Fiber-hydrogel composite materials for forming self-supporting structures, methods of forming self-supporting structures

Abstract

Some embodiments provide methods for forming self-supporting, three-dimensional structure by extrusion of a fiber-hydrogel composite material that is solid prior to extrusion, liquifies under stress during extrusion, and resolidifies after extrusion to form the self-supporting, three-dimensional structure. No support bath, no coagulating bath and no cross-linking bath are used during extrusion of the fiber-hydrogel composite material to form the three-dimensional self-supporting structure. In some embodiments, the self-supporting, three-dimensional structure is a tissue scaffold. In some embodiments, the resulting self-supporting three-dimensional structure is edible.

Description

[0001] FIBER-HYDROGEL COMPOSITE MATERIALS FOR FORMING SELF-SUPPORTING STRUCTURES, METHODS OF FORMING SELF-SUPPORTING STRUCTURES BY EXTRUSION OF FIBER-HYDROGEL COMPOSITE MATERIALS, AND METHODS OF USE THEREOF

[0002] Government Support

[0003] This invention was made with government support under HL141798 awarded by National Institutes of Health (NIH). The government has certain rights in this invention.

[0004] Related Applications

[0005] This application claims benefit of and priority to U.S. Provisional Patent Application No. 63 / 676,331, filed July 26, 2024, and U.S. Provisional Patent Application No. 63 / 676,377, filed July 27, 2024, the entire contents of each application are incorporated by reference herein in their entirely.

[0006] Background

[0007] Hydrogels are attractive materials for tissue engineering, but efforts to date have shown limited ability to produce the microstructural features necessary to promote cellular self-organization into tissues.

[0008] Prior efforts to employ a biological ink in additive manufacturing (e.g., 3-D printing) to create free-standing three-dimensional structures (e.g., tissue scaffolds) often required printing into a support material, a sacrificial material, or a bath that is later drained or removed. In some prior efforts, after printing, chemical or thermal means (e.g., for crosslinking or gelation) were required to change the material properties of the printed ink such that it could be self-supporting. This increased the number of steps, time required, and complexity of a process for forming the free-standing three-dimensional structures (e.g., tissue scaffolds).

[0009] Summary

[0010] An aspect provides a method of forming a three-dimensional, self-supporting structure of a fiber-hydrogel composite material by extrusion. The method includes providing or obtaining a fiber-hydrogel composite material that is solid prior to extrusion. The fiberhydrogel composite material includes: a plurality of polymeric fibers, an average diameter of the plurality of polymer fibers falling in a range of 3-200 microns, and a hydrogel. The method also includes extruding the fiber-hydrogel composite material through at least one aperture onto a support to form the three-dimensional self-supporting structure. Shear stress on the fiber-hydrogel composite material during extrusion liquefies the fiber-hydrogel composite material before the fiber-hydrogel composite material exits the at least one aperture and the fiber-hydrogel composite material returns to a solid state after extrusion. No support bath, no coagulating bath and no cross-linking bath are used during extrusion of the fiber-hydrogel composite material onto the support to form the three-dimensional self- supporting structure.

[0011] In some embodiments, the three-dimensional, self-supporting structure is a three- dimensional, multilayer structure.

[0012] In some embodiments, polymeric fibers in the plurality of polymeric fibers are at least partially aligned with an extrusion direction during extrusion resulting in shear thinning that liquefies the fiber-hydrogel composite material.

[0013] In some embodiments, more than 50% of the polymeric fibers in the plurality of polymeric fibers show anisotropic alignment along an extrusion direction in the formed three- dimensional self-supporting structure.

[0014] In some embodiments, the fibers of plurality of polymeric fibers of the fiber-hydrogel composite material have an aspect ratio of average length to average diameter that falls in a range of 5: 1 to 100: 1. In some embodiments, the fibers of plurality of polymeric fibers of the fiber-hydrogel composite material have an aspect ratio of average length to average diameter that falls in a range of 5 : 1 to 50: 1. In some embodiments, the fibers of the plurality of polymeric fibers of the fiber-hydrogel composite material have an aspect ratio of length to diameter that falls in a range of 10: 1 to 30: 1

[0015] In some embodiments, a weight percent of the plurality of polymeric fibers in the fiber-hydrogel composite material is in a range of 8% to 40%. In some embodiments, a weight percent of the plurality of polymeric fibers in the fiber-hydrogel composite material is in a range of 8% to 15%. In some embodiments, a weight percent of the plurality of polymeric fibers in the fiber-hydrogel composite material is in a range of 8% to 10%.

[0016] In some embodiments, the plurality of polymeric fibers comprises one or more proteins, one or more polysaccharides, or both. In some embodiments, the plurality of polymeric fibers include gelatin and the one or more proteins are one or more gelatin proteins. In some embodiments, the plurality of polymeric fibers include gelatin and the one or more proteins are gelatin proteins, and includes alginate and the one or more polysaccharides are alginate polysaccharides, or both. In some embodiments, the plurality of polymeric fibers comprises hyaluronic acid (HA) and the one or more polysaccharides include HA.

[0017] In some embodiments, the plurality of polymeric fibers comprises a synthetic biodegradable polymer.

[0018] In some embodiments, the synthetic biodegradable polymer is selecting from the group consisting of polylactic acid (PCL), poly(L-lactide-co-s-caprolactone) PLCL, and polyacrylamide (PAA).

[0019] In some embodiments, a standard distribution of lengths for the plurality of polymeric fibers is within a range of 10% of the average length. In some embodiments, a standard distribution of lengths for the plurality of polymeric fibers is within a range of 6% of the average length.

[0020] In some embodiments, the hydrogel includes one or more proteins, one or more polysaccharides, or both. In some embodiments, the hydrogel includes gelatin and the one or more proteins are proteins of gelatin, alginate and the one or more polysaccharides are alginate polysaccharides, or both.

[0021] In some embodiments, the at least one aperture is defined by an orifice of a three- dimensional printing system or an additive manufacturing system.

[0022] In some embodiments, the method further includes moving the at least one aperture relative to the support, moving the support relative to the at least one aperture, or both, during extrusion using a three-dimensional printing system or the additive manufacturing system to control relative movement of the at least one aperture and the support.

[0023] In some embodiments, the at least one aperture is defined by an extruder. In some embodiments, the at least one aperture is defined by a die of a die former, a depositer, or a screw extruder.

[0024] In some embodiments, the fiber-hydrogel composite material is edible. In some embodiments, the resulting fiber-hydrogel composite structure is a food product.

