Patent Description:
Solid organs transport fluids through distinct vascular networks that are biophysically and biochemically entangled, creating complex three-dimensional (3D) transport regimes that are difficult to produce and study. The morphologies of the circulatory and pulmonary systems are physically and evolutionarily entangled. In air-breathing vertebrates, these bounded and conserved vessel topologies interact to enable the oxygen-dependent respiration of the entire organism.

<CIT> describes a hydrogel matrix comprising a plurality of layers, each layer comprising a cross-linked polymer network formed from a photosensitive polymer; a first elongated void in the hydrogel matrix providing a first tubular channel; a second elongated void in the hydrogel matrix providing a second tubular channel; wherein the first tubular channel and the second tubular channel are perfusable; wherein the first tubular channel does not intersect the second tubular channel; and wherein the second tubular channel interpenetrates the first tubular channel.

The present invention is defined by the independent claims, while the dependent claims concern the preferred embodiments. Any "embodiment" or "example" which is disclosed in the description but is not covered by the claims should be considered as presented for illustrative purpose only. The following detailed description describes the preparation of monolithic transparent hydrogels by stereolithographic production. The cytocompatible hydrogels contain intricate and functional vascular architectures, and have functional vascular topologies for studies of fluid mixers, valves, intervascular transport, nutrient delivery, and host engraftment. These intravascular and multivascular designs are created with photopolymerizable hydrogels by using food dye additives as biocompatible photoabsorbers for the projection stereolithography. The stereolithographic process enables simultaneous and orthogonal control over tissue architecture and biomaterials for the design of regenerative tissues. The entangled vascular networks formed from space-filling mathematical topologies enable creation of complex geometries.

According to the claimed invention, a device has a photopolymerizable hydrogel matrix including a photoabsorber and a void architecture in the matrix, having a first vessel architecture and a second vessel architecture that are each tubular and branching, where the first and second vessel architectures are fluidically independent from each other, and wherein one or both of the first vessel architecture and second vessel architecture is formed from a model based on a tessellation of polyhedra.

Implementations can include one or more of the following. The device allows at least <NUM>% of visible light incident on the device to pass through and allow imaging of the device. The photoabsorber has been at least partially washed out of the device. The photoabsorber is degradable independent of any degradation of the remaining hydrogel matrix. The photoabsorber is degradable by chemical or physical processes. The photoabsorber is photobleachable, or removable by boiling. The model is based on a tessellation of polyhedra represents blood vessels. The model is based on a tessellation of polyhedra represents an airway. In various embodiments, the multi-vascular void architecture has a feature of a functional valve. In various embodiments, the valve is a bicuspid valve, tricuspid valve, monocuspid valve, or Tesla valve. In various embodiments, the multi-vascular void architecture has a feature of a fluidic static mixer. In various embodiments, the fluidic static mixer has between two and fifty fin elements. The feature is positionable at any position in the multi-vascular void architecture. The device is monolithic. The hydrogel matrix is biodegradable. The device is produced by additive manufacturing. The first vessel architecture and the second vessel architecture are entangled. The first vessel architecture is ensheathed by the second vessel architecture. The hydrogel matrix is porous. The device is implantable. The photoabsorber is hydrophilic. The photoabsorber is one of a food dye, tartrazine, Sunset Yellow FCF (Yellow No. <NUM>), Brilliant Blue FCF (FD&C Blue No. <NUM>), indigo carmine (FD&C Blue No. <NUM>), Fast Green FCF (FD&C Green No. <NUM>) anthocyanins, anthocyanidin, erythrosine (FD&C Red No.<NUM>), Allura Red AC (FD&C Red No. <NUM>), riboflavin (Vitamin B2, E101, E101a, E106), ascorbic acid (vitamin C), Quinoline Yellow WS, carmoisine (azorubine), Ponceau 4R (E124), Patent Blue V (E131), Green S (E142), Yellow <NUM> (E107), Orange GGN (E111), Red <NUM> (E128), caramel color, phenol red, methyl orange, <NUM>-nitrophenol, and NADH disodium salt. The photoabsorber is hydrophobic. The photoabsorber is one of curcumin (E100), turmeric, alpha carotene, beta carotene, canthaxanthin (keto-carotenoid), cochineal extract, paprika, saffron, ergocalciferol (vitamin D2), cholecalciferol (vitamin D3), Citrus Red <NUM>, annatto extract, and Lycopene. Three or more vessel architectures fluidically independent from each other and from the first and second vessel architectures.

In some embodiments, a pre-polymerization solution has a photosensitive polymer, and a biocompatible, light-absorbing additive material suitable to control light penetration, where the additive material is at least partially removable from a solid formed of the pre-polymerization solution. The solution include plant, bacterial, or mammalian cells.

A device according to the claimed invention can be formed by a method of fabricating a 3D hydrogel construct by creating a 3D model of the construct based on a tessellation of polyhedra having a number of faces connected by edges and vertices. Subsequently generating a vascular component of the model by removing the faces and optionally the vertices of the polyhedra, and using the remaining topology as a vascular skeleton, and skinning the skeleton with a polygonal mesh and then smoothing the result to produce cylindrical channels along the edges of the model with intervessel junctions located at each vertex location. Then generating an airway component of the model by scaling the faces of the 3D model along local face normals such that the airway is nested inside the vasculature (vascular) component, forming independent fluidic connections to the vasculature and to the airway, and performing a Boolean subtraction from a solid volume, and combining the vascular component and the airway component of the model.

In some embodiments, fabricating a hydrogel alveolar construct includes fusing multiple spheres in a radially symmetric fashion to create an airway topology, offsetting the airway topology to generate a vascular surface topology, tessellating, skelatonizing, and skinning the vascular surface topology to produce a Voronoi vascular topology, where tessellating comprises performing a tessellation of polyhedra, and performing a Boolean subtraction of the original airway and the tessellated Voronoi vascular topology from a solid volume to generate a final 3D model for additive manufacturing. The tessellation of polyhedra is a Weaire-Phelan space-filling foam model of two dodecahedron and six tetrakaidecahedron cells.

In some embodiments, a method of manufacturing a hydrogel matrix construct includes creating a 3D model of the hydrogel matrix construct using a design software, where the 3D model of the hydrogel matrix construct comprises a first computational algorithm that yields a first tubular channel network in the hydrogel matrix construct, and a second computational algorithm, different from the first computational algorithm, that yields a second tubular channel network in the hydrogel matrix, where the first and second tubular channel networks are two independent, entangled vascular networks. Then by converting the 3D model to a format suitable for use in a 3D additive manufacturing software to yield a formatted 3D model to be generated using a 3D additive manufacturing machine, and directing the additive manufacturing machine to generate the model.

Implementations can include one or more of the following. The computational algorithm is a tessellation of polyhedra. Supplying a pre-polymerization solution to the 3D additive manufacturing machine where the pre-polymerization solution includes a photoabsorber. The pre-polymerization solution comprises one or more bacterial, mammalian, and plant cells. Lining the first tubular channel network or second tubular channel network with cells. Embedding the hydrogel matrix with cells. The cells are one or more of liver, lung, bone, endothelial, cardiac, pancreas, kidney, epithelial, muscle, cartilage, stem cells, skin, or eye cells. Delivering heat while the 3D additive manufacturing machine generates the 3D model. Heat is added via a silicone heater, heat lamps, or infrared LEDs. Enclosing the 3D model in a heating enclosure during generation of the 3D model.

Also described herein, but not forming part of the claimed invention, is a method of treating a subject in need thereof comprising implanting any of the devices into the subject, and a device comprising multiple joined subunits, where each subunit is any of the devices.

Advantages of the methods and materials of this description include use of stereolithography to efficiently convert photoactive liquid resins into structured plastic parts through localized photopolymerization. Compared to extrusion 3D printing, which deposits voxels in a serial fashion, photocrosslinking can be highly parallelized via image projection to simultaneously and independently address millions of voxels per time step. In stereolithography, xy-resolution is determined by the light path while z-resolution is dictated by light attenuating additives that absorb excess light and confine the polymerization to the desired layer thickness, thereby improving pattern fidelity. In the absence of suitable photoabsorber additives, 3D photopatterning of soft hydrogels can be been limited in the types of patterns that can be generated, or has implemented complex, expensive, and low-throughput microscopy. Common light blocking chemicals utilized for photoresist patterning or plastic part fabrication are not suitable for biomanufacturing due to their known genotoxic and carcinogenic characteristics. The use of non-toxic light blockers for projection stereolithography provides a major advance to the architectural richness available for the design and generation of biocompatible hydrogels.

Devices can be formed of a photopolymerizable hydrogel that includes a photoabsorber. During fabrication, the photoabsorber can be removed from the device, whether by being washed out, boiled out, or is degradable by another physical or chemical process that allows the photoabsorber to degrade independent of any other constituents in the hydrogel matrix. The resulting biocompatible device is biodegradable and porous, and suitable for implant into a patient, or for scientific studies.

The devices are formed to include complex internal structures while still being monolithic. These internal structures form a void architecture in the hydrogel matrix that takes the form of first and second vessel architectures that are each tubular and branching, but fluidically independent from each other, e.g., a multi-vascular void architecture. The first and second vessel architectures can be complex, entangled, or the first vessel architecture ensheathed by the second vessel architecture. The multi-vascular void architecture can take many forms, whereby one or both of the first and second vessel architectures is formed from a model based on a tessellation of polyhedra that can represent blood vessels or an airway.

<FIG> describe the validation of monolithic hydrogels generated with a simple functional intravascular topology, in this case a static mixer with fins that homogenizes fluid flows entering the mixer. <FIG> is a schematic of a monolithic hydrogel's interior integrated static mixer composed of 3D twisted fin elements of alternating chirality. <FIG> pictures a fabricated hydrogel having the integrated fin element configuration of <FIG> that is <NUM> thick inside a <NUM> cylindrical channel. Two fluid streams incident on the channel (for example, laminar fluid streams at a low Reynolds number (e.g., Re = <NUM>)) are intermixed by interaction with the fins, as illustrated in the fluorescent image of <FIG>. The fluorescence imaging of <FIG> matches a computational model of flow (<FIG>). The laminar fluids' interaction with the static mixer varies with fin number, as shown in <FIG>'s stitched fluorescence images of printed monolithic hydrogels (<NUM> wt% <NUM> kDa) containing static mixers with <NUM> to <NUM> fin elements and perfused with fluorescent dyes.

<FIG> further quantifies the mixing efficiency of perfusable static mixers formed from the monolithic hydrogels described herein. <FIG> shows the normalized fluorescence intensity profiles at the outlet of a static mixer with <NUM> or <NUM> fins (the solid line represents the average intensity over a <NUM> length of the outlet immediately following the fins and the shaded regions represent standard deviation). <FIG> shows the average intensity data from <FIG> replotted to demonstrate mixing ratio calculations for static mixers. <FIG> shows a plot of mean mixing ratio by number of fins. These data illustrate that the fabricated hydrogel devices with their intravascular topology have predictable functionality.

