Additive manufacturing support material

Abstract

To provide a process of producing gel microparticles, which are consistent in size and morphology.SOLUTION: Through a process of coacervation, large volumes of gel microparticle slurry can be produced by scaling up reactor vessel size. Particles can be repeatedly dehydrated and rehydrated in accordance with their environment, allowing for storage of the particles in a non-solvent such as ethanol. Gel slurry exhibits a Bingham plastic behavior in which the slurry behaves as a solid at shear stresses that are below a critical value. Upon reaching a critical shear stress, the slurry undergoes a rapid decrease in viscosity and behaves as a liquid. A rheological behavior of these slurries can be adjusted by changing compaction processes such as centrifugation force to alter a yield-stress. A narrower distribution and reduced size of these particles allow for an increase in FRESH printing fidelity.SELECTED DRAWING: Figure 1A

Description

[Technical Field]

[0001] (Priority Claim) This application claims priority under 35 U.S.C. §119(e) to U.S. patent application Ser. No. 62 / 601,949, filed April 5, 2017, and U.S. patent application Ser. No. 62 / 606,578, filed September 28, 2017, the entire contents of which are hereby incorporated by reference.

[0002] (Government Support Section) This invention was made with government support under National Institutes of Health No. HL117750. The U.S. Government has certain rights in this invention.

[0003] (Technical field) This application relates to additive manufacturing, and more particularly to support materials for additive manufacturing. [Background technology]

[0004] Additive manufacturing can be used to create three-dimensional objects or structures. Materials can be printed onto a support skeleton that temporarily supports the structure during assembly. When assembly is complete, the support skeleton is removed. Summary of the Invention

[0005] This application describes a support material for additive manufacturing and a method for its manufacture. The fluid additive manufacturing method, called Freeform Reversible Suspension Hydrogel Embedding (FRESH), involves embedding a fluid material (e.g., alginate, collagen, fibrin, etc.) into a temporary support material (e.g., including a particulate slurry). The support material and method for its manufacture overcome the limitations imposed by conventional mechanical compounding techniques for support material manufacture. The support material includes particulates of approximately uniform size and uniform geometry. For additive manufacturing methods (e.g., embedded printing in the support material), the support material enables the generation of printed structures with higher fidelity (e.g., fewer defects, fewer voids, fewer irregularities, etc.) in the printed structure than printed structures using previous versions of the support material.

[0006] The process for providing the support material described below is more efficient than previous processes for creating polymeric micro- and nanogels for drug encapsulation and / or delivery and uses the underlying chemical principles of emulsification or coacervation. Previous processes may involve blending and emulsions. These previous processes have lower yields, are less efficient, create particles with small volumes, and rely on chemicals and polymers that are less suitable for bioprinting.

[0007] Described herein is a process for creating microparticle-containing support materials that overcomes the limitations of conventional mechanical compounding techniques for the generation of support material particles. Using scalable phase separation, known as coacervation, large quantities of monodisperse microparticles can be produced from a variety of raw, reversibly gelling materials with tight control over particle morphology and bulk rheological behavior. Phase separation is used to drive the formation of gel particles. These particles can then be further isolated to form support materials. Embedding a gelling fluid "ink" into this support material allows the ink to fuse into a three-dimensional object.

[0008] These processes rely on dissolving a gel in a mixture of a solvent (e.g., water) and a co-solvent (e.g., ethanol) under stirring. By changing the mixing conditions, the solubility of the gel decreases until gel particles nucleate from the solution. These particles can be washed and isolated to form the support material slurry.

[0009] This process creates gel microparticles in a simple, single-step, high-yield, and inexpensive manner. Furthermore, due to its chemically driven nature, the process is easily scalable to large quantities, which is challenging for other processes that rely on mechanical blending, emulsification, or ultracentrifugation. This process allows for the rapid adoption of gelling fluids for 3D printing, allowing for large-scale production of support materials.

[0010] The support material includes a slurry comprising a solution and coacervate particles in the solution, the coacervate particles being of a substantially constant geometry; wherein at least a portion of the slurry forms a rigid body when subjected to a stress below a threshold stress; and wherein at least a portion of the slurry forms a viscous fluid when subjected to a stress above the threshold stress.

[0011] In some implementations, the solution includes a surfactant configured to reduce the number of dendritic coacervate particles that form in the solution relative to the number of dendritic coacervate particles that form in a solution without the surfactant. In some implementations, each of the coacervate particles includes at least one of gelatin, alginate, and cellulose. In some implementations, the coacervate particles include two or more different polymers. In some implementations, one of the two or more different polymers includes gum arabic, and another of the two or more different polymers includes gelatin. In some implementations, the solution includes one or more of water and ethanol.

[0012] In some implementations, the harmonic mean size of the coacervate particles is from about 0.5 μm to about 60 μm. In some implementations, the harmonic mean size of the coacervate particles varies by less than about 35%.