[0025] In some embodiments, the fiber-hydrogel composite structure is a tissue scaffold.

[0026] In some embodiments, the fiber-hydrogel composite material further also includes one or more additional agents for cell programming. In some embodiments, the one or more additional agents for cell programming are disposed on or in the plurality of polymeric fibers.

[0027] In some embodiments, the fiber-hydrogel composite material also includes one or more biologically active agents. In some embodiments, the one or more biologically active agents are disposed on or in the plurality of polymeric fibers. In some embodiments, the fiber-hydrogel composite material also includes one or more pharmaceutically active agents. In some embodiments, the one or more pharmaceutically active agents are disposed on or in the plurality of polymeric fibers.

[0028] In some embodiments, the fiber-hydrogel composite material also includes cells.

[0029] In some embodiments, the fiber-hydrogel composite material also includes fluorescent molecules. In some embodiments, the fluorescent molecules are disposed on or in the plurality of polymeric fibers.

[0030] In some embodiments, the fiber-hydrogel composite material also includes nanoparticles.

[0031] Another aspect provides a method of forming an engineered food product. The method of forming an engineered food product includes providing a three-dimensional self- supporting structure that is a tissue scaffold using any of the methods disclosed herein that produce an edible three-dimensional self-supporting structure. The method also includes seeding the scaffold with muscle cells. The method also includes culturing the muscle cells under suitable conditions to form a muscle tissue, thereby forming the three-dimensional engineered food product.

[0032] Another aspect provides a method forming a three-dimensional engineered tissue. The method includes providing a three-dimensional self-supporting structure that is a tissue scaffold using any of the methods disclosed herein. The method also includes seeding the tissue scaffold with cells. The method also includes culturing the cells under suitable conditions to form a tissue, thereby forming the three-dimensional engineered tissue.

[0033] Another aspect provides a fiber-hydrogel composite material for forming a three- dimensional self-supporting structure by extrusion. The fiber-hydrogel composite material includes a plurality of polymeric fibers, with an average diameter of the plurality of polymer fibers falling in a range of 5-200 microns, and the fibers of the plurality of polymeric fibers having an aspect ratio of average length to average diameter that falls in a range of 5: 1 to 50: 1. The fiber-hydrogel composite material includes also includes a hydrogel that comprises one or more proteins, one or more polysaccharides, or both. The fiber-hydrogel composite material is solid at room temperature prior to extrusion, and the fiber-hydrogel composite material is configured to liquefy due to shear stress during extrusion and configured to return to a solid state after extrusion.

[0034] In some embodiments, plurality of polymeric fibers includes gelatin and the one or more proteins are gelatin proteins. In some embodiments, the hydrogel includes gelatin and the one or more proteins are gelatin proteins, includes alginate and the one or more polysaccharides are alginate polysaccharides, or both.

[0035] In some embodiments, a weight percentage of the polymeric fibers in the fiberhydrogel composite material is in a range of 8% to 15%.

[0036] Brief Description of the Drawings

[0037] The features and advantages of the present disclosure will be more fully understood from the following description of exemplary embodiments when read together with the accompanying drawings. The drawings are intended to illustrate the teachings taught herein and are not intended to show relative sizes and dimensions unless otherwise noted, or to limit the scope of examples or embodiments. In the drawings, the same numbers are used throughout the drawings to reference like features and components of like function.

[0038] FIG. 1 A schematically depicts a process of formation of a fiber-hydrogel composite material in accordance with some embodiments.

[0039] FIG. IB schematically depicts a method of forming a three-dimensional, self- supporting structure of a fiber-hydrogel composite in accordance with some embodiments.

[0040] FIG. 1C is a scanning electron microscopy image showing truncated gelatin fibers (Scale bar, 100 pm) in accordance with an example.

[0041] FIG. ID includes images comparing Gel-Alg ink (0 wt% fiber, left panel) or FIG ink (8 wt% fiber, right panel) in accordance with an example. The Gel-Alg hydrogel has a low- viscosity-liquid-like behavior (left). FIG inks behave solid-like at rest and extrude in a continuous stream (right) (Scale bars, 2 mm).

[0042] FIG. IE schematically depicts a process of formation of a fiber-hydrogel composite material in accordance with an example.

[0043] FIG. IF includes images of long gelatin fibers that were spun by a focused rotary jet spinning method in an example.

[0044] FIG. 1G is a graph of the dependence of length distribution of gelatin fiber after ultrasonication on the sonication time and amplitude relationship in an example.

[0045] FIG 1H includes images of printed 3D donut shapes with a rectilinear infill pattern with increasing number of stacking layers for a fiber-hydrogel composite material with 8 wt % gelatin fibers (top) and for a hydrogel material with no fibers (bottom) showing the high shape retention of using FIG inks (scale bar, 5 mm) in accordance with an embodiment. FIG. II includes images of a cone-shaped model of the self-supporting inverted left ventricle printed in the circumferential direction in accordance with an example (scale bars, 2 mm).

[0046] FIG. 1 J includes images of self-supportive dual ventricle chambers and a heart valve printed in circumferential direction and an angled left ventricle printed in diagonal (30°) titled direction in accordance with an example (scale bars, 5 mm).

[0047] FIG. IK is a micro-computed tomography image of the 3D printed ventricle scaffold after critical point drying, showing fiber structure in the 3D printed geometry in accordance with an example (scale bars, 1 mm).

[0048] FIG. IL is a scanning electron microscopy image of the 3D printed ventricle scaffold showing fiber alignments in printing direction (scale bar, 200 pm.

[0049] FIG. IM includes confocal images of the 3D printed ventricle FIG scaffolds with 5 and 8 wt% fibers and analysis of fiber alignment from confocal images of the 3D printed ventricle FIG scaffolds with 5 and 8 wt% fibers producing corresponding fiber orientation angular distribution graphs where 0° indicates printing direction in accordance with an example (scale bars, 100 pm.k).

[0050] FIG. 2A schematically depicts FIG inks as fiber concentration is diluted and concentrated in accordance with an example. At a concentrated regime, the formation of the fiber-fiber network increases the solid-like behavior of the FIG inks.

[0051] FIG. 2B is a graph of storage (G1) and loss (G") moduli at a concentrated regime in accordance with an example. The formation of the fiber-fiber network increases the solid-like behavior of the FIG inks’ moduli as a function of oscillation stress depending on the fiber weight concentration of FIG inks.