<FIG> illustrates the use of the fabricated hydrogel devices in the form of a 3D functional bicuspid venous valve. The valve leaflets are dynamic, rapidly respond to pulsatile anterograde and retrograde flows, and promote the formation of stable mirror image vortices in the valve sinuses according to established mappings of native tissue. The Particle Image Velocimetry (PIV) image at the right of <FIG> demonstrates stable mirror image vortices in the sinus region behind open valve leaflets.

<FIG> shows example compliance measurement results of horizontal channels fabricated within the hydrogels. At the left and center, vessel distension was measured upon application of pneumatic air pressure (hydrogel composition: <NUM> wt% <NUM> kDa PEGDA; channel diameter: <NUM>; channel length: <NUM>) both at rest and inflated to <NUM> mmHg. White dashed lines represent walls of the imaged channel. On the right, the graphed data indicates that compliance of vessels was found to be dependent on composition of pre-hydrogel solution. Solutions composed of lower polymer molecular weight and higher wt% resulted in hydrogels with lower compliance. The data of <FIG> illustrate the viability of the hydrogel devices for use in multivascular systems.

Solid organs contain distinct fluid networks that are physically and chemically entangled. Separate vascular networks generally do not make a direct fluid connection to prevent being topologically reduced to a single connected network. Multivascular topologies within biocompatible and aqueous environments enables design of entangled networks that are suitable blueprints for fabrication within the hydrogels.

Mathematical space-filling and fractal topology algorithms provide an efficient parametric language to design complex vascular blueprints, and a mathematical means to design a second vascular architecture that does not intersect the first. <FIG> illustrate adaptations of mathematical space-filling curves into entangled vessel topologies within hydrogels (<NUM> wt% PEGDA, <NUM> kDa), which are not according to the claimed invention and thus to be seen as reference-embodiments. These include a helix surrounding an axial vessel (<FIG>), <FIG> and <FIG> interpenetrating Hilbert curves (<FIG>), a bicontinuous cubic lattice (based on a Schwarz P surface, <FIG>), and a torus entangled with a torus knot (<FIG>). Perfusion with colored dyes and micro-computed tomography (µCT) analysis demonstrate pattern fidelity, vascular patency, and fluidic independence between the two networks.

The efficiency of the intervascular interstitial transport was evaluated by measuring the delivery of oxygen from a source vessel to perfused human red blood cells (RBCs) flowing in an adjacent 3D topology. <FIG> illustrate the tessellation of the axial vessel and its encompassing helix along a serpentine pathway while maintaining the intervessel distance at <NUM>. The top-down photograph of <FIG> (with magnified view shown in <FIG>) is of a fabricated hydrogel with oxygen and RBC delivery to respective vessels. Perfusion of deoxygenated RBCs (pO<NUM> ≤ <NUM> mmHg; sO<NUM> ≤ <NUM>%) into the helical channel during ventilation of the serpentine channel with humidified gaseous oxygen (<NUM> kPa) caused a noticeable color change of RBCs from dark red at the inlet to bright red at the outlet.

Collection of perfused RBCs showed significantly higher oxygen saturation and oxygen partial pressure compared to deoxygenated RBCs loaded at the inlet, and compared to negative control gels ventilated with humidified nitrogen gas. This is illustrated by <FIG>, for which perfused RBCs were collected at the outlet and quantified for sO<NUM> and pO<NUM>. Compared to deoxygenated RBCs perfused at the inlet (dashed line), oxygen flow increased sO<NUM> and pO<NUM> of perfused RBCs compared to nitrogen flow negative control.

Oxygenation of perfused human red blood cells in serpentine-helix gels are affected by flow rates. The experimental results of <FIG> show that slower perfusion of deoxygenated RBCs through the vascular network (during gaseous oxygen flow) resulted in higher sO<NUM> (B) and pO<NUM> of the collected RBCs. The dashed line indicates the measured sO<NUM> and pO<NUM>, respectively, of deoxygenated RBCs used for the perfusion. <FIG> shows that the fraction of oxygenated hemoglobin increased significantly with slower flow rates. FHHb = fractional deoxyhemoglobin; FMetHb = fractional methemoglobin; FCOHb = fractional carboxyhemoglobin; FO<NUM>Hb = fractional oxyhemoglobin. These data illustrate the viability of the hydrogel devices for use as channels for carrying RBCs.

The following establishes the utility of the described processes for fabricating structurally complex and functional replacement tissues for therapeutic transplantation. The liver is the largest solid organ in the human body, carrying out hundreds of essential tasks in a manner that is thought to be intimately dependent upon its structural topology, and is the organ used for these studies.

Complex structural features in hydrogels were created within the expanded design space imparted by SLATE (described below) to assemble multi-material liver tissues. Also this embodiment is to be considered as a reference-embodiment, not according to the claimed invention. Bioprinted single-cell tissues and bioprinted hydrogel 'carriers' containing hepatocyte aggregates were fabricated (<FIG>). Albumin promoter activity was enhanced in hydrogel carriers containing hepatic aggregates after implantation in nude mice. Data from all time points for each condition are shown in <FIG> with cumulative bioluminescence for each condition shown in <FIG>. The albumin promoter activity of tissue carriers loaded with aggregates was enhanced by more than sixty-fold compared to implanted tissues containing single cells.

<FIG> shows gross examination of tissues after resection, where hydrogel carrier tissues appeared to have more integration with host tissue and blood. Despite the improved utility of hepatic aggregates over single cells, aggregate size puts significant architectural limitations on 3D printing because aggregates are larger in size than typical lowest voxel resolution (<NUM>). To accommodate these design constraints, a more advanced carrier which could deliver hepatic aggregates within natural fibrin gel was used, having a vascular compartment that can be seeded with endothelial cells, and incorporates structural hydrogel anchors to physically, rather than chemically, retain the fibrin gel and facilitate remodeling between the graft and host tissue. Referring to <FIG>, microchannel networks were seeded with human umbilical vein endothelial cells (HUVECs) while hydrogel anchors physically entrap fibrin gel containing the hepatocyte aggregates (Hep) observed via confocal microscopy.

The bioengineered liver tissues survive transplantation in a model of chronic liver injury. After <NUM> days of engraftment in mice with chronic liver injury, hepatic hydrogel carriers exhibited albumin promoter activity indicative of surviving functional hepatocytes (<FIG>). Immunohistological characterization revealed the presence of hepatic aggregates adhered to printed hydrogel components that stained positively for the hepatocyte marker cytokeratin-<NUM> (<FIG>). Further characterization through gross examination and higher magnification images of hematoxylin and eosin stained slides indicated the presence of host blood in explanted tissues. Immunostaining for Ter-<NUM> confirmed erythroid identity of cells in microvessels immediately juxtapositional to hepatic microaggregates in explanted tissues (<FIG>, right). These illustrate the efficacy of the hydrogel devices for use as implantable devices.

Additional structural features of native distal lung are included in the monolithic bioinspired hydrogel models of alveolar morphology and oxygen transport. 3D hydrogels that contain branching networks and that can support mechanical distension during cyclic ventilation of a pooled airway could enable investigations of the performance of lung morphologies derived from native structure and could provide a broad workflow to develop and interrogate new functional topologies.

Complex morphology are approximated mathematically as 3D space-filling tessellations of polyhedra, which is a morphology used in devices according to the claimed invention. To manufacture a hydrogel matrix construct that models alveolar morphology and oxygen transport a 3D model of the hydrogel construct is created using a design software. The construct includes a first computational algorithm that yields a first tubular channel network and a second computational algorithm, different from the first computational algorithm, that yields a second tubular channel network. The first and second tubular channel networks are two independent, entangled vascular networks (e.g., alveolar morphology and oxygen transport morphology). The 3D model is converted to a format suitable for use in a 3D additive manufacturing software to yield a formatted 3D device to be generated using a 3D additive manufacturing machine.

Steps of creating a tessellated model include fusing multiple spheres in a radially symmetric fashion to create an airway topology, offsetting the airway topology to generate a vascular surface topology, (by moving each face in its local normal direction and having the new surface serve as the template on which a new vascular skeleton is built), tessellating, skelatonizing, and skinning the vascular surface topology to produce a Voronoi vascular topology. Tessellating includes performing a tessellation of polyhedra, and a Boolean subtraction of the original airway and the tessellated Voronoi vascular topology from a solid volume to generate a final 3D model for additive manufacturing. <FIG> illustrate these details for the design of bioinspired alveoli models. In <FIG> the fundamental Weaire-Phelan polyhedra are tessellated in 3D to fill space. <FIG> illustrates the procedural derivation of the entangled alveolar model from the Weaire-Phelan tessellation. The manifold air surface is extended in the normal direction, faces removed, and edges ensheathed in a smoothed polygonal mesh to form a highly branched vascular network (containing <NUM> vessel segments and <NUM> fluidic branch points) that encloses the airway and tracks its curvature. <FIG> are photographs of the printed alveoli model, without RBC flow in the ensheathing vasculature, highlighting that the airway is smaller at rest (left) compared to the airway during inflation and hydrogel distension (right, <NUM> kPa, <NUM>). Cyclic ventilation of the pooled airway with humidified oxygen gas (<NUM> kPa, <NUM>) results in noticeable distension and an apparent change in the curvature of concave airway regions.

<FIG> is a schematic xy cross-section view of the model with convex (blue) and concave (green) regions of the airway (white), and accompanying blood vessels (red). Normal force arrows converge at concave regions. This simplified 2D computational model of airway inflation predicts anisotropic distension of the airway and compression of adjacent blood vessels corresponding to local curvature. This is shown in <FIG>, a 2D computational model of a symmetric airway surrounded by circular vessels equidistant from convex or concave regions of the airway (black edges indicate starting positions). Upon airway inflation, concave regions displace and compress adjacent blood vessels.

In <FIG> the steps of distal lung subunit model design are illustrated. The airway was first designed by fusion of spheres with inlet and outlet channels. The airway topology was offset and used as a template for vascular generation by implementing a Voronoi 3D surface tessellation, skeletonization, and skinning. A Boolean subtraction of these two topologies from a solid volume was performed which represents the final hydrogel design.

<FIG> illustrates generation of the lung- mimetic model by populating branch tips of airway and offset vascular networks, grown within a defined computational 3D bounding volume, with the distal lung subunit model.

Alveolar morphology are approximated mathematically as 3D space-filling tessellations of polyhedra that are efficiently space-filling and also replicate an ensheathing vasculature that closely tracks the curvature of the 3D airway topography. <FIG> illustrate a bioinspired alveolar model with an ensheathing vasculature from 3D tessellations of the Weaire-Phelan topology. <FIG> shows the architectural design of an alveolar model topology based on a Weaire-Phelan 3D tessellation and a topologic offset to derive an ensheathing vasculature. The cutaway view at the bottom illustrates the model alveoli with a shared airway atrium. The 3D tessellations produce a surface containing both convex and concave regions that are reminiscent of native alveolar air sacs with a shared airway atrium supporting alveolar buds.

