Modification of aqueous phase density in fresh support baths to control rate of gelation

Abstract

Additive manufacturing methods, additive manufacturing systems (100), support material for additive manufacturing, and products thereof are provided. For example, the additive manufacturing methods includes depositing a structure material into a support material (108) based on a first computer model of an object, thereby forming a first portion of the object in the support material. The structure material exhibits a viscosity in a range of 1 mPa*s to 5000 mPa*s (1 cP to 5,000 cP) at 25 degrees Celsius. The support material exhibits a density in a range of 1.05 g / mL to 3 g / mL. The method includes repeating the depositing of the structure material as necessary to additively form the object.

Description

IN THE UNITED STATES PATENT AND TRADEMARK OFFICEPCT APPLICATION FORMODIFICATION OF AQUEOUS PHASE DENSITY IN FRESH SUPPORT BATHS TO CONTROL RATE OF GELATIONInventors: Brian Coffin, Adam Walter Feinberg, Daniel J. ShiwarskiRELATED APPLICATIONS

[0001] The present application claims priority to United States provisional patent application Serial No. 63 / 533,090, filed August 16, 2023, which is incorporated herein by reference in its entirety.BACKGROUND

[0002] Recently, 3 -dimensional (3D) bio-printing has emerged as a viable platform for engineering tissues, with exciting applications as platforms for drug discovery and disease modeling as well as new therapies for tissue regeneration. However, translation of these technologies from the laboratory into industry and the clinic has been limited, in large part due to the poor reproducibility of the 3D bio-printing process itself. There are challenges with 3D bio-printing.SUMMARY

[0003] In one general aspect, the present disclosure is related to an additive manufacturing method. The method comprises depositing a structure material into a support material based on a first computer model of an object, thereby forming a first portion of the object in the support material. The structure material exhibits a viscosity in a range of 1 cP to 5,000 cP at 25 degrees Celsius. The support material exhibits a density in a range of 1.05 g / mL to 3 g / mL. The method comprises repeating the depositing of the structure material as necessary to additively form the object.

[0004] Various embodiments and implementations of the present invention provide many benefits and improvements relative to prior additive printing techniques, such as, for example, techniques related to embedded printing. For example, the methods according to the present disclosure can enhance filament resolution, prevents formation agglomerates on the needle, control diffusion of structure material, control gelation of structure material, control of reaction rate, and enable a wider range of reactant concentrations to be utilized inthe support material. These and other benefits that are potentially realizable through various implementations of the present invention will be apparent from the description that follows.

[0005] It will be understood that the invention disclosed and described in this specification is not limited to the aspects summarized in this Summary. The reader will appreciate the foregoing details, as well as others, upon considering the following detailed description of various non-limiting and non-exhaustive aspects according to this specification.BRIEF DESCRIPTION OF THE DRAWINGS

[0006] Features and advantages of the examples presented herein, and the manner of attaining them, will become more apparent, and the examples will be better understood, by reference to the following description taken in conjunction with the accompanying drawings, wherein:

[0007] FIG. 1 is a block diagram of an example of an additive manufacturing FRE system according to the present disclosure, the X-axis is coming out of the page.

[0008] FIG. 2 is flow chart of an example of an additive manufacturing FRE method according to the present disclosure.

[0009] FIG. 3 A is a chart illustrating shear storage and loss moduli of thermally gelled Lifeink 200 high concentration collagen I bioink compatible with FRESH bioprinting, (n = 3)

[0010] FIG. 3B is a chart illustrating equilibrium storage moduli of tested Lifeink 200 compared to GelMA based hChaMP bioink and Omentum ECM moduli interpolated from published results. Data presented as mean ± standard error of mean (sem).

[0011] FIG. 4 A is a chart illustrating storage moduli of fibrin hydrogels over the course of 1 hour after preparation.

[0012] FIG. 4B is a chart illustrating equilibrium storage moduli of fibrin hydrogels after one hour of gelation. Significant differences observed when fibrinogen concentration exceeds 60 mg / mL. Comparisons between samples made by Brown-Forsythe and Welch ANOVA. Mean shear storage moduli of each condition 431, 482, 2525, and 6245 Pa respectively.

[0013] FIG. 4C is a chart illustrating comparison of 60 mg / mL fibrin hydrogels prepared with 0.1 and 1.0 U of thrombin per mg of fibrinogen. Only 2 replicates were completed for the 1.0 U condition due to depletion of the fibrinogen stock material. (*p<0.05, **p<0.01, ****p<0.0001).

[0014] FIG. 5A is images illustrating spontaneous formation of gel at extrusion needle tip and along length of needle during printing.

[0015] FIG. 5B is images illustrating wicking of fibrinogen solution up the length of the needle rather than hanging from the end of the needle during extrusion.

[0016] FIG. 6A is CAD drawings of strip model with isometric, side profile and top-down views.

[0017] FIG. 6B is a Geode preview model of strip models with a nozzle diameter of 160 pm, extrusion multiplier of 75%, print speed of 10 mm / s, 3 perimeters and single filament gap fill.

[0018] FIG. 6C is representative dark field stereomicrographs of released and washed printed strip models when fabricated with geode that added no wipe, water wipe, or a brush wipe (respectively, from left to right) between each printed layer. 60 mg / mL fibrin strips in support prepared in lx PBS with 5 U / mL thrombin.

[0019] FIG. 7A is a print pathing (geode) preview model of fibrin strip model with outer dimensions of 11 x 3 x 1 mm.

[0020] FIG. 7B is representative darkfield images of fibrin strip models printed in support compacted in 0%, 25%, and 50% iodixanol and supplemented with 0.1, 1, and 10 U / mL thrombin. Scale bars = 1 mm.

[0021] FIG. 7C is a graph illustrating strip width for fibrin strips illustrated in FIG. 7B.

[0022] FIG. 7D is a graph illustrating strip length for fibrin strips illustrated in FIG. 7B.

[0023] FIG. 7E is a graph illustrating infill filament diameter for fibrin strips illustrated in FIG. 7B. Feature dimensions compared with two-way ANOVA with Tukey’s test for interactions. Significance indicated by *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. Significance indicated for interactions driven by iodixanol concentration within one concentration of thrombin. Bar charts plotted as mean ± sem.

[0024] FIG. 8A is scanning electron microscopy images of 60 mg / mL fibrin scaffolds printed into 25% iodixanol support (density of 1.13 g / mL) supplemented with 0.01, 0.1, 1, or 10 U / mL thrombin viewed from top-down at low (600x), medium (lOOOx), and high (8000x) magnification. Scale bars = 50 pm at 600x and lOOOx and 5 pm at 8000x.

[0025] FIG. 8B is scanning electron microscopy images of 60 mg / mL fibrin scaffolds printed into 25% iodixanol support supplemented with 0.01, 0.1, 1, or 10 U / mL thrombin viewed in cross section at low (600x), medium (lOOOx), and high (8000x) magnification. Scale bars = 50 pm at 600x and lOOOx and 5 pm at 8000x.

[0026] FIG. 9A is a CAD model designed based on ASTMD-638-V with a shortened overall length of 50 mm. Other dimensions were designed according to the standard.

[0027] FIG. 9B is a geode preview model of print path with aligned 50% density infill parallel to the long axis of the model.

[0028] FIG. 9C is a brightfield image of 60 mg / mL fibrin dog bone printed in 25% iodixanol support supplemented with 10 U / mL thrombin.

[0029] FIG. 9D is top down and isometric renderings of dog bone model fixed into grips and mounting bracket.

[0030] FIG. 9E is a printed fibrin dog bone mounted in grips in an MTS tensile testing machine at initiation of tensile test.

[0031] FIG. 9F is a printed fibrin dog bone mounted in grips in an MTS tensile testing machine just prior to failure.

[0032] FIG. 10A is stress-strain diagrams calculated from raw force and displacement data for constructs printed support baths compacted in 25% iodixanol (density of 1.13 g / mL) and supplemented with 10 U / mL thrombin.

[0033] FIG. 10B is stress-strain diagrams showing low strain region of stress strain curves with linear regression fit to the raw data at strain values <1.5.

[0034] FIG. 10C is a chart illustrating Young’s moduli of 10 U / mL was 14.22 ± 1.568 kPa.

[0035] FIG. 10D is a chart illustrating the fibrin tensile specimens failed at a strain of 6.023 ± 0.54.

[0036] FIG. 10E is a chart illustrating the fibrin tensile specimens had a peak stress of 90.18 ± 13.69 kPa. Values reported as and bar charts plotted as mean ± SEM.

[0037] Corresponding reference characters indicate corresponding parts throughout the several views. The exemplifications set out herein illustrate certain embodiments, in one form, and such exemplifications are not to be construed as limiting the scope of the appended claims in any manner.DETAILED DESCRIPTION

[0038] Various examples are described and illustrated herein to provide an overall understanding of the structure, function, and use of the disclosed installation apparatus, fasteners, and methods of fastening. The various examples described and illustrated herein are non-limiting and non-exhaustive. Thus, the invention is not limited by the description of the various non-limiting and non-exhaustive examples disclosed herein. Rather, the invention is defined solely by the claims. The features and characteristics illustrated and / or described in connection with various examples may be combined with the features and characteristics of other examples. Such modifications and variations are intended to be included within the scope of this specification. As such, the claims may be amended to recite any features or characteristics expressly or inherently described in, or otherwise expressly orinherently supported by, this specification. Further, Applicant reserves the right to amend the claims to affirmatively disclaim features or characteristics that may be present in the prior art. The various examples disclosed and described in this specification can comprise, consist of, or consist essentially of the features and characteristics as variously described herein.

[0039] As used herein, “additive manufacturing” means a process of joining materials to make objects from 3D model data, usually layer upon layer, as opposed to subtractive manufacturing methodologies. For example, additive manufacturing can comprise fused deposition modeling (FDM) and Freeform Reversible Embedding (FRE). FDM can comprise extruding a material by heating it to a temperature above its melting temperature and depositing the extruded material in a pattern to form a layer of an object. Subsequent layers can be deposited on top of the previous layer as necessary to form an object.

[0040] FRE is similar to FDM, but instead of depositing a material on top of previous depositions or supports, FRE embeds structure material near other embedded deposits inside a support material and relies on the triggered assembly or reorganization of the material using targeted heating, photopolymerization, crosslinking, slow reaction kinetics, application of binders, and / or other curing technique. For example, the support material may provide divalent cations for crosslinking, such that when the structure material contact the support material, the structure material begins to cure.

