). Whereas static macroscopic mixers have found great utility in industrial processes (27Because of their unparalleled efficiency, the translation of intravascular static mixers into microfluidic systems has been difficult to implement because of their complex 3D topology. To this end, we have generated monolithic hydrogels with an integrated static mixer composed of 3D twisted fin elements (150 μm thick) of alternating chirality in a 1 mm cylindrical channel. We applied laminar fluid flow to the static mixer with a low Reynolds number (0.002) and observed rapid mixing per unit length (Fig 1A) and the number of fins (Fig S7). . The elasticity and compliance of PEG-based hydrogels (Fig. S6) allowed easy generation of a 3D functional bicuspid venous valve (Fig. 1B). We have observed that valvular leaflets are dynamic, respond rapidly to antegrade and retrograde pulsatile flow, and promote the formation of stable vortices in the valvular sinuses (Fig. 1B and film S1) according to established native tissue mapping (Fig.28, 29).

Solid organs contain distinct networks of fluids, physically and chemically entangled, constituting the rich extracellular environment that characterizes multicellular life. The possibility of making such multivascular topologies in biocompatible and aqueous environments could allow a radical change in the fields of biomaterials and tissue engineering. A first objective is to develop an effective framework for designing entangled networks capable of providing suitable plans for their manufacture in hydrogels. Separate vascular networks should not establish a direct fluid connection, otherwise they would be reduced topologically to a single connected network. We find that mathematical space filling and fractal topology algorithms provide an efficient parametric language for the design of complex vascular planes and a mathematical means for the design of a second vascular architecture that does not cut the first. (Fig 2). We present a selection of hydrogels (PEGDA at 20% by weight, 6 kDa) containing entangled vascular networks based on 3D mathematical algorithms (Fig. 2, A to D): a helix surrounding an axial vessel, curves of Hilbert at 1 ° and 2 °, a cubic lattice bicontinuous (based on a Schwarz P surface) and a torus entangled with a toric knot. An infusion with colored dyes and microcomputer tomography (μCT) analysis demonstrate pattern fidelity, vascular permeability and fluidic independence between the two networks (Fig. 2, A to D and S2 film).

We sought to evaluate the efficacy of intervessel interstitial transport by measuring the oxygen supply from a source vessel to perfused human red blood cells circulating in an adjacent 3D topology. We have paved the intertwined helical topology shown in Figure 2A along a serpentine path while maintaining the distance between intervals at 300 μm (Figure 2E). Deoxygenated red blood cell infusion[Pressionpartielled'oxygen([Oxygenpartialpressure([pressionpartielled'oxygène([oxygenpartialpressure(Po₂) ≤ 40 mmHg; Oxygen saturation (So₂) ≤ 45%]in the helical channel during ventilation of the serpentine channel with humidified oxygen gas (7 kPa) caused a noticeable color change of red blood cells ranging from dark red to bright red input to the output (Fig. 2, E and F). Collected red blood cells showed significantly higher rates So₂ and Po₂ compared to deoxygenated red cells loaded at the inlet and negative control gels ventilated with humidified nitrogen (Fig. 2G and Fig. S8).

Although this serpentine spiral design demonstrates the feasibility of oxygen transport between 3D entangled arrays, we sought to introduce additional structural features of the native distal lung into a bioinspired model of alveolar morphology and transport. l & # 39; oxygen. In particular, the production of 3D hydrogels comprising branching networks and able to withstand mechanical distension during the cyclic ventilation of a pooled airway could make it possible to investigate the performance of pulmonary morphologies derived from the native structure (30) and could provide a complete workflow for the development and review of new functional topologies. In recent decades, alveolar morphology has been approached mathematically as 3D mosaics of space-filling polyhedra (31–34). However, the translation of these ideas into useful shots has remained non-trivial because of the need for efficient space-filling mosaics and a cumbersome vascular system that closely follows the curvature of the 3D airway topography . Our solution is to calculate a 3D topological shift of the airways (by moving each side in its normal local direction) and to use the new surface as a template on which a vascular skeleton is constructed. With this approach, we have developed a bioinspired alveolar model with an enveloping vasculature from 3D tessellations of the Weaire-Phelan foam topology (35) (Fig. 3 and Fig. S9). Although the basic units of the Weaire-Phelan foam are convex polyhedra (Fig. S9), 3D mosaics can produce a surface containing both convex and concave regions reminiscent of native alveolar air sacs (Fig.30) with a shared airway atrium supporting alveolar buds (Fig. 3A). We extended the air surface of the manifold in the normal direction, the removed faces and the sheathed edges in a polygonal mesh smoothed to form a highly branched vascular network (containing 185 vessel segments and 113 fluidic connection points) which surrounds the airways and follows their curvature (Fig. S9B).