<FIG> shows printed hydrogels (<NUM> wt% <NUM> kDa PEGDA) patterned with the alveolar model topology of <FIG> at a voxel resolution of <NUM> pL and a print time of <NUM> hr. The figure is of the printed hydrogel during RBC perfusion while the air sac is ventilated with O<NUM>. Perfusion of deoxygenated RBCs at the blood vessel inlet (<NUM>-<NUM>µL/min) during cyclic ventilation led to observable compression and RBC clearance from vessels next to concave airway regions (<FIG>). With dilute RBC streams at the early stages of perfusion the cyclic compression of RBC vessels-actuated by the concave airway regions upon each inflation cycle-acted as switching valves to redirect fluid streams to neighboring vessel segments.

Analysis from a 3D computational model supports anisotropic distension of the concave regions of the airway during inflation (<FIG>). Moreover, despite the alveolar model hydrogel (<NUM>) being more than four times smaller in volume than the serpentine-helix model (<NUM>), the result is similar oxygenation efficiencies between the two designs (<FIG>). These data suggest that branching topology, hydrogel distension, and the redirection of fluid streams during ventilation may boost intravascular mixing and allow faster volumetric uptake of oxygen by the well-mixed RBCs. These data illustrate the advantage of vascular constriction during breathing actualized in biocompatible materials and within aqueous environments.

For a scalable lung-mimetic design, the location of the vascular inlet, the vascular outlet, and the air duct are consolidated such that distal lung subunits can be populated on the tips of multiscale branching architecture. <FIG> shows a branching airway within a chosen computational bounding volume. This is an elaboration of a lung-mimetic design through generative growth of the airway, offset growth of opposing inlet and outlet vascular networks, and population of branch tips with a distal lung subunit.

The centerlines of inlet and outlet blood vessel networks are grown opposing each other across and topologically offset from the airway, and the blood vessels traverse down to the tips of all daughter branches. The tips of each distal lung are populated with an alveolar unit cell (<FIG>) whose ensheathing vasculature (containing <NUM> vessel segments and <NUM> fluidic branch points) itself is an anisotropic Voronoi surface tessellation along a topological offset of its local airway. Hydrogels (<NUM> wt% <NUM> kDa PEGDA) advantageously were found to withstand more than <NUM>,<NUM> ventilation cycles (at <NUM> kPa and a frequency of <NUM>) over <NUM> hours during RBC perfusion and while switching the inflow gas between humidified oxygen and humidified nitrogen (<FIG> shows color-filtered views of the early stages of RBC perfusion and demonstrates that ventilation promotes RBC mixing and bidirectional flows within selected vessel segments near the midpoint of the distal lung subunit.

Referring back to <FIG>, <FIG> show additional imaging and development of hepatic hydrogel carriers, not according to the claimed invention. In <FIG>, <FIG> printed hydrogel carriers accommodate primary hepatocyte aggregates (green) suspended in fibrin (blue), entangled with hydrogel anchors (purple) and has an underlying patterned vascular network (red). <FIG> shows 3D printed chamber demonstrate perfusable vasculature (red), and hydrogel anchors. <FIG> is a magnified view of highlighted region from <FIG> showing hydrogel anchors and underlying perfusable vasculature (red). <FIG> is a <NUM> cross-section view demonstrates the fibrin seeding volume (blue) and underlying vasculature (red). <FIG> is a confocal maximum intensity projection (Max IP, left) and volumetric rendering of endothelial cords (red) and hepatocytes (green).

SLATE studies indicate that SLATE fabrication supports rapid biomanufacturing, can maintain the viability of mammalian cell lines, supports the normal function and differentiation of primary human stem cells, and suggests an experimentally tractable means to explore stem cell differentiation as a function of soluble factor delivery via vascular perfusion.

<FIG> is a schematized workflow for fabrication of 3D objects by projection stereolithography, in accordance with various embodiments. Photomasks corresponding to a 3D model can be projected into a vat containing a photosensitive liquid solution. Upon completion of sequential layer-by-layer photopatterning, a 3D object is obtained. <FIG> is a 3D rendering of an example projection stereolithography apparatus <NUM>. The apparatus can include a motor <NUM> (which can be attached to a frame <NUM>), controlled by an actuator <NUM>, onto which the build platform <NUM> is attached. The back of the printer can house the motherboard <NUM> that controls the motor and performs input/output with any additional sensors or switches. The front of the printer contains a mirror <NUM> placed to reflect incident patterns from a projector <NUM> onto a dish containing the photosensitive materials. All off-white colored parts correspond to parts that were 3D printed out of consumer-grade poly(lactic acid) (PLA) plastic filament. <FIG> Schematic demonstrating photomasks (left) that are generated at different locations along the height of the 3D rook model.

The device fits entirely inside common biosafety cabinets with sufficient room for cell handling. Although SLATE fabrication is rapid (at up to <NUM>/hr with voxels of <NUM> pL), mammalian cells typically settle out of suspension within tens of seconds. To prevent cells from settling and facilitate automated bioprinting, xanthan gum (a natural, biocompatible, shear-thinning carbohydrate) is added to the pre-hydrogel solution.

SLATE demonstrates the ability to generate tissue constructs containing mammalian cells using the custom lung-mimetic architectures described above. Hydrogels composed of tens of layers can be printed in a few minutes, and mammalian cells are uniformly distributed (<FIG> show that incorporation of xanthan gum into the pre-hydrogel solution resulted in homogeneous distribution of cells along the height of the gel. <FIG> is a side view (xz plane) of a fabricated <NUM> wt% hydrogel containing HEK mCherry cells (<NUM>×<NUM><NUM> cells/mL). The experimental results in <FIG>, D show total light output of HEK Luc2P cells encapsulated in PEGDA hydrogels containing a horizontal <NUM> diameter channel at different cell densities (<FIG>) and different flow rates of luciferin substrate (<FIG>).

<FIG> illustrate rapid biomanufacturing with cryopreserved cell stocks and non-invasive characterization of cellular activity. <FIG> illustrates that cryopreserved cell stocks are thawed and immediately used in projection stereolithography to yield perfusable tissues with patterned vessel architectures. The total procedure from cell thaw to perfusion experiments takes less than <NUM> hr. Referring to <FIG>, using this workflow, fabricated tissue constructs containing mammalian cells (<NUM>×<NUM><NUM> cells/mL) expressing firefly luciferase (Luc2P) were perfused with deoxygenated or oxygenated RBCs along with luciferin substrate. Luminescence quantification (<NUM> hr) demonstrates increased light emission from cells within gels perfused with oxygenated RBCs. <FIG> is a full <NUM> hour time course of the perfusion experiment from <FIG> plotted as total light flux emitted from cells within hydrogels.

Hydrogel constructs can also be populated with human lung fibroblasts in the bulk of the interstitial space and human epithelial-like cells in the airway which could facilitate the development of a hydrogel analog of a lab-on-a-chip lung design for a monolithic lung-mimetic perfusion and ventilation tissue culture system. <FIG> illustrates a bioinspired design of an alveolar sac with surrounding pulmonary vasculature. The overall size of the model is <NUM>×<NUM>×<NUM> (x,y,z) with a smallest channel diameter of <NUM> and alveolus diameter of <NUM>. <FIG> is a photo of the fluidic connections to the printed gel for perfusion through the vascular channel and ventilation of the airway vessel. <FIG> shows hydrogels that were fabricated with IMR-<NUM> fibroblasts (<NUM>×<NUM><NUM> cells/mL) encapsulated in the bulk of the hydrogel, including the interstitial region. Ventilation of the airway under perfusion tissue culture through the vasculature (<NUM>µL/min over <NUM> days) maintained patency of the airway as seen in cross-sectional imaging after staining for nuclei (Hoechst). In <FIG>, seeded A549 epithelial-like cells (expressing H2B-mVenus, center) are attached to the airway lumen of printed hydrogels containing encapsulated fibroblasts in the interstitial zone.

<FIG>, B show histological results of a related multi-week tissue culture of hMSCs with osteogenic differentiation media, in which osteogenic marker-positive hMSCs were visible throughout the perfused single channel gels. <FIG> shows alkaline phosphatase (ALP) staining of hMSC tissue cross-section from a hydrogel that underwent osteogenic media perfusion for <NUM> days, with the vascular channel highlighted with the dotted circle (<FIG> being a close-up of the section). ALP stain was found strongly along the perimeter of the perfused channel with some positive staining throughout the bulk of the gel.

As mentioned above, synthetic and natural food dyes can be used as biocompatible photoabsorbers to enable the stereolithographic production of the hydrogels containing intricate and functional vascular architectures. Aqueous pre-hydrogel solutions containing tartrazine (yellow food coloring FD&C Yellow <NUM>, E102), curcumin (from turmeric), or anthocyanin (from blueberries) can each yield hydrogels with a patent vessel. In addition to these organic molecules, inorganic gold nanoparticles (<NUM>), widely regarded for their biocompatibility and light attenuating properties, also function as an effective photoabsorbing additive to generate perfusable hydrogels (discussed below with respect to <FIG>).

Possible photoabsorbers can be one or more food dyes including tartrazine, Sunset Yellow FCF (Yellow No. <NUM>), Brilliant Blue FCF (FD&C Blue No. <NUM>), Indigo Carmine (FD&C Blue No. <NUM>), Fast Green FCF (FD&C Green No. <NUM>) anthocyanins, anthocyanidin, erythrosine (FD&C Red No.<NUM>), Allura Red AC (FD&C Red No. <NUM>), riboflavin (Vitamin B2, E101, E101a, E106), ascorbic acid (vitamin C), Quinoline Yellow WS, carmoisine (azorubine), Ponceau 4R (E124), Patent Blue V (E131), Green S (E142), Yellow <NUM> (E107), Orange GGN (E111), Red <NUM> (E128), caramel color, phenol red, methyl orange, <NUM>-nitrophenol, and NADH disodium salt. Also possible are curcumin (E100), turmeric, alpha carotene, beta carotene, canthaxanthin (keto-carotenoid), cochineal extract, paprika, saffron, ergocalciferol (vitamin D2), cholecalciferol (vitamin D3), Citrus Red <NUM>, annatto extract, and lycopene.

<FIG> illustrate the fabrication of patent vessels within monolithic hydrogels. <FIG> schematically illustrates a monolithic hydrogel with a horizontal vessel that can be fabricated by projection stereolithography with one of the suitable photoabsorbers (tartrazine is used for the experimental results shown) added to the pre-hydrogel solution to minimize excess light penetration into the nascent vessel. <FIG> shows that projection stereolithography with tartrazine yields hydrogels with minimal excess crosslinking in the vessel lumen. <FIG> is absorbance spectra of a LAP photoinitiator and the photoabsorber tartrazine. The absorbance spectra of tartrazine encompass the light source used (vertical bar at <NUM>), which can be used to initiate photocrosslinking via the LAP photoinitiator. <FIG> is photorheology during short duration (left) and long duration (right) light exposures (blue shaded region) and demonstrates that tartrazine addition (<NUM>-<NUM>) slows the induction of crosslinking but does not ultimately interfere with gelation. <FIG> shows that hydrogels release up to <NUM>% tartrazine within hours (exemplified by photos of gels above a color wheel), making fabricated hydrogels suitable for color and fluorescence imaging.