[0013] In some implementations, the threshold stress comprises a critical shear stress, wherein the adhesive force between the first and second coacervate particles of the slurry is approximately equal to an external shear force applied to the coacervate particles of the slurry. In some implementations, the value of the critical shear stress is from about 20 Pa to about 140 Pa. The value of the critical shear stress is based on the viscosity of the additive manufacturing ink in the slurry. In some implementations, the ink comprises collagen.

[0014] This document describes a process for manufacturing a support material, the process including generating a coacervate from a polymer, the coacervate including particles that are substantially uniform in geometry, and generating the coacervate including forming a solution of a solvent and a cosolvent; stirring the solution to dissolve the polymer in the solution; then adjusting the pH of the solution to a specific value based on the type of polymer; and then forming a slurry from the coacervate having a specific yield stress value, the formation including compressing the coacervate during one or more centrifugation cycles.

[0015] In some implementations, the process includes selecting one or more parameters and adjusting one or more parameters during the formation of the coacervate, wherein each of the one or more parameters includes gelatin bloom value, polymer processing method, polymer precipitation rate, polymer solubility, polymer molecular weight, polymer concentration, solvent and cosolvent volume ratio, surfactant type, surfactant concentration, cooling rate, or stirring rate.

[0016] In some implementations, the process includes selecting one or more parameters; and then adjusting the one or more parameters during shaping of the slurry, the one or more parameters including a type of washing solution, a centrifugation time for one or more centrifugation cycles, a centrifugal force for one or more centrifugation cycles, and a number of one or more centrifugation cycles.

[0017] In some implementations, the polymer is gelatin, including gelatin bloom values of 200 Bloom, 250 Bloom, and 275 Bloom. The polymer is gelatin, and the gelatin includes one or both of acid-hardened gelatin and lime-cured gelatin. The solution includes a solvent-to-cosolvent ratio of about 52.5:47.5, where the solvent includes water and the cosolvent includes ethanol.

[0018] In some implementations, the process includes adding a surfactant to the solution. This action includes dehydrating the slurry in ethanol. In some implementations, the process includes rehydrating the slurry in water, where the slurry maintains a particular yield stress value after dehydration and rehydration.

[0019] In some implementations, the polymer is a first polymer, and generating the coacervate further includes adding a second polymer to the solution, selecting an isoelectric point for either the first polymer or the second polymer, and then adjusting the pH based on the selected isoelectric point. In some implementations, the first polymer includes gelatin, the second polymer includes gum arabic, and the pH is about 5-6.

[0020] In some implementations, the process includes adjusting one or more centrifugation cycles to achieve a particular yield stress value of the slurry. In some implementations, the particular value is between about 20 Pa and about 140 Pa. In some implementations, the particular value is based on the viscosity of the additive manufacturing ink in the slurry.

[0021] In some implementations, the ink includes collagen. In some implementations, the process includes washing the coacervate in a wash solution. In some implementations, the coacervate particles are approximately monodisperse in the solution.

[0022] In some implementations, the support material comprises a colloidal solution comprising ethanol and water; a surfactant; and a gelatin slurry comprising coacervate microparticles in the colloidal solution, wherein the acervate microparticles are monodisperse in the solution, the acervate microparticles having an average size of 0.5 to 60 micrometers and a size variance of less than 35%; at least a portion of the slurry forms a rigid body when subjected to a shear stress below a yield stress value; and at least a portion of the slurry forms a viscous fluid when subjected to a shear stress above a yield stress value.

[0023] The details of one or more exemplary embodiments of the support material are set forth in the accompanying drawings and specification below. Other features, objects, and advantages will become apparent from the specification and drawings, and from the claims.

[0024] Like reference symbols in the various drawings indicate like elements. [Brief explanation of the drawings]

[0025] [Figure 1A] FIG. 1A shows a printing system. [Figure 1B] FIG. 1B shows a representative gelatin particle. [Figure 2-6] 2 to 6 each show a representative gelatin microparticle for an example of a support material. [Figure 7] FIG. 7 shows a histogram of particle size distribution for an example support material. [Figure 8] FIG. 8 shows a graph depicting gelatin concentration versus particle size for example support materials. [Figure 9-10] 9 and 10 each show an example of a support material. [Figure 11]FIG. 11 shows an example of the hydration process for the support material. [Figure 12] 12A-12B show examples of particle size and yield control for support materials. [Figure 13-15] 13 to 15 show examples of rheological data of the support material. [Figure 16] FIG. 16 shows yield stress values for example support materials. [Figure 17] Figure 17 shows the stages of the additive manufacturing process using an example support material. [Figure 18] FIG. 18 shows an example print produced for the example support material. [Figure 19] FIG. 19 shows a flow diagram of an example process for forming support material. DETAILED DESCRIPTION OF THE INVENTION

[0026] FIG. 1A illustrates a printing system 100. The printing system 100 is configured to print a material 110 (e.g., collagen, fibrin, etc.) embedded in a support material 130, such as via an injector 120. The support material 130 forms a scaffold to support the material 110 as it prints and hardens (e.g., gels). When the viscosity and yield stress of the support material 130 are similar to that of the printed object (also known as ink), the printing system 100 can print with higher precision than when the viscosity and yield stress of the support material are mismatched or not similar to those of the support material. Additionally, support materials that form slurries with smaller particles facilitate high-fidelity printing of structures in the support material by the printing system 100.