We printed hydrogels (PEGDA at 20% by weight, 6 kDa) with alveolar model topology at a vlew resolution of 5 μl and a print time of 1 hour (Fig. 3B). Cyclic ventilation of the pooled airways with humidified oxygen (10 kPa, 0.5 Hz) resulted in visible distension and apparent change in curvature of the concave airways (Figure S9C). Perfusion of deoxygenated red blood cells at the entry of the blood vessels (10-100 μm / min) during cyclical ventilation resulted in observable compression and clearance of red blood cells from vessels adjacent to concave regions of the respiratory tract ( Fig. 3, B and C). By observing diluted red blood cell fluxes in the early stages of infusion, we also found that cyclic compression of red blood vessel vessels – triggered by concave airway regions at each inflation cycle – acts as switching valves to redirect fluid flows to neighboring vessel segments (film S3). . We implemented a simplified 2D computer model of airway inflation (Fig S9D), which predicts anisotropic distension of the airways and compression of adjacent blood vessels, corresponding to the local curvature (Fig. S9E). In addition, analysis from a 3D computer model supports anisotropic distension of the concave regions of the airways during inflation (Fig. 3D). Although the volume of the alveolar model hydrogel (0.8 ml) is <25% of that of the serpentine-helix model (3.5 ml), we measured similar oxygenation yields for both models (Figure 3E). Our data suggest that branching topology, hydrogel distension and reorientation of fluid flow during ventilation may stimulate intravascular mixing and allow faster volumetric absorption of oxygen by red blood cells well. mixed. Vascular constriction during respiration has already been described as an important mechanism for fluid control in mammalian lungs (36), and we provide here a way to update these ideas in completely defined and biocompatible materials and in aqueous environments.

To extend this work to a consistent approximation of the scalable mimetic design of the lung, we must consolidate the location of the vascular input, the vascular output and the aeration duct, so that the sub – Distal lung units can be populated at the ends of the multi-scale branching architecture. Therefore, in a given computational volume, we first derive a branch airway (Fig. 3F). Then, the median lines of the input and output blood vessel networks grow 180 degrees to each other and are topologically offset from the airways, and the blood vessels pass through the blood vessels. The end of all the daughter branches. The last step is to populate the extremities of each distal lung with an alveolar unit cell (Fig. 3G and S4 film) whose enveloping vasculature (containing 354 vessel segments and 233 points of fluidic branching) is itself an anisotropic tessellation of Voronoi surface along a topological shift. local airways (Figs S9, F and G). We found that hydrogels (20% by weight of 6 kDa PEGDA) could withstand more than 10,000 ventilation cycles (at 24 kPa and at a frequency of 0.5 Hz) for 6 hours during the RBC infusion. and when switching the incoming gas between humidified oxygen and humidified nitrogen. (Fig 3, H to J). The color-filtered views of the early stages of red cell infusion (Fig. 3I) indicate that ventilation promotes mixing of red blood cells and bidirectional flows within selected vessel segments near the midpoint of the pulmonary subunit. distal (film S4).