To understand how these photoabsorbers impact the gelation kinetics of photopolymerizable hydrogels photorheological characterization was performed with short duration light exposures. The results indicate that these additives cause a dose-dependent delay in the induction of photocrosslinking (<FIG> and <FIG>). Saturating light exposures that extend beyond the reaction termination demonstrate that suitable additives did not ultimately interfere with the reaction because hydrogels eventually reached an equivalent storage modulus independent of the additive concentration (<FIG> and <FIG>).

<FIG> show experimental results indicating that fabrication orientation and layer thickness affect the fidelity of printed channels. <FIG> is a circular channel of <NUM> diameter fabricated such that the longitudinal axis of the channel is parallel with the print direction. This orientation produces no overhanging features with high circularity (as quantified in <FIG> is a square channel with <NUM> sides, fabricated in a horizontal orientation perpendicular to the light projection axis (the asterisk denotes the region where excess light penetration caused extraneous hydrogel crosslinking). <FIG> shows circular channels of <NUM> diameter in a horizontal orientation that were printed using a layer thickness of <NUM>, <NUM>, <NUM>, or <NUM>. <FIG> is a plot of channel circularity as a function of layer thickness. Horizontal channel prints are shown left of the dashed line and the vertical channel from <FIG> is shown at right. As layer height decreases, channels exhibit higher circularity and thus greater geometric fidelity to their original CAD model.

<FIG> show experimental results indicating that biocompatible photoabsorbers allow fabrication of hydrogels with open channels. <FIG> are absorbance spectra of LAP photoinitiator and biocompatible photoabsorbers tartrazine, curcumin, anthocyanin, and <NUM> gold nanoparticles. The absorbance spectra of these additives encompass the light source (vertical bar at <NUM>) that can be used to initiate photocrosslinking via the LAP photoinitiator. <FIG> shows that the amount of tartrazine was found to be lower in a printed gel (<NUM>×<NUM>×<NUM> with <NUM> diameter channel) compared to the starting pre-hydrogel solution, attributable to its degradation during the radical-mediated photochemical reaction. <FIG> is a hydrogel with the horizontal vessel geometry described in (<FIG>) but fabricated without any photoabsorber additive, results in undesired gelation within the vessel, completely occluding it. Rhodamine-dextran was incorporated into the pre-hydrogel mixture to enable fluorescence. <FIG> is fluorescence (left) and color photograph (right) images of curcumin (top), anthocyanin (middle), and <NUM> gold nanoparticles (bottom) as photoabsorbers for fabrication of open horizontal channels using stereolithography. FITC-dextran was incorporated into the pre-hydrogel mixture to obtain fluorescence images. <FIG> is the results of photorheological studies with short (left) and long (right) duration light exposures with the following additives: curcumin (top), anthocyanin (middle), and <NUM> gold nanoparticles (bottom). <FIG> are working curves for hydrogel formulations of increasing photoabsorber concentration. Each data point represents the time required for a single layer hydrogel of given thickness to reach its gel point during dynamic oscillation and irradiation and all data points are shown in table form in <FIG>.

Tartrazine is selected as an example of a preferred photoabsorber. In addition to its low toxicity in humans and broad utility in the food industry, the hydrophilic dye is easily washed out of generated hydrogels (for example, <NUM>% elutes within <NUM> hours for small gels) resulting in nearly transparent constructs which are suitable for imaging. Some tartrazine may also be degraded during polymerization as tartrazine is known to be sensitive to free radicals. Submerging gels in water or saline solution to remove soluble tartrazine also flushes the vascular topology and removes unreacted pre-hydrogel solution. In contrast to tartrazine, curcumin is lipophilic and does not wash out in aqueous solutions, anthocyanin has a peak absorbance far away from our intended <NUM> light source requiring high concentrations for suitable potency, and gold nanoparticles are physically entrapped and make transmission or fluorescence microscopy impractical. Use of tartrazine is advantageous in the hydrogel pre-polymerization solution as a light-absorbing additive material suitable to control light penetration that can be later removed from the device formed of the pre-polymerization solution, allowing it become transparent or nearly so (e.g., allowed <NUM>% or more of incident light to pass through).

The hydrogel devices discussed herein can further include cells (plant, bacterial, or mammalian) either in the pre-polymerization solution or later seeded in the porous hydrogel device. <FIG> show experimental results indicating that the inclusion of tartrazine does not affect viability and differentiation of human mesenchymal stem cells. In <FIG>, B human mesenchymal stem cells (hMSCs) incubated with <NUM>-<NUM> tartrazine (<FIG> and <NUM>-<NUM> LAP (<FIG>) for <NUM> and <NUM> resulted in high cell viability. <FIG> shows that hMSCs within bioactive cylindrical hydrogels demonstrated high cell viability. <FIG> is quantification of osteogenic differentiation of hMSCs encapsulated in hydrogels fabricated with tartrazine and show that the cells within cylindrical fabricated hydrogels remain viable and can undergo osteogenic differentiation.

More advanced photoactive materials can be used to create the hydrogel devices. <FIG> show experimental results illustrating the compatibility of photoabsorber additives with advanced materials and fabrication strategies. <FIG> shows photorheology of radical-mediated chain-growth photopolymerization with PEGDA and photoabsorber and reveals that increases in hydrogel stiffness slowly increase long after light (blue shaded region) is switched off, also known as dark reactions. In contrast, as shown in <FIG>, with PEG-norbornene and PEG- dithiol step-growth polymerization (thiol-ene click chemistry) in the presence of photoabsorber additives, an increase in hydrogel stiffness stops abruptly after light (shaded blue region) is switched off. <FIG> shows that the staircase-like appearance of step-growth reaction kinetics can be replicated in series with discrete light pulses (shaded blue regions) to yield hydrogels of variable stiffness without the complication of slowly evolving reactions as seen in chain-growth polymerization. <FIG> shows that projection stereolithography of thiol-ene materials yields hydrogels with minimal excess crosslinking in the vessel lumen. <FIG> illustrates that continuous projection stereolithography of hydrogels containing open channels can be achieved. FITC-Dextran was incorporated into the pre- hydrogel mixture to obtain the fluorescence image.

Strong lamination was observed between adjacent fabricated layers and a rapid response of the patterned hydrogel to mechanical deformations (as shown in the resting/inflated results of <FIG>). This facile generation of soft hydrogels with patent cylindrical vessels oriented orthogonal to the light projection axis suggests a significant design flexibility toward the generation of complex vascular topologies, and the optical clarity of resultant hydrogels implies imaging methodologies suitable for characterization and validation of fluid flows.

The methods and validation experiments describe how to fabricate a 3D hydrogel construct by creating a 3D model of the construct based on a tessellation of polyhedra having a number of faces connected by edges and vertices. Then by generating a vascular component of the model by removing the faces and optionally the vertices of the polyhedra, and using the remaining topology as a vascular skeleton, skinning the skeleton with a polygonal mesh, and then smoothing the result to produce cylindrical channels along the edges of the model with intervessel junctions located at each vertex location. An airway component of the model can be generated by scaling the faces of the 3D model along local face normals such that the airway is nested inside the vasculature (vascular) component, forming independent fluidic connections to the vasculature and to the airway, and performing a Boolean subtraction from a solid volume, and combining the vascular component and the airway component of the model.

These 3D models can be used for additive manufacturing with a pre-polymerization solution including a photoabsorber solution being supplied to the 3D additive manufacturing machine. The pre-polymerization solution can have one or more bacterial, mammalian, and plant cells or the architecture resulting from the 3D model can be a first tubular channel network or second tubular channel network that is lined with cells. Alternatively, the hydrogel matrix can be embedded with cells. The cells can be one or more of liver, lung, bone, endothelial, cardiac, pancreas, kidney, epithelial, muscle, cartilage, stem cells, skin, or eye cells.

While the 3D additive manufacturing machine generates the hydrogel based on the 3D model, heat can be added via a silicone heater, heat lamps, or infrared LEDs. The building hydrogel can be enclosed in a heating enclosure during generation. Once fabricated, the hydrogel device can be implanted into a subject for medical intervention, or multiple devices joined into a larger implant can be used for treatment.

In accordance with various embodiments, a device including a hydrogel matrix as defined in the appended claims is provided. The hydrogel matrix includes a photoabsorber and a void architecture in the matrix, having a first vessel architecture and a second vessel architecture that are each tubular and branching, wherein the first and second vessel architectures are fluidically independent from each other, and wherein one or both of the first vessel architecture and second vessel architecture is formed from a model based on a tessellation of polyhedra.

In accordance with various embodiments, the device allows between about <NUM>% and <NUM>% of visible light (<NUM> - <NUM> wavelength) incident on the device to pass therethrough and allow imaging of the device. In accordance with various embodiments, the device allows between about <NUM>% and <NUM>%, between about <NUM>% and <NUM>%, between about <NUM>% and <NUM>%, or between about <NUM>% and <NUM>%, of visible light incident on the device to pass therethrough and allow imaging of the device. In accordance with various embodiments, the device allows at least <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, or <NUM>%, inclusive of any percentages of visible light incident on the device to pass therethrough and allow imaging of the device. In accordance with various embodiments, the photoabsorbers might not result in transparent hydrogels.

In accordance with various embodiments, the photoabsorber has been at least partially washed out of the device. For example, some percentage of the photoabsorber may not wash out completely. This can result, for example, in gels that are transparent but still slightly "yellow" in color. In accordance with various embodiments, the photoabsorber has been washed out at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, or at least <NUM>%, inclusive of any values therebetween. In accordance with various embodiments, the photoabsorber is degradable independent of any degradation of the remaining hydrogel matrix. In accordance with various embodiments, the photoabsorber is degradable by chemical or physical processes. For example, at least <NUM>% of a photoabsorber such as tartrazine can be washed within three hours after printing is complete.

In accordance with various embodiments, the photoabsorber is photobleachable by exposure to absorbable light having a wavelength <NUM> - <NUM> wavelength, chemical degradation such as by peroxides, or any other suitable material, or removable by exposure to boiling aqueous solution, such as water, or any other suitable material.

According to the claimed invention, one or both of the first vessel architecture and second vessel architecture is formed from a model based on a tessellation of polyhedra. In accordance with various embodiments, the model based on a tessellation of polyhedra represents blood vessels. In accordance with various embodiments, the model based on a tessellation of polyhedra represents an airway.