[0041] For additive manufacturing techniques such as FDM, support materials are usually as stiff as the printed material, printed as part of the previous layer, and placed only underneath or neighboring the print layers to prevent deformations. In FRE, the support material can surround the extrusion nozzle and the print material can be deposited inside the support. The support material can be a non-newtonian fluid that allows for deposition of various materials while maintaining a buoyant, physical support for already embedded deposits of print material. When two embedded deposits of print material with a predetermined distance inside of the support material, they can fuse. After printing, the support material can be removed from the deposited print material to form a fully assembled object from the deposited print material.

[0042] In FRE, an object can be printed in any direction in 3D space and is not limited to layer-by-layer printing. For example, a structure can also be printed layer by layer in an X-Y plane, or a non-X-Y plane, such as the X-Z plane, or in a plane at any angle offset from the X-Y Plane. An object can also be printed utilizing FRE in a non-planar fashion, such as, for example, in a curved path such as a helix. Utilizing FRE can enable printing of objects with mechanical properties that are different in the plane of printing versus orthogonal to the planeof printing or other angle to the plane of printing. Additional details regarding the FRE process can be found in U.S. Patent No. 10,150,258, titled ADDITIVE MANUFACTURING OF EMBEDDED MATERIALS, filed January 29, 2016, which is hereby incorporated by reference herein.

[0043] As the demand for donor tissue and organs continues to outpace the supply, clinicians are turning to regenerative medicine and tissue engineering strategies to create tissue de novo. 3D bioprinting has emerged as a way to build these tissues using robotic control to precisely pattern cells and biological hydrogels in a layer-by-layer process. However, this technology has been slowed by the difficulty of printing these soft, deformable materials into complex 3D architectures that recapitulate anatomic structure from the micro to macro length scale. Embedded 3D bioprinting, such as FRE is a FRE of Suspended Hydrogels (FRESH), can addresses this challenge by providing a sacrificial support that prevents tissue deformation during bioprinting to produce highly relevant constructs such as heart valves, cardiac tissue scaffolds, and perfusable vascular networks. These sophisticated bioprinted constructs could prove very useful as clinical diagnostic tools or direct tissue replacements. However, for an approved therapy, ensuring accuracy of the manufactured product may be desired. To continue to move bioprinting techniques such as FRESH towards clinical application, the present disclosure provides improved printing capabilities to allow for high-fidelity reproduction of patient-specific organic shapes, and / or improved quality control of geometric fidelity. In embedded 3D bioprinting techniques such as FRESH and related approaches, a challenge can be controlling the gelation of printed structure material filaments within the support material, which may become more difficult with low viscosity structure materials.

[0044] The present disclosure provides methods, systems, and materials that can enhance quality control, and process reliability during the FRE process, other 3D bio-printing process, or other additive manufacturing process. The additive manufacturing method comprising depositing a structure material into a support material based on a first computer model of an object, thereby forming a first portion of the object in the support material. The method comprises repeating the depositing of the structure material as necessary to additively form the object. The structure material comprises a viscosity in a range of 1 cP to 5,000 cP at 25 degrees Celsius and the support material exhibits a density in a range of 1.05 g / mL to 3 g / mL. The methods according to the present disclosure can enhance filament resolution, prevents formation agglomerates on the needle, control diffusion of structure material, control gelation of structure material, control of reaction rate, and enable a wider range of reactantconcentrations to be utilized in the support material. It is also believed the present disclosure applies to other additive manufacturing techniques in addition to FRE.

[0045] A challenge in the fabrication of scaffolds in embedded 3D bioprinting can be the formation of an agglomerate at the extrusion needle tip, which effectively can clog the extruder and decrease the fidelity, uniformity, and cell viability of the biofabricated part. For example, when using fibrinogen, a fibrin plug may form due to the rapid gelation of fibrinogen into fibrin networks when activated by thrombin in the support material. The support material according to the present disclosure can enable fabrication of structurally complex 3D fibrin scaffolds and control the mechanical properties and improve print fidelity and resolution. The increase in density of the support bath can decrease diffusion of the structure material into the support bath, which may be more pronounced with low viscosity structure material, which can spatially restrict where gelation occurs, thereby enabling higher resolution 3D printing of structures (e.g., structure that are <200 pm in size).

[0046] Referring to FIG. 1, a block diagram illustrating an example of an additive manufacturing system 100 for additive manufacturing, such as, for example, FRE, according to the present disclosure is provided. The system 100 comprises an extruder assembly 102, a computer system 104, and a material deposition region 106. In various examples, a detector, additional extruders, and / or nozzles may be added to the additive manufacturing system to increase the printing capabilities of the additive manufacturing system. The system 100 can be capable to print structure material in the support material 108 in the material deposition region 106 using the extruder assembly 102 to form an object 114.

[0047] The support material 108 can mechanically support at least a portion of the embedded structure material (i.e., object 114), maintain the intended geometry of the embedded structure material, and inhibit deformation of the structure material during the FRE additive manufacturing process. For example, the embedded structure material can be held in position within the support material 108 until the structure material is cured. The support material 108 can be stationary at an applied stress level below a threshold stress level and can flow at an applied stress level at or above the threshold stress level during the FRE additive manufacturing process.

[0048] The support material 108 can exhibit a density in a range of 1.05 g / mL to 3 g / mL, such as, for example, 1.1 g / mL to 1.5 g / mL or 1.1 g / mL to 1.3 g / mL. The density of the support material can enhance filament resolution, control diffusion, prevent formation of agglomerates on the needle, and enable a wider range of reactant concentrations to be utilized in the support material. For example, the density of the support material 108 can decreasediffusion of the structure material into the support material 108, which may be more pronounced with low viscosity structure material, which can spatially restrict where gelation occurs, thereby enabling higher resolution 3D printing of structures.

[0049] The support material 108 can be a viscoplastic material with Bingham plastic-like rheological behavior. The support material 108 may demonstrate a significant shear thinning behavior such that the support material 108 acts like a solid material during deposition of the structure material and then acts like a fluid when the nozzle 110 is moved through the support material 108 such that the movement of the nozzle 110 does not disturb deposited structure material. A drop in viscosity of the support material 108 under dynamic loading can make the support material 108 suitable for FRE. For example, in FRE, the dynamic loading can be caused by the force of the nozzle 110 through the support material 108, affecting the support material 108 in a number of ways. The extruder assembly 102 can be configured to change the support material 108 by imposing a mechanical load via shear, pressure, or vibration. The extruder assembly 102 can be configured to irradiate or heat the support material 108 to thin it. In various examples, the support material 108 can reduce viscosity under vibration, heating, or irradiation that occurs locally to the extruder assembly 102.

[0050] The support material 108 can comprise other materials with viscoplastic behavior, such as Herschel-Bulkley fluid. Bingham plastics and Herschel-Bulkley fluids are viscoplastic materials included in the “shear-thinning” or “yield-stress fluid” category. Below a specific shear stress, these materials appear as a solid material. Above a threshold shear force, these materials behave as a fluid. A Bingham plastic may not necessarily “shear thin,” but rather may act much like a Newtonian fluid once it begins to flow. In contrast, the Herschel-Buckley fluid undergoes shear thinning once it begins to flow.

[0051] The support material 108 can comprise at least two phases. For example, the support material 108 can comprise particles (e.g., microparticles) dispersed in a diluent. In various examples, the support material 108 can be a hydrogel. The at least two phases can enable the Bingham plastic-like rheological behavior.

[0052] The diluent can be aqueous or non-aqueous depending on the desired properties of the support material. In various examples, where the diluent is aqueous (e.g., comprises water), and the diluent can comprise at least one additive selected from the group consisting of cesium chloride, cesium bromide, cesium iodide, cesium sulfate, cesium nitrate, rubidium chloride, rubidium bromide, rubidium iodide, rubidium sulfate, rubidium nitrate, ficoll, sucrose, lymphocyte separation medium, sodium diatrizoate, ammonium chloride, barium chloride, calcium chloride, cesium chloride, hydrogen-chloride, iron (III) chloride, lithiumchloride, magnesium chloride, potassium chloride, sodium chloride, strontium chloride, ammonium sulfate, copper (II) sulfate, magnesium sulfate, manganese (II) sulfate, nitric acid, phosphoric acid, silver nitrate, 1,2-ethanediol, b-D-fructose, a-D-glucose, glycerol, b-D- lactose, a-Maltose, D-mannitol, sucrose, urea, sodium metatungstate, sodium polytungstenate, lithium heteropolytungstates (LST), iodixanol, other additive for obtaining a desired density of the support material 108, or a combination thereof. In certain examples, the additive comprises iodixanol.

[0053] In various examples, the support material 108 can comprise 5% to 80% by weight of the additive based on a total weight of the support material 108, such as, for example, 20% to 60% by weight of the additive or 30% to 60% by weight of the additive based on a total weight of the support material 108. For example, the support material 108 can comprise 5% to 80% by weight of iodixanol based on a total weight of the support material 108, such as, for example, 20% to 60% by weight of iodixanol or 30% to 60% by weight of the iodixanol based on a total weight of the support material 108.

[0054] The particles can comprise gelatin, alginate, calcium chloride, soy lecithin, Carbopol, acrylamide, agarose, alginate, a cell spheroid, a cell organoid, gellan gum, hyaluronic acid, laponite nanoclay, nanoclay, pluronic F127, poly(ethylene oxide), oxidized bacterial cellulose, xanthan gum methacrylate, fumed silica, hyaluronic acid-norbomenefibronectin & hyaluronic acid, K-Carrageenan, decellularizaed extracellular matrix, other suitable particle forming compound, or a combination thereof. For example, the particles can comprise gelatin.

[0055] The support material 108 can comprise a reactant. The reactant can be capable to increase a viscosity of the structure material and / or cure the structure material. For example, the reactant can catalyst a reaction of the structure material. The reactant can be a catalyst, such as, for example, an enzyme. For example, the reactant can comprise thrombin, a photo initiator, a pH buffer, calcium, magnesium, sodium nitrate, a salt solution, transglutaminase, or a combination thereof.

[0056] The photo initiator can comprise be suitable for methacrylated polymers. For example, the photo initiator can comprise Irgacure-2959, lithium phenyl-2,4,6- trimethylbenzoylphosphi-nate (LAP), eosin Y, or a combination thereof.

[0057] The pH buffer can cure acidified materials. The pH buffer can comprise 2-[4-(2- hydroxyethyl)piperazin-l-yl]ethane-l -sulfonic acid (HEPES).