[0052] FIG. 2C is a stress-shear rate graph depending on the fiber concentration in accordance with an example. Low yield stress of 0% fiber Gel-Alg hydrogel inks can be measured at the plateau, d,

[0053] FIG. 2D is a graph of yield stress depending on the fiber concentration that was calculated at the cross-over point of the G' and G" shown in FIG. 2B. n=3,4,4,4,3 inks per condition. Data are presented as mean values + / - SEM. Yield stress for 0 % fiber concentration was derived from the graph of the shear rate sweep test FIG. 2C.

[0054] FIG. 2E is a graph of tan 5 (derived by G" / G') moduli as a function of cyclic strain changes for 0, 5, 6, 8, and 10 wt% gelatin fibers of FIG inks in accordance with an example. The graph shows a clear sol-gel transition between 6 and 8 wt% as the storage and loss moduli intersect depending on the applied oscillation strain. 0 wt% shows only liquid-like behavior (tan 5 >1) and 10 wt% only showed solid-like behavior due to much higher G' at rest than other compositions.

[0055] FIG. 2F is a graph of the storage modulus as a function of temperature for various concentrations of fibers in the FIG in accordance with an example. The higher G' at the lower temperature enables the printing of 3D free-standing objects.

[0056] FIG. 2G includes images of forming structures showing the effect of fiber concentration in 3D printability in accordance with an example. While the FIG inks with 8 wt% fibers maintain the 3D ventricle structure after printing at 6 °C of printing bed temperature, the structure with the 6 wt% fiber of FIG inks collapses due to its less solid-like behavior at the same printing bed temperature.

[0057] FIG. 2H is a graph of measured viscosity changes as a function of shear rate for each fiber concentration of the FIG inks in accordance with an example.

[0058] FIG. 21 is a graph of n indexes depending on the fiber concentration derived from slopes of viscosity-shear rate graph FIG. 2H by the power-law equation i](y) = K(y), where / is viscosity, K is consistency index (defined as the viscosity at the shear rate of 1 s'1), y is the shear rate, and n is the shear-thinning index. n=5,4,5,5,5 inks per condition. Data are presented as mean values + / - SEM.

[0059] FIGS. 3A-3F depicts anisotropic intra and intercellular organization of cardiac tissues cultured on printed FIG scaffolds.

[0060] FIG. 3 A schematically depicts (i) NRVM tissue formation on the 2D-printed Gel- Alg hydrogel, and includes (ii) a brightfield image of the NVRM tissue and (iii) representative immunostained images of nuclei (blue), a-actinin (grey) and F-actin (green) hydrogel in an example (scale bars, 20 pm. DAPI stain, 4',6-diamidino-2-phenylindole) in an example.

[0061] FIG. 3B schematically depicts (i) NRVM tissue formation on the 2D-printed fiberhydrogel composite material and includes (ii) a brightfield image of the NVRM tissue on the hydrogel composite material, and (iii) representative immunostained images of nuclei (blue), a-actinin (grey) and F-actin (green) in the fiber-hydrogel composite material (scale bars, 20 pm. DAPI stain, 4',6-diamidino-2-phenylindole) in an example.

[0062] FIG. 3C is a graph of measurements of normalized sarcomeric a-actinin and F-actin alignment with their alignment quantified on a scale of 0 (random) to 1 (aligned) using an orientation order parameter (OOP) in an example. Statistical analysis was performed using a two-tailed Student’s t-test with unequal variance; **P = 0.000252 and 0.00149 for sarcomere and F-actin, respectively, n = 5 tissues per scaffold condition. Data are presented as mean values ± s.e.m.

[0063] FIG. 3D is a graph of representative distribution of nuclear shape (line length to eccentricity ratio) and orientation (line angle) with printing direction at 0° in an example.

[0064] FIG. 3E is a graph of the Nuclear eccentricity ratio (n = 203 and 183 nuclei from three tissues on Gel-Alg and FIG scaffolds, respectively) in an example. Statistical analysis was performed using a two-tailed Student’s t-test with unequal variance, *P = 0.0295.

[0065] FIG. 3F is a graph of the Nuclear angle distribution from -90° to 90° (n = 203 and 183 nuclei from three tissues on Gel-Alg and FIG scaffolds, respectively) in an example. Statistical analysis was also performed using a two-sample Kolmogorov-Smirnov test,

[0066] = 3 46 x 10-10 (f); n = 203 and 183 nuclei from three independent tissues on Gel- Alg and FIG scaffolds, respectively. In the box plot, the center diamond, box limits and whiskers indicate the mean, the first and third quartiles and the maximum-minimum, respectively.

[0067] Detailed Description

[0068] I. Definitions

[0069] In order that the present invention may be more readily understood, certain terms are first defined. In addition, it should be noted that whenever a value or range of values of a parameter are recited, it is intended that values and ranges intermediate to the recited values are also part of this invention.

[0070] In the following description, for purposes of explanation, specific numbers, materials and configurations are set forth in order to provide a thorough understanding of the invention. It will be apparent, however, to one having ordinary skill in the art that the invention may be practiced without these specific details. In some instances, well-known features may be omitted or simplified so as not to obscure the present invention. Furthermore, reference in the specification to phrases such as “one embodiment” or “an embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the invention. The appearances of phrases such as “in one embodiment” in various places in the specification are not necessarily all referring to the same embodiment.

[0071] The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0072] The term “comprising” or “comprises” is used herein in reference to compositions, methods, and respective component(s) thereof, that are essential to the disclosure, yet open to the inclusion of unspecified elements, whether essential or not.

[0073] The term “consisting of’ refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.

[0074] The word “nozzle” as used herein refers to any element having an opening or orifice for extrusion of a material. The term “nozzle” as used herein, includes, but is not limited to 3D printing nozzles, additive manufacturing nozzles, hollow needles suitable for extrusion, hollow cylinders suitable for extrusion, components of 3-D printing and additive manufacturing heads suitable for extrusion, an orifice in a wall suitable for extrusion.

[0075] The terms “extrusion axis” or “extrusion direction” refer to a particular axis for each portion (e.g., cross-section) of material that was extruded through the nozzle at a particular point in time. At the time point of extrusion for a particular portion of material, the extrusion axis for that particular portion of material is generally an axis that extends through and is perpendicular to a plane of the nozzle opening. After extrusion, as that particular portion of material moves away from the nozzle and is tilted and / or rotated, the corresponding extrusion axis is tilted or rotated accordingly. Because interaction with the substrate and movement of the nozzle relative to substrate usually causes these rotations and tilts of each portion of the extruded material and the corresponding rotations and tilts of the extrusion axis for the portion, the extrusion axis for a portion of material may also be referred to as a printing direction for the portion of material.