In accordance with various embodiments, the void architecture is multi-vascular and further comprises a functional valve. In accordance with various embodiments, the valve is a bicuspid valve, tricuspid valve, monocuspid valve, or Tesla valve.

In accordance with various embodiments, the void architecture comprises a fluidic static mixer. In accordance with various embodiments, the fluidic static mixer has between two and fifty (<NUM>-<NUM>) fin elements. In accordance with various embodiments, the fluidic static mixer has between <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, <NUM>-<NUM>, or <NUM>-<NUM> fin elements, inclusive of any ranges therebetween. In accordance with various embodiments, the functional valve is positionable at any position in the multi-vascular void architecture.

In accordance with various embodiments, the device is monolithic. In accordance with various embodiments, the hydrogel matrix is a photopolymerized hydrogel matrix and/or biodegradable. In accordance with various embodiments, the device is produced by additive manufacturing.

In accordance with various embodiments, the first vessel architecture and the second vessel architecture are entangled. In accordance with various embodiments, the first vessel architecture is ensheathed by the second vessel architecture. In accordance with various embodiments, the hydrogel matrix is porous. In accordance with various embodiments, the porosity ranges from about <NUM> (<NUM> Angstroms) to about <NUM>, <NUM> to about <NUM>, <NUM> to about <NUM>, <NUM> to about <NUM>, <NUM> to about <NUM>, <NUM> to about <NUM>, <NUM> to about <NUM>, <NUM> to about <NUM>, or <NUM> to <NUM>, inclusive of any porosity ranges therebetween. In accordance with various embodiments, the device is implantable. In accordance with various embodiments, the photoabsorber is hydrophilic.

In accordance with various embodiments, the photoabsorber is one of a food dye, tartrazine, sunset yellow FCF (Yellow No. <NUM>), Brilliant Blue FCF (FD&C Blue No. <NUM>), Indigo Carmine (FD&C Blue No. <NUM>), Fast Green FCF (FD&C Green No. <NUM>) anthocyanins, anthocyanidin, erythrosine (FD&C Red No.<NUM>), Allura Red AC (FD&C Red No. <NUM>), riboflavin (Vitamin B2, E101, E101a, E106), ascorbic acid (Vitamin C), Quinoline Yellow WS, carmoisine (azorubine), Ponceau 4R (E124), Patent Blue V (E131), Green S (E142), Yellow <NUM> (E107), Orange GGN (E111), Red <NUM> (E128), caramel color, phenol red, Methyl orange, <NUM>-nitrophenol, and NADH disodium salt. In accordance with various embodiments, the photoabsorber can include a concentration of about <NUM> to about <NUM>, about <NUM> to about <NUM>, about <NUM> to about <NUM>, about <NUM> to about <NUM>, about <NUM> to about <NUM>, about <NUM> to about <NUM>, about <NUM> to about <NUM>, about <NUM> to about <NUM>, about <NUM> to about <NUM>, about <NUM> to about <NUM>, inclusive of any concentration ranges therebetween.

In accordance with various embodiments, the photoabsorber is hydrophobic. In accordance with various embodiments, the photoabsorber is one of Curcumin (E100), turmeric, alpha carotene, beta carotene, canthaxanthin (keto-carotenoid), cochineal extract, paprika, saffron, ergocalciferol (vitamin D2), cholecalciferol (vitamin D3), Citrus Red <NUM>, annatto extract, and Lycopene. In accordance with various embodiments, the hydrophobic photoabsorber can include a concentration of about <NUM> to about <NUM>, about <NUM> to about <NUM>, about <NUM> to about <NUM>, about <NUM> to about <NUM>, about <NUM> to about <NUM>, about <NUM> to about <NUM>, about <NUM> to about <NUM>, about <NUM> to about <NUM>, about <NUM> to about <NUM>, about <NUM> to about <NUM>, inclusive of any concentration ranges therebetween. In accordance with various embodiments, the hydrophobic photoabsorber can include a solvent. The solvent can be selected from the group consisting of dimethyl sulfoxide, ethanol (<NUM>-<NUM>%), isopropyl alcohol, (<NUM>-<NUM>%) dimethylformamide, N-Methyl-<NUM>-pyrrolidone, tetrahydrofuran, chloroform, methylene chloride, toluene, hexafluoro-<NUM>-propanol, and any combination thereof.

In accordance with various embodiments, the device includes three or more vessel architectures fluidically independent from each other and from the first and second vessel architectures.

In accordance with various embodiments, the fluidic static mixer includes a photoabsorber. In accordance with various embodiments, the fluidic static mixer includes tartrazine. In accordance with various embodiments, the fluidic static mixer includes fin elements with <NUM>° twists. In accordance with various embodiments, the fluidic static mixer includes fin elements having a twist angle in the of <NUM>°-<NUM>°, <NUM>°-<NUM>°, <NUM>°-<NUM>°, <NUM>°-<NUM>°, <NUM>°-<NUM>°, <NUM>°-<NUM>°, <NUM>°-<NUM>°, <NUM>°-<NUM>°, <NUM>°-<NUM>°, <NUM>°-<NUM>°, <NUM>°-<NUM>°, <NUM>°-<NUM>°, or <NUM>°-<NUM>°, inclusive of any ranges of twisting angles therebetween. In accordance with various embodiments, the fluidic static mixer is produced by additive manufacturing.

In accordance with various embodiments, the device can include a thickener to prevent cell settling. In accordance with various embodiments, the thickener includes xanthan gum having a concentrations about <NUM> wt% to about <NUM> wt%, about <NUM> wt% to about <NUM> wt%, about <NUM> wt% to about <NUM> wt%, or about <NUM> wt% to about <NUM> wt%, inclusive of any thickener values therebetween.

In accordance with various embodiments, a pre-polymerization solution is provided. In accordance with various embodiments, the pre-polymerization solution includes a photosensitive polymer, and a photoabsorber additive material suitable to control light penetration, wherein the additive material is at least partially removable from a solid formed of the pre-polymerization solution. In accordance with various embodiments, a photosentive polymer is one that can be polymerized via photosensitive reactions. In accordance with various embodiments, photoabsorbers can include photoabsorbers that are covalently or non-covalently bound to polymers. In accordance with various embodiments, the pre-polymerization solution can also include photoinitiators. In accordance with various embodiments, the photoinitiators can be covalently or non-covalently bound to polymers.

In accordance with various embodiments, the pre-polymerization solution can include a thickener to prevent cell settling. In accordance with various embodiments, the thickener includes xanthan gum having a concentrations about <NUM> wt% to about <NUM> wt%, about <NUM> wt% to about <NUM> wt%, about <NUM> wt% to about <NUM> wt%, or about <NUM> wt% to about <NUM> wt%, inclusive of any thickener values therebetween.

In accordance with various embodiments, the pre-polymerization solution includes a pH between about <NUM> to about <NUM>, about <NUM> to about <NUM>, about <NUM> to about <NUM>, about <NUM> to about <NUM>, about <NUM> to about <NUM>, about <NUM> to about <NUM>, about <NUM> to about <NUM>, or about <NUM> to about <NUM>, inclusive of any pH values therebetween.

In accordance with various embodiments, the photosensitive polymer can include polyethylene glycol diacrylate, gelatin methacrylate, collagen methacrylate, silk methacylate, hyaluronic acid methacrylate, chondroitin sulfate methacrylate, elastin methacrylate, cellulose acrylate, dextran methacrylate, heparin methacrylate, NIPAAm methacrylate, Chitosan methacrylate, polyethyelene glycol norbornene, polyethylene glycol dithiol, thiolated gelatin, thiolated chitosan, thiolated silk, PEG based peptide conjugates, or any combination thereof.

In accordance with various embodiments, the ranges of polymer in the pre-polymerization solution can range between about <NUM> wt% to about <NUM> wt%, about <NUM> wt% to about <NUM> wt%, or about <NUM> wt% to about <NUM> wt%, inclusive of any ranges therebetween. In accordance with various embodiments, the ranges of photoabsorber in the pre-polymerization solution can include a concentration of about <NUM> to about <NUM>, about <NUM> to about <NUM>, about <NUM> to about <NUM>, about <NUM> to about <NUM>, about <NUM> to about <NUM>, about <NUM> to about <NUM>, about <NUM> to about <NUM>, about <NUM> to about <NUM>, about <NUM> to about <NUM>, about <NUM> to about <NUM>, inclusive of any concentration ranges therebetween. In accordance with various embodiments, the photoinitiator can include a concentration of about <NUM> to about <NUM>, about <NUM> to about <NUM>, about <NUM> to about <NUM>, about <NUM> to about <NUM>, about <NUM> to about <NUM>, about <NUM> to about <NUM>, inclusive of any concentration ranges therebetween.

In accordance with various embodiments, the pre-polymerization solution includes plant, bacterial, or mammalian cells. In accordance with various embodiments, the ranges of cells include 1x10<NUM>-200x10<NUM> per milliliter for mammalian and plant cells. In accordance with various embodiments, the ranges of cells can include 1x10<NUM>-1x10<NUM> per milliliter for mammalian and plant cells. In accordance with various embodiments, the ranges of cells can include 1x10<NUM>-1x10<NUM>per millileter for bacteria and other archae including fungi. In accordance with various embodiments, the ranges of cells can include 1x10<NUM>-1x10<NUM> for bacteria and other archae including fungi.

In accordance with various embodiments, the photoabsorber additive material is biocompatible. In accordance with various embodiments, the photoabsorber additive material is hydrophilic. In accordance with various embodiments, the photoabsorber additive material is one of a food dye, tartrazine, sunset yellow FCF (Yellow No. <NUM>), Brilliant Blue FCF (FD&C Blue No. <NUM>), Indigo Carmine (FD&C Blue No. <NUM>), Fast Green FCF (FD&C Green No. <NUM>) anthocyanins, anthocyanidin, erythrosine (FD&C Red No.<NUM>), Allura Red AC (FD&C Red No. <NUM>), riboflavin (Vitamin B2, E101, E101a, E106), ascorbic acid (Vitamin C), Quinoline Yellow WS, carmoisine (azorubine), Ponceau 4R (E124), Patent Blue V (E131), Green S (E142), Yellow <NUM> (E107), Orange GGN (E111), Red <NUM> (E128), caramel color, phenol red, Methyl orange, <NUM>-nitrophenol, and NADH disodium salt.

In accordance with various embodiments, the photoabsorber additive material is hydrophobic. In accordance with various embodiments, the photoabsorber additive material is one of Curcumin (E100), turmeric, alpha carotene, beta carotene, canthaxanthin (keto-carotenoid), cochineal extract, paprika, saffron, ergocalciferol (vitamin D2), cholecalciferol (vitamin D3), Citrus Red <NUM>, annatto extract, and Lycopene. In accordance with various embodiments, the hydrophobic photoabsorber additive material can include a solvent. The solvent can be selected from the group consisting of dimethyl sulfoxide, ethanol (<NUM>-<NUM>%), isopropyl alcohol, (<NUM>-<NUM>%) dimethylformamide, N-Methyl-<NUM>-pyrrolidone, tetrahydrofuran, chloroform, methylene chloride, toluene, hexafluoro-<NUM>-propanol, and any combination thereof.