[0027] The support material 130 includes a material that forms skeletal support for the additive manufacturing process. The support material includes a slurry that supports an embedded material (e.g., ink) used for embedded 3D printing of a structure. The support material supports the printed ink, which is temporarily embedded in the support material. Once the structure is formed by the printing process, the support material is removed.

[0028] The support material (also called the support bath) exhibits at least some of the properties of a Bingham plastic material. For example, when the support material is not subjected to stress (e.g., shear stress) exceeding the yield stress value, the support material exhibits the properties of a solid material. When at least a portion of the support material is subjected to stress (e.g., shear stress) exceeding the yield stress value, at least a portion of the support material behaves like a viscous liquid. In some implementations, a printer injector applies stress to the support material as it moves through the support material. This allows the printer head to inject ink into the support material, holding the ink in place until a structure is formed. The ink may include tissues such as collagen, or other materials, such as materials that undergo gelation after being infused into the support material. The support material supports the ink until gelation is complete. The support material can then be removed (e.g., melted away).

[0029] The yield stress of the support material is an important factor in enabling accurate form factors to be printed in the support material. The homogeneity of the support material is also a factor in the quality of the printed structure. The support materials described herein can have a yield stress that is set to a specific value (e.g., based on the application, ink type, etc.). The support materials described herein contain particles of specific sizes that are designed to increase printing fidelity and enable accurate 3D embedded printing of structures.

[0030] Figure 1B shows an example of a gelatin support material 150 produced by a blending technique. This demonstrates that the generation of gelatin particulate supports utilizes mechanical blending of gelatin blocks to produce smaller gelatin particles. Due to the nature of the blending process, the produced gelatin particulates were random in both their size and shape distribution. The support material 150 exhibits particles with irregular size and shape distribution. These particles are not uniformly dispersed in the support bath slurry. For embedded printing applications, a nearly uniform particle size, shape, and distribution is preferred. The support material and support material manufacturing process described below overcome or reduce the limitations imposed by conventional mechanical blending techniques for support material production. The support material described below contains particulates of nearly uniform size and geometry. For additive manufacturing processes (e.g., embedded printing in a support material), the support material enables the generation of printed structures with higher fidelity (e.g., fewer defects, voids, irregularities, etc.) in the printed structure than structures printed using previous versions of the support material. As shown in Figure 1B, gelatin microparticles 150 produced by mechanical compounding exhibit a random size and shape distribution. Scale bar is 50 microns.

[0031] Figures 2-6 show representative examples of support materials 200, 300, 400, 500, and 600 produced by the coacervation process. The particles produced by the coacervation process are smaller and more consistent in morphology compared to support material 150 shown in Figure 1B, which is produced by a mechanical compounding process.

[0032] The substrate forming the support material particles comprises a polymer. The polymer can include one or more of gelatin, alginate, cellulose, and similar polymers. The substrate undergoes a coacervation process to produce a coacervate containing the material. The coacervate contains microparticles of the material with a substantially uniform geometry. Particles with a substantially uniform geometry have substantially the same shape, size, structure, and arrangement (e.g., uniformity between particles). For example, the particles have a substantially uniform size. Here, substantially uniform size means that the particles (e.g., droplets) exhibit a particle size distribution with a coefficient of variation (i.e., the population standard deviation divided by the population mean) of less than about 35%, or about 10, 15, 20, 25, or 30%. A coefficient of variation of less than about 15% is preferred. In some embodiments, about 70 percent or about 90 percent of the beads have a volume particle diameter of about 0.90 to about 1.1 times the average volume particle diameter of the particles. In some implementations, the particles are monodisperse in the coacervate.

[0033] The coacervate-support synthesis protocol for forming support materials for embedded printing applications involves the coacervation of a material and its compaction. Support material parameters can be selected based on the specific application of the support material (e.g., based on the ink used during the embedded printing process). These parameters (e.g., size, yield, etc.) affect the yield stress of the support material. The yield stress of the support material is based on the size of the particles produced during the coacervation process and can be tailored to a specific value. The properties of the substrate (e.g., polymer) can be selected and / or adjusted to adjust the size of the coacervate particles and thus the yield stress of the support material. These properties can include the type of substrate, the material processing method, the rate of polymer precipitation (e.g., during coacervation), polymer solubility, molecular weight of the material, polymer concentration, the volume ratio of the solution (e.g., solvent to cosolvent), surfactant type, surfactant concentration, cooling rate, and stirring rate.