[0076] The term “solid” as used herein refers to a material that behaves as a solid and retains its shape when subjected to the relevant or specified conditions.

[0077] The term “liquid” as used herein refers to a material that behaves as a liquid does not retain its shape when subjected to the relevant or specified conditions , instead flowing to conform to a structure containing it. II. Methods of Forming a Three-Dimensional, Self-Supporting Structure of a Fiber- Hydrogel Composite

[0078] Some embodiments provide methods of forming self-supporting structures of fiberhydrogel composite materials by extrusion of a fiber-hydrogel composite material onto a support without requiring a support bath, a coagulating bath, or a cross-linking bath after extrusion. The fiber-hydrogel composite material is solid prior to extrusion. Shear stress on the fiber-hydrogel composite material during extrusion liquefies the fiber-hydrogel composite material enabling it to flow through at least one orifice, and the fiber-hydrogel composite material solidifies after extrusion to form the self-support structure. This may be described as the initially solid fiber-hydrogel composite material exhibiting sufficient thinning due to flow shear during extrusion that the fiber-hydrogel composite material can be extruded (e.g., through a nozzle, needle, a screw extruder), and thickening after being extruded when it is no longer under flow shear such that it forms a three-dimensional self-supporting structure (e.g., a free standing structure) or product without requiring a support, a sacrificial material, or a bath.

[0079] In some embodiments the fibers improve rheological properties of the extruded materials. In some embodiments, the addition of prefabricated fibers (e.g., gelatin fibers) to a hydrogel to form a fiber-hydrogel composite material enables the tailoring of the material rheology of the fiber-hydrogel composite material for a controlled sol-gel transition to achieve extrusion of three-dimensional, free-standing structures without additional supporting materials or requiring post-printing chemical or thermal steps for the structures to be self- supporting.

[0080] Some embodiments provide fiber-hydrogel composite materials (e.g., fiber-hydrogel composite inks) including prefabricated fibers (e.g., gelatin fibers) for printing or extruding three-dimensional (3-D) self-supporting structures (e.g., tissue scaffolds) that recapitulate biological function. In some embodiments, the tissue scaffolds may be scaffolds for muscle cells.

[0081] Some embodiments provide fiber-hydrogel composite materials that that include prefabricated fibers (e.g., gelatin fibers) for printing or extruding three- dimensional self- supporting structure of materials with some properties similar to those of some biological structures or materials (e.g., muscle or meat). In some embodiments, the formed structures or products are edible. In some embodiments, the fibers add meat-like texture (e.g., anisotropic alignment) to extruded products.

[0082] Some embodiments provide a method of forming a three-dimensional, self-supporting structure of a fiber-hydrogel composite material by extrusion. The method includes providing or obtaining a fiber-hydrogel composite material that is solid prior to extrusion. The fiber fiber-hydrogel composite material includes a plurality of polymeric fibers with an average diameter of the plurality of polymer fibers falling in a range of 3-200 microns. The fiber fiber-hydrogel composite material also includes a hydrogel. The method also includes extruding the fiber-hydrogel composite material through at least one aperture onto a support to form the three-dimensional self-supporting structure. The fiber-hydrogel composite material, which is solid prior to extrusion, is configured such that shear stress on the fiberhydrogel composite material during extrusion liquefies the fiber-hydrogel composite material before the fiber-hydrogel composite material exits the at least one aperture, and the fiberhydrogel composite material returns to a solid state after extrusion. No support bath, no coagulating bath and no cross-linking bath are used during extrusion of the fiber-hydrogel composite material onto the support to form the three-dimensional self-supporting structure.

[0083] In some embodiments, the dimensions of the polymeric fibers, the hydrogel and the weight percent concentration of the plurality of polymeric fibers in the fiber-hydrogel composite material are selected such that shear stress on the fiber-hydrogel composite material during extrusion liquefies the fiber-hydrogel composite material before the fiberhydrogel composite material exits the at least one aperture, and the fiber-hydrogel composite material returns to a solid state after extrusion.

[0084] In some embodiments, the plurality of polymeric fibers are premade polymeric fibers having specified average length and diameter or having specified length and diameter distributions. In some embodiments, the method also includes making the plurality of fibers.

[0085] FIG. 1 A schematically illustrates some steps in providing or obtaining a fiberhydrogel composite material that is solid prior to extrusion in accordance with some embodiments. In FIG. 1 A the polymeric fibers are gelatin fibers and the hydrogel is a gelatin- alginate (Gel-Alg) hydrogel; however, one of ordinary skill in the art in view of the present disclosure will understand that embodiments are not limited to those materials. In the embodiment depicted in FIG. 1 A, long polymeric fibers 102 (e.g., gelatin fibers) are formed by rotary jet spinning. The long polymeric fibers 102 are processed to produce the plurality of polymeric fibers 104 (e.g., plurality of gelatin fibers) having a desired average length or a desired length distribution (not depicted) in accordance with some embodiments. See discussion of processing of polymeric fibers to obtain desired average length or desired average length distribution below with respect to FIG.1G. The plurality of polymeric fibers 104 are combined with a hydrogel 106 e.g., a Gel-Alg hydrogel) to form a fiber-hydrogel composite material 108 (e.g., a fiber-hydrogel composite ink). The addition of the polymeric fibers to the hydrogel increases the viscosity of the resulting material such that it behaves as a solid or displays solid-like behavior.

[0086] The fiber-hydrogel composite material is extruded through at least one aperture onto a support to form the three-dimensional self-supporting structure. FIG. IB schematically depicts an embodiment in which a fiber-hydrogel composite is extruded from a nozzle as a 200-300 micron in diameter filament onto a support (not shown) to form a three-dimensional self-supporting structure. In some embodiments, the three-dimensional, self-supporting structure is a three-dimensional, multilayer structure. In the embodiment of FIG. IB, the structure is a multilayer structure for tissue engineering.

[0087] Although FIG. IB schematically depicts the fiber-hydrogel composite being extruded from a nozzle (e.g., of a three-dimensional printing system), embodiments are not limited to a nozzle. In some embodiments, the at least one aperture is defined by an extruder. In some embodiments, the at least one aperture is defined by screw extruder. In some embodiments, the at least one aperture is defined by a die former. In some embodiments, the at least one aperture is defined by a depositer.

[0088] In some embodiments, the method also includes moving the at least one aperture relative to the support, moving the support relative to the at least one aperture, or both, during extrusion using a three-dimensional printing system or the additive manufacturing system to control relative movement of the at least one aperture and the support. In the embodiment, depicted in FIG. IB, relative movement between the nozzle and the support is controlled by a three-dimensional printing system to form a hollow organ shape.