In accordance with various embodiments, a method of fabricating, with a processor, a 3D hydrogel construct is provided. In accordance with various embodiments, the method includes creating a 3D model of the construct based on a tessellation of polyhedra having a number of faces connected by edges and vertices; generating a first vascular component of the model by: removing the faces and/or the vertices of the polyhedra, and using the remaining topology as a vascular skeleton, and skinning the skeleton with a polygonal mesh and then smoothing the result to produce cylindrical channels along the edges of the model with intervessel junctions located at each vertex location; generating a second vascular component of the model by: scaling the faces of the 3D model along local face normals such that the second vascular component is nested inside the first vascular component, forming independent fluidic connections to the first vascular component and to the second vascular component, and performing a Boolean subtraction from a solid volume; and combining the two vascular components of the model.

In accordance with various embodiments, the method also includes fabricating a hydrogel alveolar construct by fusing multiple spheres in a radially symmetric fashion to create an airway topology; offsetting the airway topology to generate a vascular surface topology; tessellating, skeletonizing, and skinning the vascular surface topology to produce a Voronoi vascular topology, wherein tessellating comprises performing a tessellation of polyhedra; and performing a Boolean subtraction of the original airway and the tessellated Voronoi vascular topology from a solid volume to generate a final 3D model for additive manufacturing. In accordance with various embodiments, the method can further comprise delivering heat while a 3D additive manufacturing machine generates the 3D model.

In accordance with various embodiments, the tessellation of polyhedra is a Weaire-Phelan space-filling foam model of two dodecahedron and six tetrakaidecahedron cells.

In accordance with various embodiments, a method of manufacturing a hydrogel matrix construct is provided. In accordance with various embodiments, the method includes creating a 3D model of the hydrogel matrix construct using a design software, wherein the 3D model of the hydrogel matrix construct comprises a first computational algorithm that yields a first tubular channel network in the hydrogel matrix construct, and a second computational algorithm, different from the first computational algorithm, that yields a second tubular channel network in the hydrogel matrix, wherein the first and second tubular channel networks are two independent, entangled vascular networks; converting the 3D model to a format suitable for use in a 3D additive manufacturing software to yield a formatted 3D model to be generated using a 3D additive manufacturing machine; and directing the additive manufacturing machine to generate the model.

In accordance with the claimed invention, the computational algorithm is a tessellation of polyhedra.

In accordance with various embodiments, the method also includes supplying a pre-polymerization solution to the 3D additive manufacturing machine wherein the pre-polymerization solution includes a photoabsorber. In accordance with various embodiments, the pre-polymerization solution comprises one or more types of bacterial, mammalian, and plant cells.

In accordance with various embodiments, the method includes lining the first tubular channel network or second tubular channel network with cells. In accordance with various embodiments, the method includes embedding the hydrogel matrix with cells. In accordance with various embodiments, the cells are one or more of liver, lung, bone, endothelial, cardiac, pancreas, kidney, epithelial, muscle, cartilage, stem cells, skin, or eye cells. In accordance with various embodiments, the ranges of cells can include 1x10<NUM>-200x10<NUM> per milliliter for mammalian cells. In accordance with various embodiments, the ranges of cells can include 1x10<NUM>-1x10<NUM> per milliliter for mammalian cells.

In accordance with various embodiments, the method includes delivering heat while the 3D additive manufacturing machine generates the 3D model. In accordance with various embodiments, heat is added via a silicone heater, heat lamps, or infrared LEDs. In accordance with various embodiments, the ranges of temperature from heating can include about <NUM> to about <NUM>, about <NUM> to about <NUM>, about <NUM> to about <NUM>, about <NUM> to about <NUM>, about <NUM> to about <NUM>, about <NUM> to about <NUM>, about <NUM> to about <NUM>, about <NUM> to about <NUM>, or about <NUM> to about <NUM>, inclusive of any temperature or ranges of temperature therebetween. In accordance with various embodiments, the method includes enclosing the 3D model in a heating enclosure during generation of the 3D model.

In accordance with various embodiments, a method of fabricating a 3D hydrogel construct is provided. In accordance with various embodiments, the method includes using a computer-implemented process to: create a 3D model of the construct based on a tessellation of polyhedra having a number of faces connected by edges and vertices; generate a first vascular component of the model; generate a second vascular component of the model; and combine the first and second vascular components of the model.

In accordance with various embodiments, using the computer-implemented process to generate the first vascular component of the model includes removing the faces of the polyhedra, and using the remaining vertices and edges of the topology as a vascular skeleton.

In accordance with various embodiments, using a computer-implemented process to generate the first vascular component of the model further includes skinning the skeleton with a polygonal mesh and then smoothing the result to produce cylindrical channels along the edges of the model with intervessel junctions located at each vertex location.

In accordance with various embodiments, using a computer-implemented process to generate the second vascular component of the model includes scaling the faces of the 3D model along local face normals such that the second vascular component is nested inside the first vascular component, forming independent fluidic connections to the first vascular component and to the second vascular component, and performing a Boolean subtraction from a solid volume.

In accordance with various embodiments, the method further includes a hydrogel alveolar construct by: fusing multiple spheres in a radially symmetric fashion to create an airway topology; offsetting the airway topology to generate a vascular surface topology; tessellating, skeletonizing, and skinning the vascular surface topology to produce a Voronoi vascular topology, wherein tessellating comprises performing a tessellation of polyhedral.

In accordance with various embodiments, the method includes performing a Boolean subtraction of the original airway and the tessellated Voronoi vascular topology from a solid volume to generate a final 3D model for additive manufacturing. In accordance with various embodiments, the tessellation of polyhedra is a Weaire-Phelan space-filling foam model of two dodecahedron and six tetrakaidecahedron cells.

Also described herein, but not forming part of the claimed invention, is a method of treating a subject in need thereof by implanting any of the devices into a subject.

In accordance with various embodiments, a device comprising multiple joined subunits is provided. In accordance with various embodiments, each subunit includes device as disclosed herein.

In accordance with various embodiments, an apparatus for manufacturing a hydrogel matrix construct is provided. The apparatus can comprise a frame, a stage with z-axis drive motor attached to the frame, an electronics board for controlling movement of the stage with respect to the frame, a container configured for holding a solution, a projector for projecting images, wherein the images are image slices of a 3D model of the hydrogel matrix construct, and a build platform configured for holding a substrate, wherein the hydrogel matrix construct is formed on the substrate. In accordance with various embodiments, an optional <NUM>° mirror can be configured for reflecting the images projected from the projector into the container.

In accordance with various embodiments, the substrate can include a composition having organic or inorganic glass, with or without a textured surface that is either inert or functionalized with tethered groups. In accordance with various embodiments, the tethered groups can include covalently or non-covalently assist in the attachment of the hydrogel to the substrate, such as <NUM>-(trimethoxysilyl)propyl methacrylate. In accordance with various embodiments, the substrate composition may also contain mica, polycarbonate, polysulfone, polymethyl methacrylate, polystyrene, cyclic olefin copolymer, polyethylene, polypropylene, or quartz with or without a textured surface that can optionally be modified with tethered groups that can covalently or non-covalently assist in the attachment of the hydrogel to the substrate. These various components, in accordance with various embodiments, can all be provided as part of single apparatus (as described here and exemplified in <FIG> discussed in detail below). These various components, however, can also be provided in various other configurations whereby any one or more components can be provided as part of further sub-apparatus that interact with the main apparatus to together provide fundamentally the same functionality as those embodiments where all components are provided within the single apparatus. Further, various embodiments can include only a portion of the components provided above as it should not be assumed that each and every component recited is necessary for the proper functionality of an apparatus for manufacturing a hydrogel matrix construct.

In accordance with various embodiments, the solution can be a pre-polymerization solution comprising a photosensitive polymer and a photoabsorber additive material suitable to control light penetration. The additive material can be removable from a solid formed of the pre-polymerization solution. The photoabsorber additive material can be biocompatible. The photoabsorber additive material can be hydrophilic. The photoabsorber additive material can be hydrophobic.

In accordance with various embodiments, the photoabsorber additive material can be one of a food dye, tartrazine, sunset yellow FCF (Yellow No. <NUM>), Brilliant Blue FCF (FD&C Blue No. <NUM>), Indigo Carmine (FD&C Blue No. <NUM>), Fast Green FCF (FD&C Green No. <NUM>) anthocyanins, anthocyanidin, erythrosine (FD&C Red No.<NUM>), Allura Red AC (FD&C Red No. <NUM>), riboflavin (Vitamin B2, E101, E101a, E106), ascorbic acid (Vitamin C), Quinoline Yellow WS, carmoisine (azorubine), Ponceau 4R (E124), Patent Blue V (E131), Green S (E142), Yellow <NUM> (E107), Orange GGN (E111), Red <NUM> (E128), caramel color, phenol red, Methyl orange, <NUM>-nitrophenol, and NADH disodium salt.

In accordance with various embodiments, the hydrophobic photoabsorber can include a solvent. The solvent can be selected from the group consisting of dimethyl sulfoxide, ethanol (<NUM>-<NUM>%), isopropyl alcohol, (<NUM>-<NUM>%) dimethylformamide, N-Methyl-<NUM>-pyrrolidone, tetrahydrofuran, chloroform, methylene chloride, toluene, hexafluoro-<NUM>-propanol, and any combination thereof.

In accordance with various embodiments, the photoabsorber additive material can be one of Curcumin (E100), turmeric, alpha carotene, beta carotene, canthaxanthin (keto-carotenoid), cochineal extract, paprika, saffron, ergocalciferol (vitamin D2), cholecalciferol (vitamin D3), Citrus Red <NUM>, annatto extract, and Lycopene.

In accordance with various embodiments, the apparatus can further comprise plant, bacterial, or mammalian cells. The apparatus can further comprise one or more of liver, lung, bone, endothelial, cardiac, pancreas, kidney, epithelial, muscle, cartilage, stem cells, skin, or eye cells.

Referring now to <FIG>, a schematic illustration of an example apparatus <NUM> for fabricating a hydrogel construct is provided, in accordance with various embodiments. Apparatus <NUM> can include, for example, a frame <NUM>, a stage <NUM>, an electronics board <NUM>, a container <NUM>, a projector <NUM>, an optional mirror <NUM>, and a build platform <NUM>. Stage <NUM> can be attached to frame <NUM> and can include a motor attached to frame <NUM>. The motor can be a z-axis drive motor. Electronics board <NUM> can be configured for controlling movement of the stage with respect to the frame. Projector <NUM> can be configured for projecting images. The images can be image slices of a 3D model of the hydrogel matrix construct. Mirror <NUM> can be a <NUM>° mirror. Mirror <NUM> can be configured for reflecting the images projected from projector <NUM> into the container <NUM>. Note that optional mirror <NUM> can be excluded from the apparatus if the lens on the projector is already at an angle not necessitating a mirror, e.g., the projector projecting the image directly onto the desired surface. Build platform <NUM> can be configured for holding a substrate <NUM>, wherein the hydrogel matrix construct can be formed on the substrate <NUM>. Substrate <NUM> can be integrated into apparatus <NUM> or can be component wholly separate from the apparatus and, thus, can be considered to be not part of the apparatus.