[0034] Coacervation of a support material follows the process example described below. An ethanol-water solution is created. The substrate (e.g., gelatin, alginate, cellulose, etc.) used is evaluated. The amount of material dissolved in the solution can be adjusted to alter the size and yield of support material particles. Heating and cooling rates can affect the rate of polymer precipitation into the solution, which will affect particle size. Mixing speed also affects particle size, as described in more detail below. For example, a faster mixing speed will cause precipitated particles to be smaller in average size. Particle size can be measured as the harmonic mean size, as described above. The substrate (e.g., polymer) is dissolved, and a surfactant can be added to the solution to dissolve it. Once the polymer is dissolved, the pH of the solution can be lowered (e.g., by adding an acid) until the isoelectric point of the polymer is reached, and the polymer begins to precipitate out of the solution. The solution is stirred until polymer precipitation is complete or substantially complete, such that the solution exhibits a coacervate.

[0035] The coacervate solution is compressed to form the support material. The coacervate solution is placed in a centrifuge. The number of centrifugation cycles, duration, speed (e.g., RPM setting), and other centrifuge settings are based on the desired yield stress of the support material, the amount of coacervate, the polymer used, etc. After the coacervate is compressed, it is washed in a wash solution. The type of wash solution used can depend on the material being printed in the support material (e.g., collagen, alginate, etc.).

[0036] The following is an example of a process for preparing a support material. A solution is prepared by measuring a 50:50 ethanol-water solution. The ethanol-to-water ratio can be adjusted to control the particle size of the support material. For example, the ratio can include 47.5:52.5 ethanol-to-water, or a similar ratio. For example, 500 mL of deionized (DI) water can be used, and 500 mL of 200-proof absolute ethanol (EtOH) can be used for a 50:50 ratio. For a gelatin-based support material, 20 g of Type B gelatin (2% by weight) and 2.5 g of F127 Pluronic (0.25% by weight) surfactant are measured. 500 mL of DI water is heated to 45°C. Warm water is mixed into the EtOH container. The gelatin and Pluronic powder are slowly added while stirring. Sufficient time (e.g., about 10 minutes) can be allowed for the gelatin and Pluronic powder to fully dissolve in the solution. While stirring, the pH of the solution is adjusted to 5.6-5.7 with acid (e.g., 1 M HCl). Turbidity of the solution indicates coacervation. At this stage, the stirring speed is increased to at least 500 RPM. The stirring speed should be high enough to avoid introducing air bubbles into the solution.

[0037] The following is an example of a process for completing the preparation of the support material. The "raw" coacervate solution is placed in a centrifuge. For example, the support material can be placed in a centrifuge tube at 175 G for approximately 2 minutes. The supernatant is removed. The yellow-white pellet of gelatin at the bottom of the container is left in the tube, and the tube is refilled with the raw coacervate solution. The loose pellet is broken up (e.g., by shaking the container). The solution is centrifuged, for example, at 175 G for 2 minutes. The supernatant is removed. A 2:1 ratio of DI-to-gelatin is added. The pellet is dispersed and any clumps are broken up. The solution is centrifuged, for example, at 225 G for 2 minutes. The supernatant is removed and replaced with 1X PBS containing 25 mM HEPES solution. The solution is centrifuged, for example, at 450 G for 2 minutes. The solution forms a gelatin slurry, which begins to swell and will become more neutral with washing. The swelling ratio can be as high as 3:1.

[0038] In some implementations, if printing alginate, the supernatant is replaced with DI water. The solution is centrifuged, for example, at 450G for 2 minutes. The supernatant is removed and a wash fluid is added. For alginate printing, the wash fluid can include 0.16 wt% CaCl2. For fibrin and collagen printing, other wash solutions can be used. In some implementations, the slurry is allowed to cool. In some implementations, further centrifugation can be performed, for example, at 450G for 2 minutes. In some implementations, a vacuum chamber can be used for 20-30 minutes. In some implementations, further centrifugation can be performed, for example, at 750G for 5 minutes. The supernatant is removed.

[0039] FIG. 2 shows an example of gelatin microparticles 200 formed by the coacervation process described above. The scale bar is 100 micrometers. FIG. 3 shows an example of gelatin microparticles 300 formed by the coacervation process described above. The scale bar is 25 micrometers. The microparticles have substantially the same geometry, including size, shape, etc.

[0040] The process of gelatin coacervation can be further modified by the addition of other charged polymers to the coacervate solution, otherwise known as complex coacervation. In simple coacervation with a single polyampholyte polymer, the charge on the polymer itself is completely neutralized at the isoelectric point. In complex coacervation, the charges between two separate polymers are complexed together.

[0041] For example, Figure 4 shows an example of gelatin microparticles 400 formed via complex coacervation. The addition of gum arabic allows for complex coacervation between the two polymers, producing microparticles with highly consistent size, morphology, and properties. Gelatin is more positively charged in acidic solutions, while gum arabic is negatively charged in any solution with a pH >2.2–3. As a result, these two polymers are ideal for complex coacervation, and coacervation can occur near the isoelectric point of gelatin. In Figure 4, the gelatin microparticles shown were created by complex coacervation from 2.0 wt% gelatin B and 0.1 wt% gum arabic. The scale bar is 50 micrometers.