[0089] In some embodiments, polymeric fibers in the plurality of polymeric fibers are at least partially aligned with an extrusion direction during extrusion resulting in shear thinning that liquefies the fiber-hydrogel composite material. The embodiment of FIG. IB schematically depicts an example of alignment of the polymeric fibers during extrusion.

[0090] In some embodiments, the polymeric fibers show anisotropic alignment along an extrusion direction in the formed three-dimensional self-supporting structure. The embodiment of FIG. IB schematically illustrates an example of anisotropic alignment of polymeric fibers in a formed structure. In some embodiments, more than 50% of the polymeric fibers in the plurality of polymeric fibers show anisotropic alignment along an extrusion direction in the formed three-dimensional self-supporting structure.

[0091] III. Polymeric Fibers

[0092] In some embodiments, the polymeric fibers include one or more proteins, one or more polysaccharides, or both. In some embodiments, the polymeric fibers include gelatin and the one or more proteins are one or more gelatin proteins. The Examples below describe a fiberhydrogel composite material including gelatin fibers.

[0093] In some embodiments, the polymeric fibers include hyaluronic acid (HA) and the one or more polysaccharides include HA. U.S. Patent Application Publication US 2020 / 0330644A1, which is incorporated by reference herein, describes HA fibers used in a bioink. In some embodiments, the polymeric fibers include a synthetic biodegradable polymer. In some embodiments, the synthetic biodegradable polymer is polylactic acid (PCL), poly(L-lactide-co-s-caprolactone) PLCL, or polyacrylamide (PAA). US 2020 / 0330644A1, which is incorporated by reference herein, describes PLC fibers, PLCL fibers and PAA fibers used in bioinks.

[0094] The weight percent of the polymeric fibers in the fiber-hydrogel composite ink affects the rheological properties of the fiber-hydrogel composite ink. In some embodiments, the weight percent of the polymeric fibers in the fiber-hydrogel composite ink is in a range of 8% to 40%. In some embodiments, the weight percent of the polymeric fibers in the fiberhydrogel composite ink is in a range of 8% to 20%. In some embodiments, the weight percent of the polymeric fibers in the fiber-hydrogel composite ink is in a range of 8% to 15%. In some embodiments, the weight percent of the polymeric fibers in the fiber-hydrogel composite ink is in a range of 8% to 10%. See FIGS. 1H and 2A-21.

[0095] Any suitable method may be used to prepare long polymeric fibers or a material including long polymeric fibers (e.g., a non-woven polymeric fiber sheet) that is processed to produce the plurality of polymeric fibers having a desired diameter and length distribution. Suitable methods include, but are not limited to rotary jet spinning, immersion rotary jet spinning, pull-spinning, electrospinning, solution blow spinning, melt extrusion, microfluidic extrusion, etc. These methods can be used to produce fibers with diameters in a range of 0.1 pm to 200 pm and lengths typically exceeding 1 cm.

[0096] In one embodiment, long polymeric fibers or a material including long polymeric fibers (e.g., a non-woven polymeric fiber sheet) are / is formed by ejecting a polymer solution from a reservoir onto a collector (e.g., a stationary collector, a rotating mandrel or mandrel assembly). In some embodiments, rotary jet spinning (RJS) is used to create long polymeric fibers that are collected into non-woven polymeric fiber sheets. Suitable RJS devices and uses of the devices for fabricating the long polymeric fibers and non-woven polymeric fiber sheets are described in U.S. Patent Publication No. 2012 / 0135448, U.S. Patent Publication No. 2013 / 0312638, U.S. Patent Publication No. 2014 / 0322515, the entire contents of each of which are incorporated in their entirety by reference.

[0097] In some embodiments, immersion rotary jet pinning (iRJS) is used to create long polymeric fibers as described in U.S. Patent Publication No. 2015 / 0354094, the entire content of which is incorporated by reference in its entirety.

[0098] In other embodiments, the polymeric fibers may be flung using a pull spinning technique onto a collector (e.g., a stationary collector, a rotating mandrel or mandrel assembly). Suitable pull spinning devices and uses of the devices for fabricating the nonwoven polymeric fiber sheets are described in U.S. Patent Publication No. 2014 / 0322515, the entire contents of which is incorporated in its entirety by reference.

[0099] In some embodiments, long polymeric fibers or a material including long polymeric fibers are physically broken down into smaller lengths to produce polymeric fibers having different lengths, each less than 1 mm. In some embodiments, this includes cutting the material into smaller pieces (e.g., about 1 mm in length) followed by crushing, grinding or both to reduce average fiber length. In some embodiments, the material including long polymeric fibers is freeze-dried prior to the cutting, crushing and grinding. In some embodiments, the polymeric fibers are mixed with a solvent prior to grinding. In some embodiments, grinding conditions, such as applied force and grinding time, can be used to influence a length distribution of the fibers having different lengths resulting from the grinding. In some embodiments, the long polymeric fibers are mechanically broken, at least in part, using sonication. See FIG. 1G and description below regarding an example including processing fibers using sonication.

[0100] The polymeric fibers having different lengths are filtered or fractionated to select a plurality of polymeric fibers having a desired length distribution. In some embodiments, the fibers having different lengths are in a slurry for the filtering and fractionation. In some embodiments, the filtration and / or fractionation includes sedimentation, centrifugation, passage through porous material filters and / or other known methods. In some embodiments, the filtering or fractionation includes separation using a centrifuge.

[0101] In some embodiments, the plurality of polymeric fibers are pre-made or already provided.

[0102] In some embodiments, the plurality of polymeric fibers has an average diameter in a range of 3 pm to 100 pm. In some embodiments, the plurality of polymeric fibers has an average diameter in a range of 3 to 50 pm. In some embodiments, the plurality of polymeric fibers has an average diameter in a range of 3 to 10 pm. In some embodiments, the plurality of polymeric fibers has an average diameter in a range of 3 to 7 pm.

[0103] In some embodiments, the desired length distribution will include fibers falling within a specified length range. In some embodiments, the desired length distribution includes an average fiber length falling within a selected range and a standard deviation falling within a specified range.

[0104] In some embodiments, the desired length distribution includes fibers falling within a length range of 30 microns to 2 mm. In some embodiments, the desired length distribution includes fibers falling within a length range of 40 microns to 1 mm. In some embodiments, the desired length distribution includes fibers falling within a length range of 50 microns to 500 microns. In some embodiments, the desire length distribution includes fibers falling within a length range of 50 microns to 200 microns.