[0058] The calcium and / or magnesium can cure alginate and / or other ionic materials.

[0059] The sodium nitrate can cure silk fibroin.

[0060] In various examples, the reactant can comprise thrombin. For example, the support material 108 can comprise 0.001 to 10,000 units of thrombin per mL, such as, for example, 0.01 to 10,000 units of thrombin per mL, 0.1 to 10,000 units of thrombin per mL, 1 to 10,000 units of thrombin per mL, 0.01 to 20 units of thrombin per mL, 0.01 to 10 units of thrombin per mL, 0.1 to 20 units 10 units of thrombin per mL, 0.1 to 10 units of thrombin per mL, 1 unit to 20 units of thrombin per mL, 1 unit to 10 units of thrombin per mL, 2 units to 10 units of thrombin per mL, 3 units to 10 units of thrombin per mL, 4 units to 10 units of thrombin per mL, 5 units to 20 units of thrombin per mL, or 5 units to 10 units of thrombin per mL. The density of the support material 108 can enable use of higher concentrations of reactant. In various examples, the density of the support material 108 can inhibit undesirable agglomeration of the structure material on the nozzle 110 during printing.

[0061] Depending on the printing technique, the support material 108 can be clear or opaque. The support material 108 can further comprise other components, such as, for example, a surfactant, a thickening agent, or a combination thereof. In various examples, the support material 108 does not comprise a thickening agent.

[0062] The structure material can comprise a yield stress material that transitions between a fluid (e.g., liquid) state to a solid or semi-solid state by application of a pressure. For example, the structure material can be in a solid or semi-solid state in the extruder assembly 102, a pressure can be applied to the structure material to transition the structure material to a fluid state such that the structure material can flow through the nozzle 110 and can be deposited into the support material 108. After leaving the nozzle 110, the applied pressure to the structure material is removed and the structure material can transition into a solid or semisolid state and thereby resisting deformation while in the material deposition region 106. The density of the support material 108 can enhance the structure material’s resistance to deformation in the support material 108 and / or decrease diffusion of the structure material into the support material 108.

[0063] The structure material can comprise a polymer, such as, for example, a hydrogel, a thermoset polymer, a thermoplastic, or a combination thereof. The polymer can comprise a polymeric resin (e.g., a pre-polymer resin), a curing agent, a contrast agent, and / or other additives. For example, the polymer can comprise a collagen material, an alginate material, a decelluarized extracellular matrix material, a fibrinogen material, a fibrin material, a hyaluronic acid material, a protein material, a polysaccharide hydrogel material, a synthetic gel material, an elastomeric polymer material, a rigid polymer material, a Matrigel, or acombination thereof. In various examples, the polymer can comprise a fibrinogen material. In certain examples, the polymer can comprise a fibrin material.

[0064] The structure material can comprise at least 70% polymer based on the total weight of the structure material, such as, for example, at least 80% polymer based on the total weight of the structure material or at least 90% polymer based on the total weight of the structure material. In various examples, the structure material comprises a fluid that transitions to a solid or semi-solid state after deposition.

[0065] The structure material and / or support material 108 can comprise a contrast agent.

[0066] The structure material can exhibit a viscosity in a range of 1 cP to 5,000 cP at 25 degrees Celsius as measured using a discovery hybrid rheometer 2 (DHR-2, TA Instruments) having a 40 mm 1 degree cone geometry, temperature-controlled stage, and solvent trap, such as, for example, 1 cP to 4,000 cP, 1 cP to 3,000 cP, 1 cP to 2,000 cP, or 1 cP to 1,000 cP as measured using a discovery hybrid rheometer 2 (DHR-2, TA Instruments) having a 40 mm 1 degree cone geometry, temperature-controlled stage, and solvent trap. The density of the support material 108 can enable lower viscosity structure material to be printed with high fidelity and / or accuracy.

[0067] The extruder assembly 102 may be a syringe-based extruder, which can include a reservoir 112 (e.g., a barrel of a syringe) for receiving and storing structure material or support material, and a nozzle 110 (e.g., a needle) which can be in fluid communication with the reservoir 112 and can receive the structure material or the support material from the reservoir 112. For example, the reservoir 112 can comprise structure material and the structure material can be extruded through the nozzle 110 can be configured to deposit the extruded structure material in the support material 108 disposed in the material deposition region 106.

[0068] In various examples, the first extruder assembly 102 and / or additional components can comprise a gantry or other robotic device to support and / or move the extruder assembly 102 relative to the material deposition region 106. Optionally, the extruder assembly 102 can comprise a motor assembly or other movement assembly configured to translate and / or rotate the gantry and / or robotic device. In various examples, the extruder assembly comprises an actuator (e.g., a motor) configured to depress a plunger into the reservoir 112 to extrude material through the nozzle 110 into material deposition region 106 as nozzle 110 is translated through the material deposition region 106 to additively form an object 114.

[0069] The computer system 104 is in signal / data communication with the extruder assembly 102 (such as via a wired and / or wireless data bus or link). The computer system 104 can beconfigured through programming to control the operation of the extruder assembly 102. The computer system 104 can also receive data from and send data (e.g. control data) to the extruder assembly 102a. The components in the additive manufacturing system 100 may be in communication with the computer system 104 via any suitable type of data bus (e.g., parallel or bit serial connections).

[0070] The computer system 104 comprises one or more processors 120 operatively coupled to one or more non-transitory memories 122 (only one processor 120 and one memory 122 are shown in FIG. 1 for simplicity). The processor 120 may comprise one or multiple processing cores. The memory 122 can comprise primary storage (e.g., main memory that is directly accessible by the processor 120, such as RAM, ROM processor registers or processor cache); secondary storage (e.g., SSDs or HDDs that are not directly accessible by the processor); and / or off-line storage. The memory 122 stores computer instructions (e.g., software) that are executed by the processor 120. The processor 120 can be configured (through execution of the software stored in the memory 122) to control operation of the extruder assembly 102 to thereby control the deposition of the structure material through the nozzle 110. For example, the processor 120 can control the flow rate of material through the nozzle 110 (e.g., by the actuation rate of a plunger in the extruder assembly 102) and / or the pose of the extruder assembly 102 relative to the material deposition region 106.

[0071] The memory 122 can store a digital or electronic computer model 124 of the object 114 to be manufactured by the additive manufacturing process. The computer model 124 can be loaded locally into the memory 122 or can be downloaded from another device (e.g., another computer device, cloud) that is in data communication with the computer system 104. To that end, the computer system 104 may comprise a network interface controller (NIC) that connects the computer system 104 to a computer network. The computer model 124 can be in a variety of different digital or electronic formats, such as an STL file, a OBJ file, a FBS file, a COLLADA file, a 3DS file, an IGES file, a STEP file, a VRML / X3D file, a point cloud, or another 3D model file format type. The computer model 124 can be generated from image data of a biological structure, an engineered structure, a computationally derived structure, or a combination thereof. In various examples, the biological structure can be generated from the image data of a patient.

[0072] The optional detector can comprise a light-based camera, a brightfield microscope, a fluorescence microscope, CT scanner, a MRI scanner, an OCT scan, a laser scan, an ultrasound scan, or a combination thereof.

[0073] The processor 120 can be configured to separate (e.g., slice (utilizing Slic3r, Cura, Simplify3D, Skeinforge, KISSlicer software, etc.)) the computer model 124 into different segments 126, each segment containing a portion of the computer model 124. In various examples, the processor 120 can be configured to convert the computer model 124 to a different 3D model file format prior to separating.

[0074] Each segment 126 can be a layer, 114a and 114b, of the object 114 to be deposited, a portion of a layer, 114a and 114b, of the object 114 to be deposited, or other geometry of the object 114. The segments 126 can be created based on a design of the computer model 124. For example, a segment of segments 126 can comprise an overlapping region, an overhang region, an infill region, a perimeter region, another region of the object 114, or a combination thereof. Each segment 126 may or may not be in the X-Y plan and a segment can be in a non-X-Y plane, such as the X-Z plane, the Y-Z plane, other plane offset from the X-Y plane, or a non-planar segment, such as, for example, a curve. Utilizing various segments 126 for different regions of the object 114 can enable variations of machine path instructions and / or print parameters for each segment 126. Therefore, the machine path instructions and / or print parameters can be selected to suit the particular geometry to be printed in the respective segment 126.

[0075] From the segments 126, the processor 120 can be configured to create machine path instructions (e.g., G-code instructions) 132 for the segments 126 based on the design of the portion of the computer model 124 in the respective segment 126. The machine path instructions 132 can be stored in the memory 122. The machine path instructions 132 can comprise print parameters 134 and can be executed by the processor 120 to cause the processor 120 to control the operation (e.g., pose, extrusion, suction, cure) of the extruder assembly 102 or other device (e.g., structure material removal device such as a suction tube).

[0076] The nozzle 110 can be configured to deposit a structure material into the support material 108 by applying a force to the structure material in the reservoir 112 such that the structure material can flow from the reservoir 112 through the nozzle 110. The structure material can comprise a yield stress, a thixotropic property, an increased viscosity, or a combination thereof. In examples where the structure material comprises a yield stress, the force applied can be at least the yield stress. In certain examples, applying the force to the structure material can cause the structure material to flow through the nozzle 110. For example, with an increase viscosity, the force can overcome the increased viscosity and cause the material to flow through the nozzle 110. In examples wherein the structure materialcomprises a thixotropic property, the thixotropic property can cause the time scale to start flow of the structure material to be longer than the printing process.

[0077] In various examples, a plunger can be translated through the reservoir 112. In various examples, the force can be pneumatically applied or the deposition can be controlled by a cavity pump. The application of the force can cause the material in the reservoir 112 to change form a solid or semi-solid state into fluid state (e.g., liquid), so that the material can be deposited into the material deposition region 106. The structure material can be suspended in the support material 108 at a location where the structure material was deposited by the nozzle 110 within the support material 108. Since the processor 120 can control the extruder assembly 102 and nozzle 110, the deposition of the structure material by the nozzle 110 can be based on the machine path instructions 132 and associated print parameters 134 as executed by the processor 120.