The container <NUM> can be configured to receive a solution <NUM>. As discussed above, solution <NUM> can be a pre-polymerization solution comprising a photosensitive polymer and a photoabsorber additive material suitable to control light penetration.

Referring back to the example apparatus <NUM> of <FIG> discussed above, apparatus <NUM> can include a motor <NUM> (which can be attached to a frame <NUM>), controlled by an actuator <NUM>, onto which the build platform <NUM> is attached. The back of the printer can house the motherboard <NUM> that controls the motor and performs input/output with any additional sensors or switches. The front of the printer contains an optional mirror <NUM> placed to reflect incident patterns from a projector <NUM> onto a dish containing the photosensitive materials. Note that optional mirror <NUM> can be excluded from the apparatus if the lens on the projector is already at an angle not necessitating a mirror, e.g., the projector projecting the image directly onto the desired surface.

In accordance with various embodiments, the various apparatus embodiments can be incorporated as part of a system. An example system <NUM> for manufacturing a hydrogel matrix construct is illustrated in <FIG> and will discussed in greater detail below.

The system for manufacturing a hydrogel matrix construct can comprise a processor. The processor can include a modeling engine. The modeling engine can be configured to create a 3D model of the hydrogel matrix construct based on a tessellation of polyhedra having a number of faces connected by edges and vertices. The modeling engine can be configured to generate a first vascular component of the model. The modeling engine can be configured to generate a second vascular component of the model. The modeling engine can be configured to combine the first and second vascular components of the model.

The system can further comprise an apparatus configured for manufacturing the hydrogel matrix construct. The apparatus can comprise a frame, a stage with z-axis drive motor attached to the frame, an electronics board for controlling movement of the stage with respect to the frame, a container configured for holding a solution, a projector for projecting images, wherein the images are image slices of a 3D model of the hydrogel matrix construct, an optional <NUM>° mirror for reflecting the images projected from the projector into the container, and a build platform configured for holding a substrate, wherein the hydrogel matrix construct is formed on the substrate. Note that the optional <NUM>° mirror can be excluded from the apparatus if the lens on the projector is already at an angle not necessitating a mirror, e.g., the projector projecting the image directly onto the desired surface. These various components, in accordance with various embodiments, can all be provided as part of single apparatus. These various components, however, can also be provided in various other configurations whereby any one or more components can be provided as part of further sub-apparatus that interact with the main apparatus to together provide fundamentally the same functionality as those embodiments where all components are provided within the single apparatus. Further, various embodiments can include only a portion of the components provided above as it should not be assumed that each and every component recited is necessary for the proper functionality of an apparatus for manufacturing a hydrogel matrix construct.

In accordance with various embodiments, the hydrophobic photoabsorber additive material can include a solvent. The solvent can be selected from the group consisting of dimethyl sulfoxide, ethanol (<NUM>-<NUM>%), isopropyl alcohol, (<NUM>-<NUM>%) dimethylformamide, N-Methyl-<NUM>-pyrrolidone, tetrahydrofuran, chloroform, methylene chloride, toluene, hexafluoro-<NUM>-propanol, and any combination thereof.

In accordance with various embodiments, the solution can include a thickener to prevent cell settling. In accordance with various embodiments, the thickener includes xanthan gum having a concentrations about <NUM> wt% to about <NUM> wt%, about <NUM> wt% to about <NUM> wt%, about <NUM> wt% to about <NUM> wt%, or about <NUM> wt% to about <NUM> wt%, inclusive of any thickener values therebetween.

In accordance with various embodiments, the generating the first vascular component of the model can comprises removing the faces and/or the vertices of the polyhedra, and using the remaining topology as a vascular skeleton. The generating the first vascular component of the model can further comprise skinning the skeleton with a polygonal mesh and then smoothing the result to produce cylindrical channels along the edges of the model with intervessel junctions located at each vertex location.

In accordance with various embodiments, the generating the second vascular component of the model can comprises scaling the faces of the 3D model along local face normals such that the airway is nested inside the vasculature component, forming independent fluidic connections to the vasculature and to the airway, and performing a Boolean subtraction from a solid volume.

In accordance with various embodiments, the processor can be further configured to fabricate a hydrogel alveolar construct. The processor can fabricate a hydrogel alveolar construct by fusing multiple spheres in a radially symmetric fashion to create an airway topology and offsetting the airway topology to generate a vascular surface topology. The fabrication of the hydrogel aveolar construct can further include tessellating, skeletonizing, and skinning the vascular surface topology to produce a Voronoi vascular topology. The tessellating includes performing a tessellation of polyhedral. The tessellation of polyhedra can be a Weaire-Phelan space-filling foam model of two dodecahedron and six tetrakaidecahedron cells. The fabrication of the hydrogel aveolar construct can further include performing a Boolean subtraction of the original airway and the tessellated Voronoi vascular topology from a solid volume to generate a final 3D model for additive manufacturing.

Referring now to <FIG>, a schematic illustration of an example system <NUM> for fabricating a hydrogel construct is provided, in accordance with various embodiments. System <NUM> can comprise an apparatus and processor. The example apparatus illustrated from <FIG> is apparatus <NUM> illustrated in <FIG>. Refer above for detailed discussion of apparatus <NUM> of <FIG>. Processor <NUM> can include a modeling engine <NUM>.

Processor <NUM> can be communicatively connected to apparatus <NUM>. In various embodiments, processor <NUM> can be communicatively connected to apparatus <NUM> via a network connection that can be either a "hardwired" physical network connection (e.g., Internet, LAN, WAN, VPN, etc.) or a wireless network connection (e.g., Wi-Fi, WLAN, etc.). In various embodiments, processor <NUM> can be part of a workstation, mainframe computer, distributed computing node (part of a "cloud computing" or distributed networking system), personal computer, mobile device, etc..

In accordance with various embodiments, the processor <NUM> and apparatus <NUM> can be part of an integrated apparatus. As such, in <FIG>, processor <NUM> can be provided within apparatus <NUM>. Alternatively, processor <NUM> can be hosted by a different device than the apparatus <NUM>. Moreover, processor <NUM> can be part of a distributed network system.

As discussed above, and in accordance with various embodiments, build platform <NUM> can be configured for holding a substrate <NUM>, wherein the hydrogel matrix construct can be formed on the substrate <NUM>. Substrate <NUM> can be integrated into apparatus <NUM> or can be component wholly separate from the apparatus and, thus, can be considered to be not part of the apparatus.

As discussed above, and in accordance with various embodiments, container <NUM> can be configured to receive a solution <NUM>. As discussed above, solution <NUM> can be a pre-polymerization solution comprising a photosensitive polymer and a photoabsorber additive material suitable to control light penetration.

As discussed above, and in accordance with various embodiments, modeling engine <NUM> can be configured to create a 3D model of the hydrogel matrix construct based on a tessellation of polyhedra having a number of faces connected by edges and vertices. The modeling engine can be configured to generate a first vascular component of the model. The modeling engine can be configured to generate a second vascular component of the model. The modeling engine can be configured to combine the first and second vascular components of the model.

It should be understood that, in accordance with various embodiments, that modeling engine <NUM> can be configured to handle all the necessary steps accomplished within processor <NUM>. This is what is illustrated by example processor <NUM> and associated modeling engine <NUM> in <FIG>.

Further, in accordance with various embodiments, the functions and steps described in association with modeling engine <NUM> can be divided among any number of separate engines or modules within processor <NUM>. For example, the steps of creating a 3D model, generating a first and second vascular component of the model, and combining the vascular components can each be accomplished by separate engines (e.g., creation engine, generation engine and combination engine) and/or by separate processors including one or more separate engines. Moreover, at least a portion of these steps can be combined into a single engine (e.g., a component engine that provides for the generation and combination steps).

<FIG> is a flowchart for an example method S100 of fabricating a 3D hydrogel construct, in accordance with various embodiments. As shown in <FIG>, the method S100 includes creating a 3D model of the construct based on a tessellation of polyhedra having a number of faces connected by edges and vertices at step S110. The method S100 also includes generating a first vascular component of the model at step S120. In accordance with various embodiments herein, generating of the first vascular component of the model includes removing the faces and/or the vertices of the polyhedra, and using the remaining topology as a vascular skeleton, and skinning the skeleton with a polygonal mesh, and then smoothing the result to produce cylindrical channels along the edges of the model with intervessel junctions located at each vertex location.

The method S100 further includes generating a second vascular component of the model at step S130. In accordance with various embodiments, generating the second vascular component of the model includes scaling the faces of the 3D model along local face normals such that the second vascular component is nested inside the first vascular component, forming independent fluidic connections to the first vascular component and to the second vascular component, and performing a Boolean subtraction from a solid volume.

As shown in <FIG>, the method S100 includes combining the first and second vascular components of the model at step S140.

In accordance with various embodiments, the method S100 optionally includes fabricating a hydrogel alveolar construct at step S150. In accordance with various embodiments, the fabrication of the hydrogel alveolar construct includes fusing multiple spheres in a radially symmetric fashion to create an airway topology, offsetting the airway topology to generate a vascular surface topology, tessellating, skeletonizing, and skinning the vascular surface topology to produce a Voronoi vascular topology, wherein tessellating comprises performing a tessellation of polyhedral, and performing a Boolean subtraction of the original airway and the tessellated Voronoi vascular topology from a solid volume to generate a final 3D model for additive manufacturing. In accordance with various embodiments, the tessellation of polyhedra is a Weaire-Phelan space-filling foam model of two dodecahedron and six tetrakaidecahedron cells.

<FIG> is a flowchart for an example method S200 of manufacturing a hydrogel matrix construct, in accordance with various embodiments. As shown in <FIG>, the method S200 includes, at step <NUM>, creating a 3D model of the hydrogel matrix construct using a design software. In accordance with various embodiments, the 3D model of the hydrogel matrix construct includes a first computational algorithm that yields a first tubular channel network in the hydrogel matrix construct, and a second computational algorithm, different from the first computational algorithm, that yields a second tubular channel network in the hydrogel matrix, wherein the first and second tubular channel networks are two independent, entangled vascular networks.

At step S220, the method S200 includes converting the 3D model to a format suitable for use in a 3D additive manufacturing software to yield a formatted 3D model to be generated using a 3D additive manufacturing machine. The method S200 further includes directing the additive manufacturing machine to generate the model at step S230.

According to the claimed invention , the computational algorithm is a tessellation of polyhedra.