[0042] As discussed above with respect to Figure 2, stirring speed affects the size of gelatin particle formation by applying higher shear forces to the particles as they form. Higher shear forces hinder the formation of gelatin particles above a critical size by making larger particles less stable. As a result, higher stirring speeds produce smaller particles. Stirring at nearly four times the standard speed (e.g., 100 RPM) produced particles that were less than one-third the size. Rapid stirring of the coacervate reduced the average particle size from 42.85 ± 13.89 μm to 13.66 ± 4.41 μm. This represents less than 35% dispersion in particle size.

[0043] FIG. 5 shows an example of the formation of smaller gelatin particles 500 relative to gelatin microparticles 200, 300 and 400.

[0044] Altering other chemical parameters, such as the solvent / nonsolvent ratio, can also be used to control particle size. Initial coacervation processes used a 50:50 ratio of water to ethanol. Changing the ratio to 52.5:47.5 water to ethanol resulted in a reduced particle size of 6.42 ± 1.68 μm and a narrower size distribution.

[0045] For example, Figure 6 shows gelatin particles 600 made from a 52.5:47.5 ratio of water to ethanol. The scale bar is 100 micrometers. Other ratios of water to ethanol can be used, such as 60:40, 55:45, 50:50, etc. Figure 7 shows a histogram of particle size distribution. The mixed gelatin particles (labeled Mixed) show a broad distribution of particle sizes. The particles from the 50:50 water to ethanol coacervation (labeled Coacervate) show a narrower distribution of smaller particles. The 52.5:47.2 water to ethanol coacervation (labeled Modified Coacervate) shows an even narrower particle size distribution of smaller particles (e.g., less than about 10 micrometers in diameter).

[0046] The average particle size remains consistent while varying the weight percentage of gelatin when using the same manufacturing conditions. As long as critical manufacturing conditions are consistent, increasing the weight percentage of gelatin increases the overall particle yield from the coacervation process but not their morphology. Figure 8 shows a graph 800. Graph 800 demonstrates that increasing the gelatin concentration during coacervation does not have a significant effect on particle size (<10 microns). Several manufacturing conditions that affect particle size include the bloom and processing method of gelatin (e.g., acid hardening, lime hardening, etc.), the pH of the coacervation solution, the water-to-ethanol volume ratio, the use of surfactants (F127 Pluronic), the cooling rate of the coacervate, and the stirring rate of the coacervate. By controlling these conditions in the coacervation solution, the solubility of gelatin (and particle geometry) can be controlled. Coacervation formation is highly dependent on controlling the solubility of gelatin between the fluid and gel phases after it has been dissolved in solution. The decrease in gelatin solubility subsequently prevents the formation of a single matrix of gelatin, instead forming particles of gelatin as the solubility continues to decrease.

[0047] Gelatin bloom strength depends on the average molecular weight of the gelatin molecules. Higher molecular weight gelatin is less soluble in water-ethanol solutions than its lower molecular weight counterpart and thus precipitates out of solution more easily. As the system cools and the solubility of the gelatin decreases, the gelatin molecules will precipitate out of solution in order of their molecular weight, starting with the highest. Thus, high bloom gelatin with a higher average molecular weight will precipitate out of solution at a different time than lower molecular weight gelatin, affecting the time and temperature at which stable particles form.

[0048] Treating animal tissue with acid or base produces acidic (A) or basic (B) gelatin, respectively. These gelatins have different isoelectric points, which affect their solubility at a given pH. Adjusting the solution pH to the molecule's isoelectric point (pI) represents a minimum in solubility. At the isoelectric point, gelatin molecules undergo a series of charge neutralizations with their own charged residues and those of other gelatin molecules, disrupting the molecules and removing them from solution. The pH of the coacervation solution also determines the solubility of the gelatin and the formation of microparticles.

[0049] Gelatin is soluble in water and nearly insoluble in organic solvents such as alcohol. If gelatin is first dissolved in water above its melting temperature and then cooled, it will form a continuous gel. In a coacervation solution containing an approximately 50:50 mixture of ethanol and water, as the temperature of the coacervation solution drops, the alcohol becomes more strongly associated with the water, making the gelatin less soluble. As a result, gelatin cannot form a continuous matrix in a water-ethanol solution at lower temperatures due to its insolubility in alcohol. Controlling the water-to-ethanol ratio in the coacervation solution significantly determines the solubility of gelatin when forming a coacervate.

[0050] The use of a surfactant (e.g., F127 Pluronic) prevents clumping of gelatin particle formation. Gelatin particles without surfactant tend to stick together, forming large, coarse, dendritic aggregates such as particle 900 shown in Figure 9. The use of a surfactant prevents or reduces the formation of these dendritic particles, resulting in smooth, round particles such as particles 200, 300, 400, 500, and 600 in Figures 2-6. Figure 9 shows the formation of large dendritic particles in the absence of surfactant. Scale bar is 100 micrometers.