[0078] The extruder assembly 102 can move the nozzle 110 in two-dimensions when depositing structure material similar to FDM or in three-dimensions when depositing material, i.e., simultaneously in the X, Y, and Z directions. Further, the extruder assembly 102, nozzle 110, and / or material deposition region 106 can be rotatable. The machine pathing instructions 132 can be defined according to both Cartesian and polar coordinates, which can allow for the production of objects having complex geometries or very specific mechanical properties. 3D movement of the nozzle 110 during deposition of the structure material can enable, for example, additive manufacture of a helical spring in one constant motion. In various examples, other complex geometries are achievable with robotic arm assemblies capable of simultaneously controlling movement with six degrees of freedom (i.e., in any Cartesian or rotational direction).

[0079] The depositing of the structure material 108 can be repeated as necessary to additively form an object. For example, the processor 120 can control the nozzle 110 to deposit the structure material in layers, such as layers 114a and 114b, in order to additively form the object 114 in the support material 108 based on the computer model 124, another plane, and / or non-planar movement. In some examples, layer 114a can be deposited prior to layer 114b. Layer 114a may not be partially and / or fully cured prior to deposition of layer 114b. Thus, the processor 120 can control the nozzle 110a to deposit layer 114b proximal to (e.g., adjacent, in contact with, directly on top of) the layer 114a such that the deposition of the layer 114b contacts the layer 114a. For example, deposition of layer 114b can change the shape of at least a portion of layer 114a. The changed shape of at least a portion of layer 114a can be one that cannot be achieved by simple extruding out of the nozzle 110.Changing the shape of the layer 114a by deposition of layer 114b can increase contact surface area between the layers, 114a and 114b, decrease void space between the layers, 114a and 114b, improve adhesion between the layers, 114a and 114b, or a combination thereof.

[0080] The material deposition region 106 can be configured for mechanically supporting and / or holding the support material 108 during FRE additive manufacturing. For example, the material deposition region 106 can comprise a vessel in which the support material 108 is disposed and a platform on which the vessel is supported. The material deposition region can comprise a motor and / or actuator that can move the platform in 3D space as needed.

[0081] The structure material can be curable and after curing, the structure material can be considered cured. The object 114 can be at least partially cured in the support material 108 after deposition of the structure material. In various examples, the structure material can be at least partially cured prior to removing the support material 108. In some examples, the structure material may not be cured until after removing the support material 108. As used in this specification, the terms “cure” and “curing” refer to the chemical crosslinking of components in the structure material. Accordingly, the terms “cure” and “curing” do not encompass solely physical drying of structure material through solvent or carrier evaporation. In this regard, the term “cured,” as used in this specification, refers to the condition of the structure material in which a component of the structure material forming the object 114 has chemically reacted to form new covalent bonds in the structure material (e.g., new covalent bonds formed between a polymeric resin and a curing agent), new ionic bonds, new hydrogen bonds, new Vander walls bonds, or combinations thereof.

[0082] For example, curing of the object 114 can comprise cross-linking. The object 114 can be treated through various cross-linking techniques to selectively increase the rigidity of the overall object 114 or portions thereof. Cross-linking can be induced by various mechanisms such as, for example, photo mechanisms (e.g., exposing the structure material to UV light), ionic mechanism, enzymatic mechanism, pH mechanisms (e.g., exposing the structure material to a different pH) or thermally driven mechanisms (e.g., cooling, heating). In various examples, the support material 108 can include a cross-linking agent or pH suitable for curing the structure material as it is deposited into the support material 108.

[0083] The mechanical properties of the object 114 can be controlled by controlling the amount of curing that occurs within the object 114. For example, the machine pathing instructions 132 can be modified to control the amount of crosslinking that occurs within the object 114. For example, the extruder assembly 102 and / or other assembly can comprise aUV light and can selectively subject the embedded structure material to the UV light as desired.

[0084] The object 114 can be at least partially removed from the support material 108. Removing the support material 108 may include heating the support material 108, cooling the support material 108, removing cations to disrupt crosslinking of the support material 108, physically removing the support material 108, vibration, irradiation with ultraviolet, infrared, or visible light, application of a constant or oscillating electric or magnetic field, other mechanism, or a combination thereof. For example, the support material can comprise a thermoreversible material and removing the support material can comprise heating the support material to a threshold temperature at which the support material transitions from a solid or semi-solid state to a liquid state.

[0085] The methods for additive manufacturing herein, such as those illustrated in described in FIG. 2 below, can be implemented in whole or in part as computer-executable instructions stored in the memory 122 of the computer system 104 that, when executed by a processor 120 of the computer system 104, cause the computer system 104 to perform the enumerated steps. The computer instructions can be implemented as one or more software modules 116 stored in the memory 122 that are each programmed to cause the processor 120 to execute one or more discrete steps of the processes described herein or other functions. For example, the software modules 116 can comprise a separation module programmed to convert the computer model 124 into segments; a conversion module programmed to convert the segments 126 into computer instructions (e.g., G-code) for controlling the movement of the extruder assembly 102 to fabricate the object 114; an imaging module for controlling imaging parameters and movement of a detector; a modeling module programmed to receive, store, create, and / or modify part files of objects to be fabricated; and a robotic control module programmed to control the extruder assembly 102 according to the instructions generated by the conversion module to fabricate the object 114. Various other modules can be implemented in addition to or in lieu of the aforementioned modules. In certain examples, the processes described herein can be executed across multiple computer systems that are communicably connected together in a network, a computer system communicably connected to a cloud computing system configured to execute one or more of the described steps, and so on.

[0086] Referring to FIG. 2, a flow chart illustrating an additive manufacturing method according to certain implementations of the present disclosure is provided. The method can comprise receiving, by the processor 120, a computer model 124 of an object 114 at step 202.At step 204, the processor 120, executing the separation module software, can separate (e.g., slice) the computer model into different part segments and the processor 120, executing the conversion module, can create machine path instructions (e.g., G-code instructions) based on the design computer model. The machine path instructions can be stored in memory 122. The support material 108 can be placed in the material deposition region 106 prior to printing of the object.

[0087] The method can comprise, at step 208, depositing a structure material, by the nozzle 110, into the support material 108 such as, for example, a first portion (e.g., the first layer 114a) of the object 114 can be deposited in the support material 108a. The structure material and the support material together are also referred to herein as an assembly.

[0088] The depositing of structure material at step 208 can be repeated over as many iterations as necessary to additively form the object 114. Each iteration can deposit portions (e.g., the second layer 114b) of the structure material and the iterations can be repeated until additive formation of the object 114 is complete (if not aborted earlier).

[0089] Thereafter, at step 210, the structure material can be at least partially cured after depositing and then, at step 212, the support material can be at least partially removed from the object 114. The curing can occur prior to, during, after, or a combination thereof, removal of the support material at step 212.

[0090] The methods for additive manufacturing and systems for additive manufacturing described herein can be used to create various products. The products can be various product types, such as, for example, a soft structure, a bioprosthetic, a scaffold, a medical device, an implantable device, a gasket, a tube, a seal, an aerospace part, an automotive part, a building component, or other structures that may be additively manufactured. In various examples, the product (e.g., object 114) can be surgically fit into a patient.

[0091] EXAMPLES

[0092] Various aspects, benefits and features that are potentially realizable through implementation of the present invention will be more fully understood by reference to the following examples, which provide illustrative non-limiting aspects of the invention. It is understood that the invention described in this specification is not necessarily limited to the examples described in this section.

[0093] 3D Bioprinting of Fibrin Using FRESH

[0094] Introduction

[0095] Fibrin in an attractive material for tissue engineering applications for its ability to bind cells and growth factors, promote angiogenic activity, undergo degradation, and highmechanical extensibility. Fibrin networks form when fibrinogen molecules circulating throughout blood plasma are exposed to the enzyme thrombin which cleaves fibrinopeptides from fibrinogen chains to form fibrin monomers which undergo a modified polycondensation reaction to form 3D hydrogel networks. Fibrin hydrogels form in vivo following traumatic injury and bind platelets to staunch blood flow to maintain hemostasis. Each repeat unit in the fibrin network has four RGD cell-integrin, two AGDV, leukocyte integrin axiP?, and vascular endothelial cadherin binding sites to promote cell attachment. The fibroblasts, macrophages, and endothelial cells attached to the fibrin network act in concert to restore function to the damaged tissue. Fibrin networks are broken down through plasmin-mediated fibrinolysis and replaced by cell secreted extracellular matrix (ECM) which is integrated into the newly formed tissue. Endothelial cells in the presence of proangiogenic growth factors actively invade the fibrin network to create new microvascular networks during the fibrinolytic process. The mechanical properties of fibrin hydrogels are significantly different than other hydrogel ECM proteins commonly used in tissue engineering applications such as collagen I, alginate, and methacrylated gelatin (GelMA). For example, fibrin networks are highly elastic with strain at failure >226% whereas collagen isolated from tendon fractures at a strain of -12%. Fibrin is one of the softest materials in the body with a Young’s modulus of fibrin is on the order of 0.1-5 kPa. This is three to four orders of magnitude lower than the Young’s moduli of other structural ECM proteins including collagen (300-500 MPa), a-keratin (2,000 MPa), and actin (1,800-2,500 MPa). The abundance of cell binding motifs, soft material properties and degradability make fibrin an ideal cell scaffold as it promotes rapid cell attachment and remodeling of the tissue according to the needs of the cells attached to the scaffold. However, the soft nature of fibrin scaffolds limits the number of compatible biofabrication methods, and most fibrin scaffolds are fabricated by casting thrombin and fibrinogen solutions into a mold to form an isotropic scaffold. To build larger architecturally complex models that recapitulate the structural anisotropy observed in natural organs, advanced biofabrication techniques such as 3D bioprinting need to be employed. However, fibrinogen is very difficult to 3D bioprint and has not been utilized to fabricate stand-alone fibrin scaffolds or cell laden tissues to date. The lack of adoption in extrusion bioprinting can be attributed to its low viscosity, low gel strength, short degradation time of 3 - 4 weeks in vitro, and difficulty of initiating enzymatic gelation under atmospheric printing conditions. These constraints severely limit the complexity of parts that can be 3D printed from unmodified fibrinogen bioinks.