[0105] In some embodiments, the plurality of polymeric fibers has an aspect ratio of average length to average diameter falling within a specified range. In some embodiments the aspect ratio falls in a range of 5: l to 100: 1. In some embodiments, the aspect ratio falls in a range of 5: 1 to 50: 1. In some embodiments, the aspect ratio falls in a range of 10: 1 to 30: 1.

[0106] In some embodiments, the distribution of lengths of polymeric fibers in the plurality of polymeric fibers has a standard deviation within a specified percentage of the average fiber length. In some embodiments, the distribution of lengths of polymeric fibers in the plurality of polymeric fibers has a standard deviation within 10% of the average fiber length. In some embodiments, the distribution of lengths of polymeric fibers in the plurality of polymeric fibers has a standard deviation within 6% of the average fiber length.

[0107] In some embodiments, the plurality of polymeric fibers has an aspect ratio of average length to average diameter falling within a specified range. In some embodiments the aspect ratio falls in a range of 5: l to 100: 1. In some embodiments, the aspect ratio falls in a range of 5: 1 to 50: 1. In some embodiments, the aspect ratio falls in a range of 10: 1 to 30: 1.

[0108] In some embodiments, one or more additional agents for cell programming are disposed on or in the plurality of polymeric fibers. In some embodiments, one or more pharmaceutically active agents are disposed on or in the plurality of polymeric fibers. In some embodiments, one or more biologically active agents are disposed on or in the plurality of polymeric fibers. In some embodiments, fluorescent molecules are disposed on or in the plurality of polymeric fibers. In some embodiments, nanoparticles are disposed on or in the plurality of polymeric fibers.

[0109] IV. Hydrogel

[0110] In some embodiments, the hydrogel includes one or more proteins, one or more one or more polysaccharides, or both. In some embodiments, the plurality of polymeric fibers includes gelatin and the one or more proteins are one or more gelatin proteins. In some embodiments, the plurality of polymeric fibers includes alginate, and the one or more polysaccharides are alginate polysaccharides. The Examples below describe a hydrogel including gelatin and alginate.

[0111] In some embodiments, the hydrogel may include one or more additional agents, e.g., a plurality of living cells, e.g., muscle cells, neuron cells, endothelial cells, and epithelial cells; biologically active agents, e.g., lipophilic agents, peptides, lipids, nucleotides, small molecules; fluorescent molecules, metals, ceramics, nanoparticles, and pharmaceutically active agents.

[0112] V. Fiber-Hydrogel Composite Material

[0113] The fiber-hydrogel composite material is solid at room temperature prior to extrusion. Shear stress on the fiber-hydrogel composite material during extrusion liquefies the fiberhydrogel composite material before the fiber-hydrogel composite material exits the at least one aperture. The polymeric fibers in the plurality of polymeric fibers are at least partially aligned with an extrusion direction during extrusion resulting in shear thinning that liquefies the fiber-hydrogel composite material. The fiber-hydrogel composite material returns to a solid state after extrusion is sufficiently fast to form a three-dimensional, self-supporting structure of a fiber-hydrogel composite material such that no support bath, no coagulating bath and no cross-linking bath are used during extrusion of the fiber-hydrogel composite material onto the support to form the three-dimensional self-supporting structure. The control of the sol-gel transition is determined at least in part, by a volume fraction of polymeric fibers in the fiber-gel composite materials.

[0114] In some embodiments, a weight percent of the plurality of polymeric fibers in the fiber-hydrogel composite material is in a range of 8% to 40%. In some embodiments, the weight percent of the plurality of polymeric fibers in the fiber-hydrogel composite material is in a range of 8% to 15%. In some embodiments, the weight percent of the plurality of polymeric fibers in the fiber-hydrogel composite material is in a range of 8% to 10%. See FIGS. 1H and 2B-2I and accompanying discussion below explaining a transition from a fiberhydrogel composite material that does not result in a self-supporting structure at 6 wt% fibers and a fiber-hydrogel composite material that does result in a self-supporting structure at 8 wt% fibers. See FIGS. 2A-2L and accompanying discussion below regarding how a wt% of fibers in the fiber-hydrogel composite material influences rheological properties of the fiberhydrogel composite material.

[0115] Some embodiments provide a fiber-hydrogel composite material for forming a three- dimensional self-supporting structure by extrusion, n some embodiments, the fiber-hydrogel composite material includes a plurality of polymeric fibers with an average diameter of the plurality of polymer fibers falling in a range of 5-200 microns. The fibers of the plurality of polymeric fibers having an aspect ratio of average length to average diameter that falls in a range of 5: 1 to 50: 1. The fiber-hydrogel composite material also includes a hydrogel including one or more proteins, one or more polysaccharides, or both. The fiber-hydrogel composite material is solid at room temperature prior to extrusion, and the fiber-hydrogel composite material is configured to liquefy due to shear stress during extrusion and configured to return to a solid state after extrusion.

[0116] In some embodiments, the hydrogel includes gelatin, and the one or more proteins are gelatin proteins. In some embodiments, the hydrogel includes alginate and the one or more polysaccharides are alginate polysaccharides. In some embodiments, the hydrogel includes both gelatin and alginate.

[0117] In some embodiments, a weight percentage of the polymeric fibers in the fiberhydrogel composite material is in a range of 8% to 15%. VI. Methods of Use - Tissue Scaffold and Engineered Food Product

[0118] Some embodiments include methods of forming an engineered food product. In some embodiments, the fiber-hydrogel composite material may be edible (e.g., for a human, a specified animal, or a specified family of animals or genus of animals).

[0119] In some embodiments, the three-dimensional self-supporting structure may be a tissue scaffold for an engineered food product. Some embodiments include methods of forming an engineered food product including providing a three-dimensional self-supporting structure that is a tissue scaffold using any of the methods disclosed herein. In some embodiments, the method also includes seeding the scaffold with muscle cells. In some embodiments, the method also includes culturing the muscle cells under suitable conditions to form a muscle tissue, thereby forming a three-dimensional engineered food product.

[0120] As noted above, the fibers and / or hydrogel may also include one or more additional agents, e.g., a plurality of living cells, e.g., muscle cells, neuron cells, endothelial cells, and epithelial cells; biologically active agents, e.g., lipophilic agents, peptides, lipids, nucleotides, small molecules; fluorescent molecules, metals, ceramics, nanoparticles, and pharmaceutically active agents.