[0051] Rapid cooling of the coacervate solution immediately reduces the solubility of gelatin. Rapid cooling of the solution (e.g., faster than 1°C / min) results in the gelatin precipitating out of solution more quickly. If the gelatin precipitates out of solution too quickly, it cannot slowly attach to existing gelatin particles, resulting in rapid buildup of gelatin on a single particle and the formation of rough, dendritic particles. Figure 10 shows particle 1000. Particle 1000 is larger and more irregularly dendritic than the particles in Figures 2-6. Particle 1000 is formed by rapidly cooling the coacervate solution with ice. The scale bar is 100 micrometers.

[0052] Hydration control Figure 11 shows an example process 1100 for controlling particle hydration. Particle hydration can be manipulated by the composition of the support material solution. Gelatin particles hydrate according to osmotic pressure and pH. Utilizing the same dehydration principles that govern their formation, coacervate-driven gelatin particles can be dehydrated by transferring them from water (shown as particle 1110) to ethanol (shown as particle 1120). Hydrophilic gelatin particles 1120 aggregate together to reduce their surface energy. Because gelatin is insoluble in ethanol, particles 1120 can be stored in a dehydrated state and cannot be dissolved in solution. Storing particles 1120 in ethanol also allows for storage below 0°C without the risk of ice crystal formation in the support material. Transferring the gelatin slurry back to water rehydrates particles 1130, allowing them to separate from each other. The dehydration / hydration process is repeatable, allowing for long-term storage of coacervate-driven gelatin particles in a dehydrated state that can be easily reversed to the original state.

[0053] FIG. 12A shows examples of gelatin support particles formed using varying pH values and varying gum arabic concentrations. The scale bar is 50 micrometers for each example. Particle 1200 is formed with a solution pH of 6 and a 0.1% gum arabic concentration. Particle 1210 is formed with a solution pH of 6.5 and a 0.1% gum arabic concentration. Particle 1220 is formed with a solution pH of 7 and a 0.1% gum arabic concentration. Particle 1230 is formed with a solution pH of 6.5 and a 0.1% gum arabic concentration. Particle 1240 is formed with a solution pH of 6.5 and a 0.25% gum arabic concentration. Particle 1250 is formed with a solution pH of 6.5 and a 0.5% gum arabic concentration. Particle 1260 is formed with a solution pH of 6.5 and a 0.75% gum arabic concentration. As can be seen in Figure 12A, as the pH of the solution moves away from the isoelectric point for gelatin (pH 5-6), the particle size decreases. As can be seen in Figure 12A, as the gum arabic concentration increases, the particle size decreases. This is because there are more nucleation sites at higher concentrations, and the same amount of gelatin precipitates from the water / ethanol solution.

[0054] FIG. 12B shows an example of controlling particle size and yield in the support material. Particle 1270 is formed with a solution pH of 5.5 and a 0.1% concentration of gum arabic. Particle 1275 is formed with a solution pH of 6.0 and a 0.1% concentration of gum arabic. Particle 1280 is formed with a solution pH of 6.5 and a 0.1% concentration of gum arabic. Particle 1285 is formed with a solution pH of 6 and a 1% concentration of gum arabic. Particle 1290 is formed with a solution pH of 6.5 and a 1% concentration of gum arabic. As seen in FIG. 12B, the pH and gum arabic concentration can be simultaneously adjusted to control particle size and slurry yield. A pH of 6 and a 1% gum arabic concentration maintains a particle size of approximately 8-12 μm but produces a relatively thicker slurry for the support material. Comparing particle yields for approximately the same size, a 1% gum arabic concentration at pH 6 gives approximately 57.15 million particles / mL, and a 0.1% gum arabic concentration at pH 6.5 gives approximately 18.98 million particles / mL. If the gum arabic-driven support material contains any debris, a brief centrifugation step can be used to remove these dense and generally large particles, while the remainder is washed away prior to cell seeding.

[0055] Rheology After compression, the particles of the support material form a slurry that behaves as a yield stress fluid. After a threshold stress is applied to the slurry, it begins to flow. This behavior can be analyzed using a rheometer to precisely deform a sample of the slurry, monitor the deformation in terms of parameters such as shear stress and shear rate, and calculate viscosity. A yield stress fluid exhibits a constant instantaneous viscosity profile when subjected to too little stress to initiate flow. This is due to the yield stress fluid behaving as a solid for stresses below the critical yield stress required to initiate flow. Once the critical yield stress is achieved, the material rapidly transitions from behaving like a solid to a fluid. At this level of applied stress, the material undergoes a rapid decrease in viscosity with increasing shear rate, evidenced by a deviation from a high instantaneous viscosity. The transition from solid to fluid behavior is initiated by a critical yield stress applied to the material to induce particle motion. Below the critical shear stress, the adhesive forces between particles are greater than the external shear force applied to them, resulting in particles at rest and solid-like behavior. Particle motion is initiated at a critical yield stress when the force applied to the particles overcomes the total adhesive forces and the particles begin to slip past one another. If the shear stress is maintained above the critical yield stress, the particles will continue to slide past one another, exhibiting fluid-like behavior. If the shear force is reduced below the critical yield stress, the particles will reattach to one another by the same forces that initially held them together.