[0096] Freeform reversible embedding of suspended hydrogels (FRESH) is an embedded bioprinting technique developed to print soft hydrogel materials and has the potential to overcome many limitations posed by traditional extrusion bioprinting techniques to fabricate fibrin scaffolds. Unlike traditional bioprinting techniques FRESH is compatible with low viscosity bioinks and has been utilized to print blended fibrinogen, collagen I, Matrigel, hyaluronic acid, alginate, and gelatin methacryloyl bioinks into acellular and cell-laden scaffolds. Despite the progress in fabricating self-supporting soft biologic scaffolds, FRESH 3D bioprinting has not been able to fabricate self-supporting structures from unmodified fibrinogen bioinks to date. Printed fibrin scaffolds utilize secondary supporting materials either blended with fibrinogen directly to form interpenetrating networks on extrusion or codeposited with a second material, such as collagen I, to impart mechanical reinforcement to the scaffold. FRESH is a complex manufacturing process with interactions between the bioink, print pathing parameters, gelatin microparticle support, and the composition of the fluid phase in the support bath playing roles in determining the success of printing.

[0097] Here, we sought to improve FRESH printing of fibrin scaffolds by understanding how interactions between fibrinogen concentration, composition of the support bath fluid phase and thrombin concentration impacts print fidelity, microstructure, and mechanical properties of printed fibrin scaffolds. To achieve this, we first evaluated the gel strength of materials known to fabricate self-supporting scaffolds with FRESH 3D bioprinting to establish a threshold gel strength requirement. Second, we characterized the relationship between fibrinogen and thrombin concentration on the storage modulus of cast gels to select a fibrinogen concentration which should be compatible with FRESH bioprinting. Third, we used this information to investigate how the thrombin concentration and fluid density in the support bath can be modified to improve the resolution of printed fibrin scaffolds. Our results show that increasing the density of the support baths fluid phase enhances filament resolution, prevents formation of a fibrin gel on the extrusion needle, and enables a wider range of thrombin concentrations to be utilized in the support bath. This suggests that by altering fluid density of the prepared support controls the rate at which initiators in the fluid phase interact with printed filaments which is likely applicable to other embedded bioprinting material systems. Finally, we evaluate the effect of printing parameters on the microstructure and the tensile strength of printed fibrin. We combine this knowledge to print large fibrin scaffolds ~20 x 7 x 15 mm which is beyond the geometric complexity and scale previously achieved.

[0098] Materials and Methods

[0099] Rheological Characterization of Cast Fibrin

[0100] The rheological properties of cast fibrin was investigated with methods adapted from Zuidema for characterization of hydrogels for tissue engineering strategies. A discovery hybrid rheometer 2 (DHR-2, TA Instruments) was utilized to characterize all samples. A 40 mm 1 degree cone geometry, temperature-controlled stage, and solvent trap were installed on the DHR-2. To characterize mechanical properties of bioink gels, approximately 350 pl of bioink was loaded onto the rheometer platform chilled to 4°C. Fibrinogen stock was prepared by dissolving bovine fibrinogen (Sigma, 341573) in lx PBS without calcium or magnesium. Acellular fibrin samples were prepared by diluting fibrinogen stock in lx PBS with calcium in a microcentrifuge tube to concentrations of 30, 60, 90, and 120 mg / mL. The thrombin was deposited on the rheometer stage then fibrinogen. The geometry was lowered to trim gap and excess material was removed. The geometry was lowered to the test gap and a solvent trap installed to limit evaporation during the test. The stage temperature was raised to 37°C while applying a 1% oscillatory straining at a frequency of 1Hz for one hour. Once the sample fully gelled the linear viscoelastic regime for the sample was confirmed by performing a strain amplitude sweep from 0.01-100% strain and a frequency sweep from 0.01-10 Hz at 1% strain. These conditions are known to be within the linear viscoelastic regime required for small angle oscillatory strain measurements. The equilibrium storage modulus, loss modulus, crossover modulus and gel fracture point were determined based on these tests and three replicates were completed for each condition. Raw data was imported into GraphPad Prism for statistical analysis and plot generation.

[0101] Gelatin Microparticle Synthesis

[0102] Gelatin microspheres were synthesized using a complex coacervation process according to published methods. To prepare a batch of gelatin microparticles the following reagents were dissolved in 450 mL of room temperature absolute ethanol and 450 mL distilled water preheated to 65°C under stirring: 27 g (3% w / v) gelatin type B (Fisher G7- 500), 1.125 g (0.125% w / v) pluronic F-127 (Sigma, P2443), and 1.35 g (0.15% w / v) gum arabic from acacia (Sigma, G9752). The solution was adjusted to pH 5.65 - 5.8 by dropwise addition of 1 N HC1, covered and mixed for >8 hours at 450 RPM. The solution was evenly distributed between 250 mL containers and compacted at 300 ref for 2 minutes. The isolated gelatin microparticles were washed three times in room temperature distilled water and stored at 4°C prior to use.

[0103] FRESH Support Preparation

[0104] Gelatin microparticles were prepared for FRESH printing by isolating microparticles at 1000 ref for 3 minutes and washed in 50 mM 4-( 2-hydroxy ethyl)- 1- piperazineethanesulfonic acid (HEPES) in distilled water adjusted to pH 7.4. Support was compacted at 500 ref for 3 minutes then washed in lx PBS two times. Support with fluid phase densities (p) of 1.0, 1.15, and 1.28 g / mL was prepared by adjusting the concentration of iodixanol in the fluid phase to 0%, 25%, and 50% (w / v) respectively. Support with 0% iodixanol was washed once more in lx PBS. Support with 25% iodixanol was prepared by compacting support for 5 minutes at 2000 ref, pouring off the supernatant and taking the mass of the compacted support. For every 1 gram of compacted support, 0.714 g of OptiPrep (StemCell Technologies, 07820) and mix with a vortex mixer for 60 seconds. Support with 50% iodixanol was prepared from 25% iodixanol support by isolating particles for 5 minutes at 4000 ref, and adding 2.5 g OptiPrep per gram of compacted support. Support solutions were supplemented with thrombin (Sigma, T4648) at 0.01, 0.10, 1.00 or 10.00 U / mL according to the experimental protocol. The solutions were homogenized with a vortex mixer for 60 seconds and transferred to a 10 mL BD syringe and degassed in a vacuum chamber for 30 minutes. Support in lx PBS was compacted at 1750 ref for 5 minutes. Support in 25% and 50% iodixanol was compacted at 4000 ref for 5 and 15 minutes respectively. Compacted slurry qualitatively evaluated for yield stress behavior by pouring off the excess fluid phase then tipping 10 mL syringe inlet to -20° below the horizon. If material flows, then it is not compacted sufficiently for printing and recentrifuged at the set rate for an additional 5 minutes to ensure adequate compaction. Compacted gelatin microparticles are transferred to a print container by inserting a paperclip into the barrel followed by the plunger.

[0105] FRESH Printing of Fibrin

[0106] 3D bioprinting of acellular fibrin was performed using FRESH microparticle support on a custom, open-source 3D bioprinter. The 3D bioprinter used in this study was a MakerBot Replicator 2 printer converted into a low cost bioprinter. The printer was converted by replacing the control board with a Duet 2 Wifi 3D printer board (www.duet3d.com), Replistruder v4 syringe pumps, and a gantry mount designed for the Replicator 2 printer. All digital models were created in Fusion360 (AutoDesk) computer aided design (CAD) software, exported in 3D manufacturing format (.3mf), and imported into Simplify3D to generate print pathing files (.geode). In general, fibrin scaffolds were printed with a 160 pm blunt tip needle (Jensen Global, JG30HP), layer height of 60 pm, extrusion multiplier of 90%, 1-2 perimeters, speed of 10-50 mm / s, and 10-30% rectilinear infill. Prior to printing,fibrinogen stock was diluted in lx PBS without calcium or magnesium to 60 mg / mL. Bioinks (e.g., structure material) used to characterize feature size with optical microscopy were supplemented with 0.05% (w / v) titanium dioxide to enhance the optical contrast of the printed model. Prepared fibrinogen bioinks were transferred to 500 or 2500 pL gastight syringes (Hamilton) then installed in a Replistruder v4 syringe pump. Support (e.g., support material) was prepared as previously described above and transferred to print container large enough to print the model into and secured to the printer stage with double sided tape. The needle was positioned in the desired xy location within the support bath and lowered to a depth ~0.5 mm above the bottom of the printing dish. The 3D print was started using the Duet wireless user interface. All fibrin constructs were printed at room temperature (22°C) and allowed to gel at room temperature for a minimum of 15 minutes prior to release in a 37°C incubator. Melted support was removed and retrieved constructs were washed in prewarmed lx PBS three times for 30 minutes each.

[0107] Identifying Key Extrusion and Support Material Conditions

[0108] To develop a process for printing fibrin with the large number of interdependent variables in FRESH bioprinting, 1 utilized a small strip model with outer dimensions of 11 x 3 x 1 mm and sliced with 15-30% rectilinear infill to assess the ability to print single filaments, multi-perimeter walls, and maintain the structure of the model. This calibration construct was designed to be quickly printed (-5 minutes per sample) while providing adequate feedback required to fabricate larger models. I qualitatively assessed the formation of a gel on the extrusion needle and quantitatively assessed the thickness of single filaments, tissue wall, and overall construct dimensions of printed models. Single printed strands represent the most simplistic geometric feature in 3D printing and understanding how the filament size changes in response to bath conditions and printing parameters is desirable to the fabrication of larger constructs. Multi perimeter walls are typically required in 3D printed parts to maintain structural integrity by anchoring infill to a continuous surface. The overall model dimensions were assessed to characterize the ability of clot inhibition and removal treatments to enable fabrication of 3D models. Immediately after printing (<10 minutes after print completion), darkfield images of the embedded constructs were captured with a Leica M125 FC stereomicroscope, lx objective, and a Prime 95B CMOS scientific camera. After an initial round of imaging samples were released in a 37°C incubator for 30 minutes then washed three times in prewarmed lx PBS to remove gelatin. Darkfield micrographs of washed scaffolds were utilized to characterize dimensions of printed models. Three samples wereprinted per condition (n = 3) and five measurements were made for each dimension of interest per sample.

[0109] Scanning Electron Microscopy Imaging of FRESH Printed Fibrinogen

[0110] Simple slab models with outer dimensions of 5 x 5 x 1 mm were printed in 25% iodixanol support supplemented with thrombin at concentrations of 0.01, 0.1, 1, and 10 U / mL. Samples were printed from 60 mg / mL fibrinogen allowed to gel for 30 minutes then released for 1 hour at 37C. Samples were washed 3 times in prewarmed lx PBS to remove entrapped gelatin from the fibrin construct. Samples were prepared for scanning electron microscopy (SEM) by snap-freezing utilizing methods developed for biologic tissue specimens. Briefly, washed samples were transferred to a cryotube and submerged in liquid nitrogen for 60 seconds. The cryogenic tube caps were removed, and the samples were lyophilized for 12 hours at -50°C under 0.059 mBar of vacuum in a freeze drier (Labconco, Freezone). Freeze dried samples were cut to expose the cross section of the scaffold then sputter coated in 5 nm of gold (50 m A, Quorum EMS 300T D) for SEA4 imaging. Top-down and cross section images were taken from a 15 mm working distance at 7.5 keV accelerating voltage (Quanta 600 SEM) at magnifications of 600x, lOOOx, and 8000x with 15 ps double line integration.