[0121] The one or more additional agents may be added to a polymer solution used to fabricate the fibers (e.g., enabling the agent to be incorporated into the fibers themselves); one or more fibers may be coated (e.g., fully or partially) with one or more additional agents prior to being combined with the hydrogel, the hydrogel may include one or more additional agents; and / or a tissue scaffold may be coated (e.g., fully or partially) with one or more additional agents.

[0122] For example, by including one or more additional agents within the fibers as they are produced, e.g., in the solution of the polymer used to produce the fibers, including them after production of the fibers, and / or including them in the hydrogel, and forming a cross-linked scaffold including the one or more additional agents, ECM-inspired nanofibrous scaffolds with controlled-release of cell-instructive factors may promote beneficial immune system reactions and endogenous repair mechanisms within regenerative medicine applications. They may promote cell programming, cell reprogramming, and tissue genesis within broader tissue engineering applications (e.g., in vitro disease models). In some embodiments, additional agents for cell programming include the so-called “Yamanaka factors” (transcription factors Oct4, S0X2, cMyc, and Klfj), which are used to induce pluripotency in somatic cells.

[0123] Another use of the three-dimensional tissue scaffolds in some embodiments is the delivery of one or more substances to a desired location and / or in a controlled manner. In some embodiments, the tissue scaffold is used to deliver the materials, e.g., a pharmaceutically active substance. In other embodiments, the tissue scaffold is used to deliver substances that are contained in the polymeric fibers or that are produced or released by substances contained in the polymeric fibers materials. For example, polymeric fibers containing cells can be implanted in a body and used to deliver molecules produced by the cells after implantation. The present compositions can be used to deliver substances to an in vivo location, an in vitro location, or other locations.

[0124] The ability to seed the tissue scaffolds of some embodiments with living cells also provides the ability to build tissue, organs, or organ-like tissues. Cells included in such tissues or organs can include cells that serve a function of delivering a substance, seeded cells that will provide the beginnings of replacement tissue, or both.

[0125] In some embodiments of the invention, a tissue scaffold is treated with a plurality of living cells and cultured under appropriate conditions to produce a bioengineered tissue.

[0126] In some embodiments, the tissue scaffolds contacted or seeded with living cells incorporate a drug such that the function of the implant will improve. For example, antibiotics, anti-inflammatories, local anesthetics or combinations thereof, can be incorporated into the tissue scaffold for a bioengineered organ to speed the healing process.

[0127] Examples of bioengineered tissue include, but are not limited to, bone, dental structures, joints, cartilage, (including, but not limited to articular cartilage), skeletal muscle, smooth muscle, cardiac muscle, tendons, menisci, ligaments, blood vessels, stents, heart valves, corneas, ear drums, nerve guides, tissue or organ patches or sealants, a filler for missing tissues, sheets for cosmetic repairs, skin (sheets with cells added to make a skin equivalent), soft tissue structures of the throat such as trachea, epiglottis, and vocal cords, other cartilaginous structures such as articular cartilage, nasal cartilage, tarsal plates, tracheal rings, thyroid cartilage, and arytenoid cartilage, connective tissue, vascular grafts and components thereof, and sheets for topical applications, and repair of organs such as livers, kidneys, lungs, intestines, pancreas visual system, auditory system, nervous system, and musculo skeletal system. In one particular embodiment, a tissue scaffold is contacted with a plurality of living muscle cells and cultured under appropriate conditions to guide cell growth with desired anisotropy to produce a muscle thin film (MTF) or a plurality of MTFs prepared as described in U.S. Patent Publication Nos. 20090317852 and 20120142556, and PCT Application No. PCT / US2012 / 068787. The entire contents of each of the foregoing are incorporated herein by reference.

[0128] Tissue scaffolds contacted with living cells can also be used to produce prosthetic organs or parts of organs. Mixing of committed cell lines in a three-dimensional tissue scaffold can be used to produce structures that mimic complex organs. The ability to shape the tissue scaffold and control fiber anisotropy enables preparation of complex structures to replace organs such as liver lobes, pancreas, other endocrine glands, and kidneys. In such cases, cells are implanted to assume the function of the cells in the organs. Preferably, autologous cells or stem cells are used to minimize the possibility of immune rejection.

[0129] In some embodiments, tissue scaffolds contacted with living cells are used to prepare partial replacements or augmentations. For example, in certain disease states, organs are scarred to the point of being dysfunctional. A classic example is hepatic cirrhosis. In cirrhosis, normal hepatocytes are trapped in fibrous bands of scar tissue. In one embodiment, the liver is biopsied, viable liver cells are obtained, cultured in the tissue scaffold, and reimplanted in the patient as a bridge to or replacement for routine liver transplantations.

[0130] In another example, by growing glucagon secreting cells, insulin secreting cells, somatostatin secreting cells, and / or pancreatic polypeptide secreting cells, or combinations thereof, in separate cultures, and then mixing them together with a tissue scaffold, an artificial pancreatic islet is created. One or more artificial pancreatic islets are then placed under the skin, retroperitoneally, intrahepatically or in other desirable locations, as implantable, long-term treatments for diabetes.

[0131] In other examples, hormone-producing cells are used, for example, to replace anterior pituitary cells to affect synthesis and secretion of growth hormone secretion, luteinizing hormone, follicle stimulating hormone, prolactin and thyroid stimulating hormone, among others. Gonadal cells, such as Leydig cells and follicular cells are employed to supplement testosterone or estrogen levels. Specially designed combinations are useful in hormone replacement therapy in post and perimenopausal women, or in men following decline in endogenous testosterone secretion. Dopamine-producing neurons are used and implanted in a matrix to supplement defective or damaged dopamine cells in the substantia nigra. In some embodiments, stem cells from the recipient or a donor can be mixed with slightly damaged cells, for example pancreatic islet cells, or hepatocytes, and placed on a tissue scaffold and later harvested to control the differentiation of the stem cells into a desired cell type. In other embodiments thyroid cells can be seeded and grown to form small thyroid hormone secreting structures. This procedure is performed in vitro or in vivo. The newly formed differentiated cells may be introduced into the patient.

[0132] VII. Examples

[0133] Three-dimensional, self-supporting structures of fiber-hydrogel composite materials were formed by extruding the fiber-hydrogel composite material through at least one aperture onto a support to form the three-dimensional self-supporting structure. Shear stress on the fiber-hydrogel composite material during extrusion liquefies the fiber-hydrogel composite material before the fiber-hydrogel composite material exits the at least one aperture and the fiber-hydrogel composite material returns to a solid state after extrusion. No support bath, no coagulating bath and no cross-linking bath were used during extrusion of the fiber-hydrogel composite material onto the support to form the three-dimensional self-supporting structures.