We are using our custom tissue engineering stereolithography (SLATE) apparatus to demonstrate the production of tissue constructs containing mammalian cells (Figs S1, S10 and S11 and film S5). Pulmonary mimetic architectures may also be populated with human lung fibroblasts in most of the interstitial space and human epithelial cells in the airways (Fig. S12), which may facilitate the development of the lungs. a hydrogel analogue of a laboratory on laboratory flea lung design (37). Finally, we subjected human primary mesenchymal stem cells (HMSC) to the manufacture of SLATE (with mixtures of PEGDA and gelatin methacrylate) and showed that the cells contained in cylindrical hydrogels remain viable and can undergo differentiation. osteogenic (Fig S13D). In tissue cultures of hMSCs associated with multi-week perfusion tissues with osteogenic differentiation media, CSMs labeled with an osteogenic marker were visible throughout the gel (Figure S14). These studies indicate that SLATE manufacturing enables rapid biofabrication, maintains the viability of mammalian cell lines, supports normal function and differentiates primary human stem cells, and provides an experimentally exploitable way to explore stem cell differentiation. function of soluble factor release via vascular perfusion.

We then sought to establish the utility of this process for the manufacture of structurally complex and functional tissues for therapeutic transplantation. In particular, the liver is the largest solid organ of the human body. It performs hundreds of essential tasks in a way that is dependent on its structural topology. We have created complex structural features in the hydrogel in the expanded design space offered by SLATE for the assembly of multimaterial liver tissue. Bi-printed single-celled tissues and bi-printed hydrogel carriers containing hepatocyte aggregates were made (Fig. 4, A-C). The albumin promoter activity of the aggregate-loaded tissue carriers was 60-fold greater than that of implanted tissues containing single cells (Fig. 4, B and C). In addition, during the global examination of tissues after resection, the hydrogel-bearing tissues seemed to integrate better with the host tissue and blood (Fig. 4D). Despite the improvement in the usefulness of hepatic aggregates over individual cells, the size of the aggregates imposes significant architectural limitations on 3D printing because the aggregates are larger in size than the of our lowest voxel resolution (50 μm). To address these design constraints, we constructed a more advanced carrier capable of delivering hepatic aggregates into a natural fibrin gel, with a vascular compartment that can be seeded with endothelial cells and incorporating anchors. Structural hydrogels to physically retain rather than chemically the fibrin gel. and facilitate remodeling between the graft and the host tissue (Fig. 4E and Fig. S15). Microchannel arrays were seeded with human umbilical vein endothelial cells (HUVEC) because our previous studies had shown that the inclusion of endothelial cords improved tissue grafting (38). We then assessed whether optimized biological liver tissue could survive transplantation in a model of chronic rodent liver injury. After 14 days of engraftment in mice with chronic liver injury, hepatic hydrogel transporters showed albumin promoter activity indicating survival of functional hepatocytes (Figure 4F) . Immunohistological characterization revealed the presence of hepatic aggregates adhering to printed hydrogel components that stained positively for the cytokeratin-18 marker (Figure 4, F and G). Further characterization by coarse examination and higher magnification images of stained slides with hematoxylin and eosin (H & E) indicated the presence of host blood in the explanted tissues. Immunostaining using a monoclonal antibody directed against Ter-119 confirmed the erythroid identity of the cells in the microvessels adjacent to the hepatic microaggregates in the explanted tissues (Figure 4G, right). This work provides an approach to address long-standing design limitations in tissue engineering that have hindered advances in preclinical studies.

Nous avons identifié des colorants alimentaires facilement disponibles qui peuvent servir de photoabsorbants puissants pour la production biocompatible et cytocompatible d’hydrogels contenant des topologies vasculaires fonctionnelles pour des études sur les mélangeurs de fluides, les valves, le transport intra-vasculaire, la délivrance de nutriments et la prise d’hôte. Avec notre processus stéréolithographique, il existe un potentiel pour un contrôle simultané et orthogonal de l’architecture tissulaire et des biomatériaux pour la conception de tissus régénératifs.