[0111] Tensile Testing of FRESH Printed Fibrinogen

[0112] To assess the tensile strength of FRESH printed fibrin, the soft materials dog bone standard ASTM D-638-V was designed in CAD with shortened grip regions. The samples were printed from 60 mg / mL fibrinogen bioinks in support prepared with 25% iodixanol. The goal of this study was to provide additional insight into the interdependence of thrombin concentration (0.1, 1, 10 U / mL) and fluid phase density (p = 1, 1.15, 1.28) on the tensile strength of printed fibrin. However, this experiment was not completed due to unavailability of the tensile testing equipment during relocation of the instrument. I will detail the developed method and present preliminary results from tensile specimens printed in support prepared with 25% iodixanol (p=l .15) and 10 U / mL thrombin. Fibrin dog bones models were printed in support prepared as previously described. Printed dog bone samples (n>6) were released, washed then stored at 4°C in lx PBS 2-3 days until preforming the tensile testing. On the day of testing samples were mounted into custom grip adapters by removing the sample from water, blotting off excess water with a nonwoven lab wipe and mounting the grip region of the sample into the disposable clamp adapter with cyanoacrylate glue. The grip adapter was slotted into a fixture mounted on the base and travel arm of the Criterion 42 (Materials Test Systems) equipped with a 50-newton load cell and biobath. The biobath wasused to prevent dehydration of the fibrin sample during printing and was filled with lx PBS at 37°C during testing. Samples were pulled at a rate of 1 mm / min until mechanical failure of the sample. After pulling the test specimen the grips were lowered to the original zero strain position and the sample was pulled at 1 mm / min to record the buoyant force of the attached grips as they travel through the biobath. The force on the sample reported is the difference between the raw data and the buoyant force of the grips. Six samples were tested per condition.

[0113] Statistical Analysis

[0114] Statistical analysis was performed using GraphPad Prism 9 software and tests were selected according to the experimental conditions and data requirements. All experiments performed in triplicate unless otherwise stated. A one-way ANOVA test was used to compare terminal fibrinogen storage modulus by bioink concentration. A students t-test was used to compare the storage modulus of cast fibrin prepared with 0.1 or 1 U thrombin per mg of fibrinogen. Two-way ANOVA with Tukey’s multiple comparisons test was used to characterize the effect of the bath thrombin concentration and fluid density on printed scaffold features. Statistical significance was considered a p-value of <0.05.

[0115] Results and Discussion

[0116] Selecting Fibrinogen Concentration for FRESH Bioprinting

[0117] To select the appropriate fibrinogen concentration for FRESH bioprinting self- supporting structures, we first reviewed literature in the fields of bioprinting, tissue engineering, and biofabrication to compile properties of hydrogels used across fabrication modalities (Table 1). We observed that the hydrogels strength increases as the fabrication shifts from casting, embedded printing, and to platform bioprinting. Cast hydrogels have storage moduli (G’) < 3,000 Pa while direct ink write hydrogels represent the other extreme are at least -10,000 Pa and can reach up to -150,000 Pa. There are not a significant number of publications utilizing FRESH embedded bioprinting where the gel strength of hydrogels was reported with many reports utilizing similar materials such as 35 mg / mL Collagen I (Lifeink 200 or 240) or 20, 30 or 40 mg / mL Alginate. However, Kupfer et al developed a hydrogel blend to support proliferation of human induced pluripotent stem cells (hiPSCs) on FRESH printed scaffolds. The bioink was used to fabricate self-supporting scaffolds that promoted cell proliferation across the printed tissue and had a gel storage modulus of 6,140 ± Pa. In another example, Noor et al developed a bioink from decellularized omentum ECM to fabricate vascularized cardiac scaffolds using embedded bioprinting. The final concentration of omentum bioink was selected in order for the printed scaffolds to maintain their structuralintegrity and had a gel storage modulus of -1,000 Pa. These two studies provide a gel storage modulus threshold of G’ -1,000 Pa to fabricate self-supporting structures with FRESH bioprinting.Table 1 Strength and fabrication method of hydrogels commonly used in biofabricationconcentration neutralized 35 mg / mL collagen I (Advanced Biomatrix, Lifeink 200) has been FRESH bioprinted nasal cartilage strips for tissue engineering applications. Lifeink 200 formed a hydrogel when the temperature was increased to 37C after loading however the gelation profile was abnormal as the storage and loss moduli decreased over gelation time indicating that the gel may have required additional time to set prior to interrogation (FIG.3 A). The observed equilibrium storage moduli of Lifeink 200 was l,557 ± 335 Pa (Mean ± sem) which falls between the reported gel storage moduli of Ometnum ECM and the hChaMP bioinks for FRESH bioprinting (FIG. 3B). We postulate that fibrinogen bioinks with a gel storage modulus > 750 Pa should be suitable for FRESH 3D bioprinting.

[0119] Previously described rheological methods were used to characterize the effect of fibrin concentration on the shear storage moduli from 30 - 120 mg / mL with 0.1 U ofthrombin per mg of fibrinogen to initiate gelation. The physical properties of biologic materials are highly variable between lots and all gels were prepared from the same high concentration 120 mg / mL fibrinogen stock solution. We observed that all preparations rapidly gelled and reached >90% of the equilibrium storage modulus within 10 minutes after mixing fibrinogen and thrombin precursor solutions (FIG. 4A). The mean equilibrium shear storage modulus of each condition was 431, 482, 2525, and 6245 Pa at fibrinogen concentrations of 30, 60, 90, and 120 mg / mL respectively (FIG. 4B). Significant differences were observed between gels formed from low concentration fibrinogen (30 and 60 mg / mL) and gels formed from fibrinogen concentrations >90 mg / mL. The lack of differences between 30 and 60 mg / mL preparations could be attributed to operating in the lower attenable signal of the rheometer or improper dilution of stock solutions as we would expect to observe significant differences between these conditions. Another confounding factor could be the activity of thrombin used to prepare the fibrin gels. While the 60 mg / mL conditions only produced hydrogels with a storage modulus of 482 Pa from this lot, alterative fibrinogen lots produced 60 mg / mL gels with storage moduli >5,000 Pa. The gel strength of fibrinogen could also be altered by increasing the ratio of thrombin units per mg of fibrinogen. We observed that gels formed with 1 U of thrombin per mg were significantly different (p<0.05) to those formed with 0.1 U thrombin per mg (FIG. 4C). Due to the nature of FRESH bioprinting in an aqueous bath, the entirety of the bath is supplemented with thrombin while only a scant amount of fibrinogen bioink is deposited within the bath. The high surface area of the extruded filament allows the surrounding environment to rapidly gel the extruded filament as there is excess thrombin relative to the surface area of extruded fibrinogen filament. These observations led us to use 60 mg / mL fibrinogen bioinks as a baseline concentration for printing experiments.

[0120] Effect of Print Pathing on Scaffold Quality

[0121] Based on the observation that gel strength is affected by fibrin and thrombin concentration we started printing fibrin scaffolds in support baths washed in lx PBS and supplemented with 1-5 U / mL thrombin. Using this approach, we could fabricate simple rings that were <600 pm thick. When we used similar bath and bioink conditions to fabricate strip models to characterize the size of filaments and scaffolds produced, a clot formed at the needle tip or along the length of the needle (FIG. 5A). We first attempted to resolve this issue by supplementing the support bath with 50 mM EDTA, treating the needle with surfactants to prevent attachment to the needle, or reducing the concentration of thrombin in the support bath. Adding EDTA to the support bath inhibited the formation of clots on the needle tip butalso arrested the fibrin coagulation cascade and prints broke apart when released from the support bath. We treated needles with 1% (w / v) pluronic F-127 to alter the hydrophilicity of the needle surface and inhibit the needle from attaching to the printed fibrin. Treating the needles did not alter the droplet formation and fibrin extruded in air proceeded to wick up the needle rather than form a hanging droplet out of the extrusion needle (FIG. 5B). Alternative treatments could be selected to improve the efficacy of the treatment however this is unlikely the underlying cause of the issue. Finally, we attempted to modify the bath by supplementing the prepared support material with EDTA to chelate calcium present in the bath to prevent the coagulation of fibrinogen at the extrusion needle. Support baths prepared with 10 mM EDTA resulted in the high geometric fidelity of the printed part while embedded. However, when the support melted the scaffold broke apart. Since none of these treatments were effective, we considered simply removing the clot by mechanical means between each printed layer.

[0122] A custom script was written in Slimplify3D that removed the extrusion needle out of the support to a second petri dish container filled with water between each printed layer. An additional condition of passing the needle through a plastic filament brush was also considered to fully remove any material from the extrusion needle between layers. A strip CAD model with outer dimensions of 11 x 3 x 1 mm with 1.82 mm square holes was designed in Fusion 360 (FIG. 6A). The print pathing file (.geode) was created in Simplify3D with and without the intralayer needle wiping script with an infill of 30%, print speed of 10 mm / s, and three perimeters (FIG. 6B). Fibrin scaffolds were printed from 60 mg / mL bioinks into support prepared with lx PBS and 5 U / mL thrombin. Scaffolds printed without wiping formed tissues without key structural features such as visible infill lines or square holes from the geode preview. Rinsing the needle with water slightly improved the printed part fidelity as evidenced by the darker region where the holes should be located. However, no infill filaments are visible suggesting that the clot was still present and diffused the material rather than forming defined filaments. When we added a brush to physically remove the clot from the extrusion needle, we were able to print strips that were dimensionally accurate to the model with well-defined holes. While the infill pattern was somewhat recognizable the individual filaments appeared fuzzy and poorly defined (FIG. 6C). Wiping the extrusion nozzle in water or through a brush between layers is not desirable as it roughly doubles the time required to print a single tissue by ~8 minutes per tissue. Printing two or more scaffold simultaneously to decrease the impact of the added time per layer rendered the treatment ineffective. These findings led us to seek alternative approaches to prevent the formation of a clot at the extrusion needle and improve the fidelity of printed fibrin scaffolds.