As shown in <FIG>, the method S200 optionally includes supplying a pre-polymerization solution to the 3D additive manufacturing machine wherein the pre-polymerization solution includes a photoabsorber at step S240. In accordance with various embodiments, the pre-polymerization solution includes one or more types of bacterial, mammalian, and plant cells.

The method S200 further optionally includes comprising lining the first tubular channel network or second tubular channel network with cells at step S250.

At step S260, the method S200 optionally includes embedding the hydrogel matrix with cells. In accordance with various embodiments, the cells are one or more of liver, lung, bone, endothelial, cardiac, pancreas, kidney, epithelial, muscle, cartilage, stem cells, skin, or eye cells.

The method S200 optionally includes comprising delivering heat while the 3D additive manufacturing machine generates the 3D model at step S270. In accordance with various embodiments, heat is added via a silicone heater, heat lamps, or infrared LEDs.

The method S200 optionally includes enclosing the 3D model in a heating enclosure during generation of the 3D model at step S280.

<FIG> is a flowchart for an example method S300 of fabricating a 3D hydrogel construct, according to various embodiments. As shown in <FIG>, the method S300 includes using a computer-implemented process to create a 3D model of the construct based on a tessellation of polyhedra having a number of faces connected by edges and vertices at step S310. At step S320, the method S300 includes using the computer-implemented process to generate a first vascular component of the model. The method S300 further includes using the computer-implemented process to generate a second vascular component of the model at step S330. The method S300 includes using the computer-implemented process to combine the first and second vascular components of the model at step S340.

As shown in <FIG>, the method S300 optionally includes using the computer-implemented process to generate the first vascular component of the model by removing the faces of the polyhedra, and using the remaining vertices and edges of the topology as a vascular skeleton at step S350.

The method S300 optionally includes, at step S360, using the computer-implemented process to generate the first vascular component of the model by skinning the skeleton with a polygonal mesh and then smoothing the result to produce cylindrical channels along the edges of the model with intervessel junctions located at each vertex location.

The method S300 optionally includes, at step S370, using a computer-implemented process to generate the second vascular component of the model by scaling the faces of the 3D model along local face normals such that the second vascular component is nested inside the first vascular component, forming independent fluidic connections to the first vascular component and to the second vascular component, and performing a Boolean subtraction from a solid volume.

As shown in <FIG>, the method S300 optionally includes, at step S380, fabricating a hydrogel alveolar construct by fusing multiple spheres in a radially symmetric fashion to create an airway topology, offsetting the airway topology to generate a vascular surface topology, tessellating, skeletonizing, and skinning the vascular surface topology to produce a Voronoi vascular topology, wherein tessellating comprises performing a tessellation of polyhedral.

The method S300 optionally includes, at step S390, performing a Boolean subtraction of the original airway and the tessellated Voronoi vascular topology from a solid volume to generate a final 3D model for additive manufacturing. In accordance with various embodiments, the tessellation of polyhedra is a Weaire-Phelan space-filling foam model of two dodecahedron and six tetrakaidecahedron cells.

In accordance with various embodiments, a non-transitory computer-readable medium in which a program is stored for causing a computer to perform a method of fabricating a 3D hydrogel construct is provided. In accordance with various embodiments, the method includes creating a 3D model of the construct based on a tessellation of polyhedra having a number of faces connected by edges and vertices. In accordance with various embodiments, the method includes generating a first vascular component of the model, generating a second component of the model, and combining the first and second vascular components of the model. In accordance with various embodiments, the method includes generating the first vascular component of the model by removing the faces and/and or the vertices of the polyhedra, and using the remaining topology as a vascular skeleton. In accordance with various embodiments, the method includes generating the first vascular component of the model further comprises by skinning the skeleton with a polygonal mesh and then smoothing the result to produce cylindrical channels along the edges of the model with intervessel junctions located at each vertex location.

In accordance with various embodiments, the method includes generating the airway component of the model comprises by scaling the faces of the 3D model along local face normals such that the airway is nested inside the vasculature (vascular) component, forming independent fluidic connections to the vasculature and to the airway, and performing a Boolean subtraction from a solid volume. In accordance with various embodiments, the method includes fabricating a hydrogel alveolar construct by fusing multiple spheres in a radially symmetric fashion to create an airway topology, offsetting the airway topology to generate a vascular surface topology, tessellating, skeletonizing, and skinning the vascular surface topology to produce a Voronoi vascular topology, wherein tessellating comprises performing a tessellation of polyhedral, and performing a Boolean subtraction of the original airway and the tessellated Voronoi vascular topology from a solid volume to generate a final 3D model for additive manufacturing. In accordance with various embodiments, the tessellation of polyhedra is a Weaire-Phelan space-filling foam model of two dodecahedron and six tetrakaidecahedron cells.

<FIG> is a block diagram that illustrates a computer system <NUM>, in accordance with various embodiments, that can be implemented to perform the various embodiments recited herein including, for example, various non-transitory computer-readable media in which a program is stored for causing a computer to perform various methods herein. In various embodiments, the computer system <NUM> can be a workstation, mainframe computer, distributed computing node (part of a "cloud computing" or distributed networking system), personal computer, mobile device, etc..

In accordance with various embodiments, the computer system <NUM> can include a bus <NUM> or other communication mechanism for communicating information, and a processor <NUM> coupled with bus <NUM> for processing information. In various embodiments, computer system <NUM> can also include a memory, which can be a random access memory (RAM) <NUM> or other dynamic storage device, coupled to bus <NUM> for determining instructions to be executed by processor <NUM>. Memory also can be used for storing temporary variables or other intermediate information during execution of instructions to be executed by processor <NUM>. In various embodiments, computer system <NUM> can further include a read only memory (ROM) <NUM> or other static storage device coupled to bus <NUM> for storing static information and instructions for processor <NUM>. A storage device <NUM>, such as a magnetic disk or optical disk, can be provided and coupled to bus <NUM> for storing information and instructions.

In various embodiments, computer system <NUM> can be coupled via bus <NUM> to a display <NUM>, such as a cathode ray tube (CRT) or liquid crystal display (LCD), for displaying information to a computer user. An input device <NUM>, including alphanumeric and other keys, can be coupled to bus <NUM> for communicating information and command selections to processor <NUM>. Another type of user input device is a cursor control <NUM>, such as a mouse, a trackball or cursor direction keys for communicating direction information and command selections to processor <NUM> and for controlling cursor movement on display <NUM>. This input device <NUM> typically has two degrees of freedom in two axes, a first axis (i.e., x) and a second axis (i.e., y), that allows the device to specify positions in a plane. However, it should be understood that input devices <NUM> allowing for <NUM> dimensional (x, y and z) cursor movement are also contemplated herein.

Consistent with certain implementations of the present teachings, results can be provided by computer system <NUM> in response to processor <NUM> executing one or more sequences of one or more instructions contained in memory <NUM>. Such instructions can be read into memory <NUM> from another computer-readable medium or computer-readable storage medium, such as storage device <NUM>. Execution of the sequences of instructions contained in memory <NUM> can cause processor <NUM> to perform the processes described herein. Alternatively hard-wired circuitry can be used in place of or in combination with software instructions to implement the present teachings. Thus, implementations of the present teachings are not limited to any specific combination of hardware circuitry and software.

The term "computer-readable medium" (e.g., data store, data storage, etc.) or "computer-readable storage medium" as used herein refers to any media that participates in providing instructions to processor <NUM> for execution. Such a medium can take many forms, including but not limited to, non-volatile media, volatile media, and transmission media. Examples of non-volatile media can include, but are not limited to, optical, solid state, magnetic disks, such as storage device <NUM>. Examples of volatile media can include, but are not limited to, dynamic memory, such as memory <NUM>. Examples of transmission media can include, but are not limited to, coaxial cables, copper wire, and fiber optics, including the wires that comprise bus <NUM>.

Common forms of computer-readable media include, for example, a floppy disk, a flexible disk, hard disk, magnetic tape, or any other magnetic medium, a CD-ROM, any other optical medium, punch cards, paper tape, any other physical medium with patterns of holes, a RAM, PROM, and EPROM, a FLASH-EPROM, any other memory chip or cartridge, or any other tangible medium from which a computer can read.

In addition to computer readable medium, instructions or data can be provided as signals on transmission media included in a communications apparatus or system to provide sequences of one or more instructions to processor <NUM> of computer system <NUM> for execution. For example, a communication apparatus may include a transceiver having signals indicative of instructions and data. The instructions and data are configured to cause one or more processors to implement the functions outlined in the disclosure herein. Representative examples of data communications transmission connections can include, but are not limited to, telephone modem connections, wide area networks (WAN), local area networks (LAN), infrared data connections, NFC connections, etc..

It should be appreciated that the methodologies described herein flow charts, diagrams and accompanying disclosure can be implemented using computer system <NUM> as a standalone device or on a distributed network of shared computer processing resources such as a cloud computing network.

The methodologies described herein may be implemented by various means depending upon the application. For example, these methodologies may be implemented in hardware, firmware, software, or any combination thereof. For a hardware implementation, the processing unit may be implemented within one or more application specific integrated circuits (ASICs), digital signal processors (DSPs), digital signal processing devices (DSPDs), programmable logic devices (PLDs), field programmable gate arrays (FPGAs), processors, controllers, micro-controllers, microprocessors, electronic devices, other electronic units designed to perform the functions described herein, or a combination thereof.

In various embodiments, the methods of the present teachings may be implemented as firmware and/or a software program and applications written in conventional programming languages such as C, C++, Python, etc. If implemented as firmware and/or software, the embodiments described herein can be implemented on a non-transitory computer-readable medium in which a program is stored for causing a computer to perform the methods described above. It should be understood that the various engines described herein can be provided on a computer system, such as computer system <NUM>, whereby processor <NUM> would execute the analyses and determinations provided by these engines, subject to instructions provided by any one of, or a combination of, memory components <NUM>/<NUM>/<NUM> and user input provided via input device <NUM>.

While this specification contains many specific implementation details, these should not be construed as limitations on the scope of what is claimed, but rather as descriptions of features that can be specific to particular implementations. Certain features that are described in this specification in the context of separate implementations can also be implemented, in combination, in a single implementation. Conversely, various features that are described in the context of a single implementation can also be implemented in multiple implementations, separately, or in any suitable sub-combination.

While operations are depicted in the drawings or claims in a particular order, this should not be understood as requiring that such operations be performed in the particular order shown or in sequential order, or that all illustrated operations be performed (some operations can be considered optional), to achieve desirable results. In certain circumstances, multitasking or parallel processing (or a combination of multitasking and parallel processing) can be advantageous and performed as deemed appropriate.

Claim 1:
A device, comprising:
a hydrogel matrix including a photoabsorber; and
a void architecture in the matrix, having a first vessel architecture and a second vessel architecture that are each tubular and branching;
wherein the first and second vessel architectures are fluidically independent from each other and wherein one or both of the first vessel architecture and second vessel architecture is formed from a model based on a tessellation of polyhedra.