[0056] Figure 13 shows a graph 1300 of the yield stress profile for a support material produced from the coacervate described above. If allowed to deform for a longer period of time, the viscosity of the material eventually begins to level off at a much lower viscosity as it reaches its fluid-like state viscosity at high shear stress. Viscometer data can be obtained from a sample of gelatin microparticle slurry produced by the outlined coacervation process. In Figure 13, the circle indicates the moment when a critical yield stress is applied, resulting in a rapid drop in sample viscosity, confirming that the support exhibits shear-thinning behavior.

[0057] The deviation from the continuous region of viscosity can be related to the instantaneous shear stress required to initiate flow of the Bingham plastic fluid. Further flow tests, such as amplitude sweeps and frequency sweeps, can more accurately determine the linear viscoelastic region (LVR) and yield stress of the non-Newtonian fluid, respectively. The LVR is determined from the linear region of the elastic modulus (G'). A frequency sweep is then performed, selecting a strain from the LVR, which for this sample was chosen to be 0.035.

[0058] FIG. 14 shows an example graph 1400 of amplitude sweep data for a gelatin support material. The linear plateau of the elastic storage modulus (G') indicates the LVR, from which the strain for the frequency sweep can be selected. FIG. 15 shows an example graph 1500 of frequency sweep data for a gelatin support material. The storage modulus (G') and viscous loss modulus (G'') are measured. The crossover point indicates the frequency at which the support material experiences and transitions from behaving as an elastic solid to a viscous fluid. Thus, the crossover point corresponds to the yield stress of the support material.

[0059] The yield stress of this slurry has been shown to be modifiable through the centrifugal compaction process. Higher centrifugal forces bring the particles together, thereby compressing them. The yield stress of the slurry can be adjusted by changing the degree of particle compaction through altering these centrifugal forces. As higher G-forces further compress the slurry, the yield stress of the slurry increases. This behavior can be seen across various brands of gelatin, which can be used to create particulate slurries via the coacervation process outlined above.

[0060] Figure 16 shows an example graph 1600 of yield stress for support materials centrifuged at different speeds, or RPM. Slurries were centrifuged at a final centrifugation speed of either "low" (1100 RPM = 227 G), "medium" (2000 RPM = 751 G), or "high" (4500 RPM = 3803 G), and their yield stresses were measured using a rheometer. The yield stresses of various brands of gelatin are shown in Figure 16. As the centrifugation RPM speed increased, the sample yield stress increased across all types of gelatin, regardless of the gelatin's Bloom value. Controlling the support's yield stress broadens the spectrum of materials that can be used with the slurry. Tuning the slurry's yield stress allows for a wider range of ink compatibility by more closely matching the ink's viscosity to the support's viscosity and yield stress. When the viscosity of the ink is similar to the viscosity and yield stress of the support, the precision of printing is increased, as opposed to the lower precision of embedded printing in support materials with different viscosities and yield stresses.

[0061] FRESH 3D Printing Increased Fidelity Because coacervate-driven microparticles can form Bingham plastic fluids, they can be utilized in FRESH printing. One of the limitations to extrusion accuracy and precision in FRESH printing is the size and shape distribution of particles in the sacrificial support bath. Irregular particle size and shape (e.g., particles in Figure 1B) hinder consistent extrusion and lead to lower print fidelity. When the support material is removed, the particles act as porogens, leaving behind void defects in the print (Figure 17B).

[0062] Because coacervate-driven gelatin particles are both smaller and more consistent in size and shape relative to gelatin particles produced from previous techniques (e.g., blending techniques, emulsification techniques, etc.), extrusion accuracy and precision are increased, and void defects in the print are smaller. The result is a significant increase in print fidelity. To demonstrate this, a "window frame" model was cut using standard 3D printing software.

[0063] Figure 17 shows an example of high print fidelity using the support materials described herein compared to the lower print fidelity of the previous support material. Image 1700 shows a 3D printed mesh model. The model was then printed using collagen ink in a mixed support bath, shown in image 1710, and a coacervate-driven gelatin particle bath, shown in image 1720. Confocal images of the labeled collagen ink highlight increased feature resolution for collagen printed on the coacervate support in image 1740 compared to that printed in the mixed support bath in image 1730. The collagen structures printed on the coacervate support have fewer voids and a more ordered structure compared to those printed on the mixed support material.

[0064] Because coacervation is a scalable chemical process, large amounts of gelatin support can be created more efficiently than mechanical mixing. This allows larger FRESH prints to be produced more rapidly and with less effort. Large prints (e.g., structures) still benefit from the improvements made to FRESH print fidelity to the submillimeter scale shown in Figure 17. The result is the ability to print macroscale objects with higher fidelity than ever before.