[0123] Effect of Bath Fluid Phase Density on Scaffold Quality

[0124] An alternative approach to modifying the needle or mechanical removal of clot formation is to alter the diffusion rate of materials through the aqueous phase of the compacted support bath. Increasing the fluid phase density (p) should simultaneously reduce the rate of diffusion flux vector (J) of printed fibrin into the fluid phase of support and reduce the rate at which thrombin diffuses through the support to extruded filaments based on Fick’s first law of diffusion (Equation 1). Altering diffusivity with the fluid density in prepared support is potentially an effective strategy to slow down the rate of fibrin polymerization and allow larger constructs to be fabricated without forming a clot at the extrusion nozzle.

[0125] Equation 1 Variation of Fick’s first law written with respect to the mass fraction gradient (Vyi)

[0127] To test this theory, we prepared support baths with densities (p) of 1, 1.15, and 1.28 g / mL by compacting gelatin support in aqueous solutions containing 0, 25, and 50% (w / v) iodixanol. lodixanol is a low viscosity density gradient media used previously to adjust the refractive index of FRESH support baths to image scaffolds with optical coherence tomography during printing to detect defects. These baths were supplemented with 0.1, 1, and 10 U / mL of thrombin to characterize the impact on the fidelity of printed fibrin scaffolds. We utilized the same strip model as previously described but prepared print pathing files with a lower infill (15%) to better visualize internal fibrin filaments (FIG. 7A). We assessed the print fidelity by measuring the width (i), length (ii) and infill extrusion width (iii) from darkfield stereomicrographs of printed, released, and washed fibrin scaffolds (FIG. 7B). Support baths supplemented with 0.1 U / mL thrombin produced scaffolds which appeared fuzzy suggesting that the fibrin diffused into the aqueous phase before gelling. In the support bath condition with 25% iodixanol supplemented with 0.1 U / mL thrombin, the fibrin diffused away from the printed scaffold and formed a very weak structure that did not maintain the structure of the model. We confirmed the qualitative assessment with dimensional data and found that this condition results in less dimensional accuracy (FIGs. 7C and 7D). The width and length of the fibrin scaffold was best controlled by increasing the thrombin concentration to 10 U / mL and no statistically significant differences were observed under this condition with respect to fluid density (FIG. 7C). Considering this we turned our attention to the differences in scaffolds printed in support with 1 and 10 U / mL thrombin. Within these conditions, the only structural difference was observed between support compacted in 25%and 50% iodixanol was the length of the fibrin scaffold (p < 0.05) printed in support with 1 U / mL thrombin. The consistency between models printed in 25% and 50% iodixanol suggests that the density of the fluid phase is helping preserve the structure of the printed model and that there is a threshold fluid density that improves the printing of fibrin. Subsequent experiments utilized support prepared in 25% iodixanol and focused on the effect of thrombin concentration on the micro and macrostructural properties of printed fibrin.

[0128] While the higher density support baths improved the macrostructural accuracy of printed fibrin scaffolds, we note that all infill filament diameters were significantly greater than the intended extrusion width of 160 pm (FIG. 7E). However, printing parameters such as extrusion multipliers or increasing the time allowed for the printed part to gel prior to release could be optimized to improve the dimensional accuracy of small features in the future.

[0129] Effect of Thrombin Concentration on Filament Microstructure

[0130] Fibrin rectangular prisms 5 x 5 x 1 mm were printed from 60 mg / mL fibrinogen in support baths prepared with 25% iodixanol supplemented with 0.01, 0.1, 1, or 10 U / mL thrombin to investigate the effect of thrombin concentration on the microstructure of printed fibrin scaffolds. Scaffolds were released, washed, and prepared for SEM as previously described. Low magnification top-down SEM images reveal that all conditions have entrapped circular pores consistent with prior results showing the porosity imparted by gelatin microparticles during FRESH printing (FIG. 8A). All images taken from the top of the scaffold seem somewhat homogenous regardless of thrombin concentration or magnification of the image. This effect could be attributed to the solid infill of the top and bottom of the scaffolds. Low magnification images of the cross section reveal qualitative differences in the microstructure of printed fibrin scaffolds (FIG. 8B). Lower thrombin concentrations (0.01 and 0.1 U / mL) in the support produce fibrin scaffolds with high porosity indicative of diffusion of printed material through the print bath. Scaffolds printed in support baths supplemented with 1 and 10 U / mL produce scaffolds with well aggregated filaments in each layer of printed fibrin. In 10 U / mL support baths high magnification images shows the formation of fibrin lamellar sheets < 1 pm thick suggesting that the material gelled almost instantaneously after extrusion. The cross-sectional images reveal that thrombin qualitatively influences the microstructure of printed fibrin. The changes in microstructure could potentially be used to alter cellular attachment and remodeling of printed fibrin scaffolds in future studies.

[0131] Effect of Thrombin Concentration on Effective Young’s Modulus

[0132] A tensile testing dog bone CAD model based on the ASTMD-638-V standard was modified to have a truncated grip region (FIG. 9A). The print pathing file was prepared with a print speed of 20 mm / s, 3 perimeters, and 50% aligned infill parallel to long axis of the model (FIG. 9B). Fibrin dog bone tensile testing specimens were printed in support prepared with 25% iodixanol in the aqueous phase of the support and was supplemented with lOU / mL thrombin. Up to 4 samples were printed sequentially at a time without formation of a clot at the tip of the extrusion needle (FIG. 9C). Tensile testing samples were mounted with cyanoacrylate adhesive to disposable polylactic mounts fabricated with fused deposition modeling 3D printing (FIG. 9D). Samples were strong enough to be transferred to and installed in the MTS tensile testing machine (FIG. 9E) and the grips effectively constrained the material even when subjected to high strain (FIG. 9F).

[0133] A total of 6 printed samples replicates were tested for each condition. The net force of the sample, stress, and strain was calculated in excel based on raw data exported from the MTS user interface and the cross-sectional area and gauge length of the CAD model.Calculated stress and strain data from each run was plotted in GraphPad Prism (FIG. 10A). A linear regression was calculated from the 0 - 150% strain for each sample (FIG. 10B). The printed fibrin had an effective Young’s modulus of 14.22 ± 1.568 kPa (mean ± SEM) (FIG. 10C). This value is 2 orders of magnitude lower than the Young’s modulus calculated by Collet et al from straining non-crosslinked fibrin fibers with laser tweezers (E = 1.7 ± 1.3 MPa). This may be attributed to the porosity of the FRESH printed fibrin and low infill used to fabricate the sample. The sample also deforms outside of the gauge length and a dog bone tensile sample may not be the most representative model for materials as extensible as fibrin. Future studies could utilize a rectangular tensile model with parallel sides as the printed fibrin samples should deform enough to break within the sample gauge length rather than fail at the grip interface using the tensile testing method defined in this study. The peak strain and stress of the printed fibrin samples was SFaiiure = 6.023 ± 0.54 and opeak = 90.18 ± 13.69 kPa (FIGs. 10D and 10E). The observed strain at failure is substantially greater than the strain at failure of individual fibrin fibers was characterized by atomic force microscopy as reported by Liu et al of 226 ± 52%. While the average strain for 36 samples was reported the author noted that several samples failed at strains exceeding 500%. The porous nature of the printed fibrin, sample geometry, assumed gauge length and gelation conditions in the embedded printing environment could account for some of these discrepancies as the 3D networks can compact and align before undergoing any strain at the molecular level.

[0134] Conclusion

[0135] We developed an optimized protocol for printing fibrin using FRESH 3D bioprinting. We investigated how the concentration of fibrin and ratio of thrombin impacts the strength of cast gels and applied this to FRESH bioprinting. We tested various methods to improve the print fidelity of fibrin scaffolds by mechanical removal of debris between layers and showed that this improved the quality of printed scaffolds. We then developed an alternative method to prevent the formation of a clot at the interface of the extrusion needle and support by increasing the fluid density of the aqueous phase of the support bath. Increasing the fluid density simultaneously prevented the formation of gels on the extrusion needle, improved the dimensional accuracy of printed models, and enabled higher concentrations of thrombin to be utilized in the support bath. Utilizing higher thrombin concentrations produced more dimensionally accurate printed parts than low thrombin concentration support baths and alleviated the presence of a hazy fibrous capsule around the printed model. Finally, we demonstrated that the developed printing method could fabricate fibrin dog bones that could be subjected to tensile testing. These printed parts were able to withstand strains >400% and failed at a peak stress of 90 kPa. Overall, this work established a process to fabricate unmodified fibrin scaffolds with extrusion bioprinting that has never been previously achieved.

[0136] Various aspects of the invention include, but are not limited to, the aspects listed in the following numbered clauses.

[0137] Clause 1. An additive manufacturing method comprising: depositing a structure material into a support material based on a first computer model of an object, thereby forming a first portion of the object in the support material, wherein the structure material exhibits a viscosity in a range of 1 cP to 5,000 cP at 25 degrees Celsius and the support material exhibits a density in a range of 1.05 g / mL to 3 g / mL; and repeating the depositing of the structure material as necessary to additively form the object.

[0138] Clause 2. The method of clause 1, wherein the support material exhibits a density in a range of 1.1 g / mL to 1.5 g / mL.

[0139] Clause 3. The method of clause 1, wherein the support material exhibits a density in a range of 1.1 g / mL to 1.3 g / mL.

[0140] Clause 4. The method of any of clauses 1-3, wherein the support material comprises: a diluent; particles dispersed in the diluent; and a reactant.

[0141] Clause 5. The method of clause 4, wherein the diluent is aqueous.

[0142] Clause 6. The method of clause 4, wherein the diluent comprises water and at least one additive selected from the group consisting of cesium chloride, cesium bromide, cesium iodide, cesium sulfate, cesium nitrate, rubidium chloride, rubidium bromide, rubidium iodide, rubidium sulfate, rubidium nitrate, ficoll, sucrose, lymphocyte separation medium, sodium diatrizoate, ammonium chloride, barium chloride, calcium chloride, cecium chloride, hydrogen-chloride, iron (III) chloride, lithium chloride, magnesium chloride, potassium chloride, sodium chloride, strontium chloride, ammonium sulfate, copper (II) sulfate, magnesium sulfate, manganese (II) sulfate, nitric acid, phosphoric acid, silver nitrate, 1,2- ethanediol, b-D-fructose, a-D-glucose, glycerol, b-D-lactose, a-Maltose, D-mannitol, sucrose, urea, sodium metatungstate, sodium polytungstenate, lithium heteropolytungstates (LST), iodixanol, or a combination thereof.

[0143] Clause 7. The method of clause 6, wherein the support material comprises 5% to 80% by weight of the additive based on a total weight of the support material.

[0144] Clause 8. The method of clause 6, wherein the support material comprises 20% to 60% by weight of the additive based on a total weight of the support material.

[0145] Clause 9. The method of clause 8, wherein the additive comprises iodixanol.

[0146] Clause 10. The method of clause 4, wherein the diluent is non-aqueous.

[0147] Clause 11. The method of any of clauses 4-10, wherein the particles comprise gelatin, alginate, calcium chloride, soy lecithin, Carbopol, acrylamide, agarose, alginate, a cell spheroid, a cell organoid, gellan gum, hyaluronic acid, laponite nanoclay, nanoclay, pluronic F127, polyethylene oxide), oxidized bacterial cellulose, xanthan gum methacrylate, fumed silica, hyaluronic acid-norbomenefibronectin & hyaluronic acid, K-Carrageenan, decellularizaed extracellular matrix, or a combination thereof.

[0148] Clause 12. The method of any of clauses 4-11, wherein the reactant comprises thrombin, a photo initiator, a pH buffer, calcium, magnesium, sodium nitrate, a salt solution, transglutaminase, or a combination thereof.

[0149] Clause 13. The method of any of clauses 4-12, wherein the support material comprises 0.001 to 10,000 units of thrombin per milliliter.

[0150] Clause 14. The method of any of clauses 4-13, wherein the support material further comprises a surfactant, a thickening agent, or a combination thereof.

[0151] Clause 15. The method of any of clauses 4-9 and 11-14, wherein: the diluent comprises water and iodixanol; the support material comprises 20% to 60% by weight of iodixanol based on a total weight of the support material; the particles comprise gelatin; thereactant comprises thrombin; the support material exhibits a density in a range of 1.1 g / mL to 1.3 g / mL; and the structure material comprises a polymer comprising a fibrinogen material.

[0152] Clause 16. The method of any of clauses 1-15, further comprising removing the support material.

[0153] Clause 17. The method of any of clauses 1-16, wherein the structure material comprises a polymer.

[0154] Clause 18. The method of clause 17, wherein the polymer comprises a hydrogel, a thermoset polymer, thermoplastic polymer, or a combination thereof.

[0155] Clause 19. The method of any of clauses 17-18, wherein the polymer comprises a collagen material, an alginate material, a decelluarized extracellular matrix material, a fibrinogen material, a fibrin material, a hyaluronic acid material, a protein material, a polysaccharide hydrogel material, a synthetic gel material, an elastomeric polymer material, a rigid polymer material, a Matrigel, or a combination thereof.

[0156] Clause 20. The method of any of clauses 17-18, wherein the polymer comprises a fibrinogen material.

[0157] Clause 21. A product produced by the method of any one of clauses 1-20.

[0158] Clause 22. A support material comprising: a diluent; particles dispersed in the diluent; and 1 to 20 units of thrombin per milliliter, wherein the support material exhibits a density in a range of 1.05 g / mL to 3.0 g / mL.

[0159] Any patent, publication, or other disclosure material identified herein is incorporated herein by reference in its entirety unless otherwise indicated but only to the extent that the incorporated material does not conflict with existing definitions, statements, or other disclosure material expressly set forth in this specification. As such, and to the extent necessary, the express disclosure as set forth in this specification supersedes any conflicting material incorporated by reference herein. Any material, or portion thereof, that is said to be incorporated by reference into this specification, but which conflicts with existing definitions, statements, or other disclosure material set forth herein, is only incorporated to the extent that no conflict arises between that incorporated material and the existing disclosure material.Applicant reserves the right to amend this specification to expressly recite any subject matter, or portion thereof, incorporated by reference herein.

[0160] In this specification, unless otherwise indicated, all numerical parameters are to be understood as being prefaced and modified in all instances by the term “about,” in which the numerical parameters possess the inherent variability characteristic of the underlying measurement techniques used to determine the numerical value of the parameter. At the veryleast, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter described herein should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0161] Also, any numerical range recited herein includes all sub-ranges subsumed within the recited range. For example, a range of “1 to 10” includes all sub-ranges between (and including) the recited minimum value of 1 and the recited maximum value of 10, that is, having a minimum value equal to or greater than 1 and a maximum value equal to or less than 10. Any maximum numerical limitation recited in this specification is intended to include all lower numerical limitations subsumed therein and any minimum numerical limitation recited in this specification is intended to include all higher numerical limitations subsumed therein. Accordingly, Applicant reserves the right to amend this specification, including the claims, to expressly recite any sub-range subsumed within the ranges expressly recited. All such ranges are inherently described in this specification.

[0162] Any references herein to “various examples”, “some examples”, “one example”, “an example”, or like phrases mean that a particular feature, structure, or characteristic described in connection with the example is included in at least one embodiment. Thus, appearances of the phrases “in various examples”, “in some examples”, “in one example”, “in an example”, or like phrases in the specification do not necessarily refer to the same examle. Furthermore, the particular described features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. Thus, the particular features, structures, or characteristics illustrated or described in connection with one embodiment may be combined, in whole or in part, with the features, structures, or characteristics of one or more other embodiments without limitation. Such modifications and variations are intended to be included within the scope of the present embodiments.

[0163] As used herein, “at least one of’ A and B, means only A, only B, or both A and B. Additionally, there may be multiple As and / or multiple Bs.

[0164] One skilled in the art will recognize that the herein described articles and methods, and the discussion accompanying them, are used as examples for the sake of conceptual clarity and that various configuration modifications are contemplated. Consequently, as used herein, the specific examples / embodiments set forth and the accompanying discussions are intended to be representative of their more general classes. In general, use of any specific exemplar is intended to be representative of its class, and the non-inclusion of specific components, devices, operations / actions, and objects should not be taken to be limiting. While the present disclosure provides descriptions of various specific aspects for the purposeof illustrating various aspects of the present disclosure and / or its potential applications, it is understood that variations and modifications will occur to those skilled in the art.Accordingly, the invention or inventions described herein should be understood to be at least as broad as they are claimed and not as more narrowly defined by particular illustrative aspects provided herein.

Claims

CLAIMSWhat is claimed is:

1. An additive manufacturing method comprising: depositing a structure material into a support material based on a first computer model of an object, thereby forming a first portion of the object in the support material, wherein the structure material exhibits a viscosity in a range of 1 cP to 5,000 cP at 25 degrees Celsius and the support material exhibits a density in a range of 1.05 g / mL to 3 g / mL; and repeating the depositing of the structure material as necessary to additively form the object.

2. The method of claim 1, wherein the support material exhibits a density in a range of 1.1 g / mL to 1.5 g / mL.

3. The method of claim 1, wherein the support material exhibits a density in a range of 1.1 g / mL to 1.3 g / mL.

4. The method of claim 1, wherein the support material comprises: a diluent; particles dispersed in the diluent; and a reactant.

5. The method of claim 4, wherein the diluent is aqueous.

6. The method of claim 4, wherein the diluent comprises water and at least one additive selected from the group consisting of cesium chloride, cesium bromide, cesium iodide, cesium sulfate, cesium nitrate, rubidium chloride, rubidium bromide, rubidium iodide, rubidium sulfate, rubidium nitrate, ficoll, sucrose, lymphocyte separation medium, sodium diatrizoate, ammonium chloride, barium chloride, calcium chloride, cecium chloride, hydrogen-chloride, iron (III) chloride, lithium chloride, magnesium chloride, potassium chloride, sodium chloride, strontium chloride, ammonium sulfate, copper (II) sulfate, magnesium sulfate, manganese (II) sulfate, nitric acid, phosphoric acid, silver nitrate, 1,2- ethanediol, b-D-fructose, a-D-glucose, glycerol, b-D-lactose, a-Maltose, D-mannitol, sucrose,urea, sodium metatungstate, sodium polytungstenate, lithium heteropolytungstates (LST), iodixanol, or a combination thereof.

7. The method of claim 6, wherein the support material comprises 5% to 80% by weight of the additive based on a total weight of the support material.

8. The method of claim 6, wherein the support material comprises 20% to 60% by weight of the additive based on a total weight of the support material.

9. The method of claim 8, wherein the additive comprises iodixanol.

10. The method of claim 4, wherein the diluent is non-aqueous.

11. The method of claim 4, wherein the particles comprise gelatin, alginate, calcium chloride, soy lecithin, Carbopol, acrylamide, agarose, alginate, a cell spheroid, a cell organoid, gellan gum, hyaluronic acid, laponite nanoclay, nanoclay, pluronic Fl 27, poly(ethylene oxide), oxidized bacterial cellulose, xanthan gum methacrylate, fumed silica, hyaluronic acid-norbornenefibronectin & hyaluronic acid, K-Carrageenan, decellularizaed extracellular matrix, or a combination thereof.

12. The method of claim 4, wherein the reactant comprises reactant can comprise thrombin, a photo initiator, a pH buffer, calcium, magnesium, sodium nitrate, a salt solution, transglutaminase, or a combination thereof13. The method of claim 4, wherein the support material comprises 0.001 to 10,000 units of thrombin per milliliter.

14. The method of claim 4, wherein the support material further comprises a surfactant, a thickening agent, or a combination thereof.

15. The method of claim 4, wherein: the diluent comprises water and iodixanol; the support material comprises 20% to 60% by weight of iodixanol based on a total weight of the support material; the particles comprise gelatin;the reactant comprises thrombin; the support material exhibits a density in a range of 1.1 g / mL to 1.3 g / mL; and the structure material comprises a polymer comprising a fibrinogen material.

16. The method of claim 1, further comprising removing the support material.

17. The method of claim 1, wherein the structure material comprises a polymer.

18. The method of claim 17, wherein the polymer comprises a hydrogel, a thermoset polymer, thermoplastic polymer, or a combination thereof.

19. The method of claim 17, wherein the polymer comprises a collagen material, an alginate material, a decelluarized extracellular matrix material, a fibrinogen material, a fibrin material, a hyaluronic acid material, a protein material, a polysaccharide hydrogel material, a synthetic gel material, an elastomeric polymer material, a rigid polymer material, a Matrigel, or a combination thereof.

20. The method of claim 17, wherein the polymer comprises a fibrinogen material.

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