[0065] Figure 18 shows an example of a high-fidelity structure printed using the support materials described herein. An adult heart model derived from patient-specific MRI data was converted into a 3D printable format shown in image 1800. A to-scale version of this heart model was then FRESH printed in pure, unmodified bovine collagen, shown in Figure 1810.

[0066] Figure 19 shows a flow diagram 1900 of an example process for manufacturing a support material. A solution is formed with a specific solvent and cosolvent ratio and a specific pH (1910). After selecting a polymer (e.g., gelatin, alginate, fibrin, etc.), the polymer is dissolved in the solution in the specified ratio (1920). The pH is adjusted based on the polymer type to precipitate a coacervate from the polymer (1930). The coacervate is compressed during one or more centrifugation cycles (1940). The coacervate is washed in a wash solution selected based on the type of polymer in the coacervate (1950).

[0067] A number of embodiments have been described. However, it will be understood that various modifications can be made without departing from the spirit and scope of the appended claims. Accordingly, other embodiments are within the scope of the following claims.

Claims

1. A method for forming an additive manufacturing support material, Form a solution of the solvent and co-solvent; The solution is stirred to dissolve the polymer in the solution, forming particles having substantially constant geometry; and Based on particles in the solution that are substantially constant in geometry, a slurry having a specific yield stress value is formed from the solution, wherein the formation comprises compressing the slurry during one or more centrifugal cycles at 1100 RPM to 4500 RPM, and the yield stress value is 20 to 140 Pascals (Pa). The forming method, including the following.

2. Select one or more parameters, then During the generation of the slurry, one or more parameters are adjusted, where each of the one or more parameters is: This includes the gelatin bloom value, polymer processing method, polymer precipitation rate, polymer solubility, polymer molecular weight, polymer concentration, volume ratio of solvent and co-solvent, surfactant type, surfactant concentration, cooling rate or stirring rate, The forming method according to claim 1, further comprising the following:

3. Select one or more parameters; then Adjust one or more parameters during slurry formation. This further includes, The above one or more parameters are: Type of washing solution, centrifugation time for one or more centrifugation cycles, centrifugation force for one or more centrifugation cycles, and number of centrifugation cycles. The method according to claim 1, including the method described in claim 1.

4. The method according to claim 3, further comprising adding a surfactant to the solution.

5. The method according to claim 3, further comprising dehydrating the slurry in ethanol.

6. The method according to claim 5, further comprising rehydrating the slurry in water, wherein the slurry maintains a specific yield strength value after dehydration and rehydration.

7. The polymer is a first polymer, and the formation of the slurry further, The second polymer is added to the solution. Select the isoelectric point of either the first polymer or the second polymer, then Adjust the pH based on the isoelectric point. The method according to claim 1, including the following:

8. The method according to claim 1, further comprising adjusting one or more centrifugal separation cycles to bring a specific yield stress value of the slurry to a specific value.

9. The method according to claim 1, wherein the average size of the particles varies by less than about 35% for particles having substantially constant geometry in the solution.

10. The method according to claim 1, wherein the solution contains a surfactant configured to suppress dendritic formation in the solution.

11. The method according to claim 1, wherein the particles comprise two or more different polymers.

12. The method according to claim 11, wherein one of the two or more different polymers comprises gum arabic, and the other of the two or more different polymers comprises gelatin.

13. The method according to claim 1, wherein the solution comprises water as a solvent and ethanol as a co-solvent, and the solution comprises a solvent-to-co-solvent ratio in the range of 30:70 to 70:

30.

14. The method according to claim 1, wherein the average size of the particles is about 0.5 μm to about 60 μm.

15. The method according to claim 1, wherein the specific yield stress value includes a critical shear stress at which the adhesive force between the first and second particles of the solution is substantially equal to the external shear force applied to the particles of the solution.

16. The method according to claim 15, wherein the critical shear stress is based on the viscosity of the ink for additive manufacturing in the solution.

17. A method for forming an additive manufacturing support material, Form a solution of the solvent and co-solvent; The solution is stirred to dissolve the polymer in the solution, forming particles having substantially constant geometry; and A forming method comprising forming a slurry having a specific yield stress value from the solution based on particles that are substantially constant in geometry in the solution, wherein 70 to 90% of the particles have a volume particle diameter of 0.90 to 1.1 times the average volume particle diameter and have a yield stress value of 20 Pa to 10,000 Pa.

18. The method according to claim 17, wherein the control of the hydration of particles in the solution includes controlling the pH of the solution and the concentration of the polymer in the solution.

19. The method according to claim 18, wherein the pH of the solution is a comprehensive 5.5 to 6, and the concentration of the polymer is 0.1% of the concentration of gum arabic.

20. The method according to claim 18, wherein the pH of the solution is inclusively 6 to 6.5, the concentration of the polymer in the solution is 0.1% of gum arabic, and the particle size is about 8 to 12 microns.

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