[0134] The inventors reasoned that printing 3D scaffolds containing prefabricated microscale fibers would provide self-supportive scaffolds for cardiomyocytes to self-organize to form a cardiac chamber. Inspired by the ECM networks of the heart, the fiber-hydrogel composite material for extrusion (also referred to as “ink” or “FIG ink” herein) was formed by infusing gelatin fibers into a gelatin and alginate (Gel-Alg) hydrogel matrix (see FIG. 1 A). In contrast to cellulose nanofibers and carbon nanotubes previously used in inks, fibronectin-coated gelatin fibers contain arginine-glycine- aspartic acid (RGD) peptide binding domains that promote matrix-cell adhesion through integrins. Furthermore, incorporating fibers into the hydrogel modifies the ink rheology, enabled formation of accurate and complex 3D scaffolds without any supporting structures or materials. The inventors hypothesized that by incorporating both topological and chemical cues in the form of aligned gelatin microfibers, 3D ventricle scaffolds printed in this manner would promote the self-organization of cardiomyocytes into anisotropic muscle tissues (see FIG. IB). Results

[0135] Printed 3D hydrogel scaffolds provide microscale anisotropy

[0136] Fiber-infused gel (FIG) inks for printing 3D scaffolds were fabricated. The fiberhydrogel composite material (e.g., inks) included of gelatin fibers (FIG. 1C) and Gel-Alg hydrogel (FIG. ID). FIG. IE schematically depicts steps in formation of the fiber-hydrogel composite inks. Gelatin fibers were produced by rotary jet spinning (FIG. IF) and were truncated by ultrasonication to a length of 85.9 ± 2.53 pm with a diameter of 4.20 ± 0.23 pm (mean ± standard error of the mean (s.e.m.)), reducing the variance in length and enabling passage through a printing nozzle with a diameter of 200 pm. FIG. 1G is the length distribution of gelatin fiber after the ultra- sonication process depending on the sonication time and amplitude. Afterward, the fibers were chemically crosslinked with N-(3- dimethylaminopropyl)- N'-ethylcarbodiimidehydrochloride N-hydroxysuccinimide (EDC / NHS) and fibronectin. Unlike Gel-Alg hydrogels where 3D printing is limited due to their fluidity, incorporating 8 wt% prefabricated gelatin fibers into Gel-Alg hydrogels rendered the hydrogels into a predominantly elastic gel (8 wt% fiber, 2.4 wt% gelatin, 2.4% alginate in phosphate buffered saline (PBS); FIGS. ID, 1H and 2A-2I). During printing, shear stresses in the nozzle were sufficient to turn the elastic FIG ink into a liquid, and its elastic stability was restored upon extrusion (see FIGS. 1D„ 1H- II and 2B-2I). These nonlinear rheological properties allow for the continuous extrusion of FIG inks with minimal post-extrusion swelling and spreading (See FIGS. ID, 1H, II and 2E) and enable the printing of layered structures for increasingly complex geometries without sacrificial layers or a sacrificial bath as supporting structures (see Fig. 1H and 2G). The rheological properties of the inks were characterized for different concentrations of gelatin fibers in the fiber-hydrogel inks (see FIGS. 2A-2I. and “Rheological test of FIG inks" section below. This enabled printing self- supportive hollow 3D structures such as a cardiac ventricle with a broad range of design parameters, reproducibility and accuracy (see FIGS. 1H and 21). More complex structures, such as heart valves, dual -chambered hearts and left ventricles with varying angular orientations, could also be printed using FIG inks (see FIG. 1 J). The high elastic modulus of gel-like FIG inks provided sufficient gel stability to print self-supportive walls with thicknesses ranging between 200 pm and 350 pm without the need for scaffolds or sacrificial baths (see FIGS. 1H-1K and 2G). Inducing cardiac muscle cells to recapitulate the structure-function relationships of heart muscle requires structural anisotropy and biochemical guidance cues with micrometrescale precision. To achieve this, the shear thinning properties of FIG inks were exploited (see FIGS. 2A-2I), which is not only essential for printing, but also for inducing fiber alignment along the direction of printing. This enabled the formation of aligned-fiber-embedded scaffolds, which can be used to direct cardiomyocyte alignment. Following this process, shear-induced fiber alignment on the micrometer scale was achieved across the entire length scale of the printed structure (FIGS. IK and IL). Alignment of the incorporated fibers was well-maintained post extrusion due to the gel stability of the FIG inks (FIG. IM), which was further enhanced by lowering the printing bed temperature to 6 °C compared to printing at room temperature (FIG. 2F). By the ionic crosslinking of alginate (1 wt% calcium chloride) and enzymatic crosslinking of gelatin (~8 wt% microbial transglutaminase), the printed scaffolds and their microscale architecture remained intact in water. The resulting printed scaffolds had a resolution closer to the fiber diameter (~5 pm) and acted as geometric cues for cell alignment despite the printing resolution being limited by the nozzle diameter (200- 300 pm).

[0137] Fiber-infused gel scaffolds potentiate cellular alignment

[0138] To evaluate the ability of FIG printed scaffolds to potentiate the intra- and intercellular organization of cardiomyocytes, myofibrillar organization and alignment, neonatal rat ventricular cardiomyocytes (NRVMs) were seeded on printed two-dimensional (2D) FIG scaffolds (8 wt% fiber, 2.4 wt% gelatin and 2.4 wt% alginate; FIG. 3B). This resulted in the formation of highly aligned cardiac tissues (FIG. 3B), which retained comparable cell viabilities relative to pleomorphic culture conditions). The degree of structural tissue anisotropy was then measure by computing the orientational order parameter (OOP), which quantifies sarcomeric a-actinin and cytoskeletal actin filament (F-actin) alignment, representative of intra- and intercellular organization (FIG. 3C). In physiologically healthy cardiac tissues, cytoskeletal structures including sarcomeres and actin filaments are highly aligned, with OOP values close to unity. Here, both sarcomeric and F-actin OOP values were significantly higher for tissues on FIG scaffolds (0.578 ± 0.060 for a-actinin, 0.657 ± 0.021 for F-actin, mean ± s.e.m., n = 5) in comparison to control Gel-Alg scaffolds that contain a similar chemical composition to FIG scaffolds without micro- scale topologies (4 wt% gelatin and 4 wt% alginate, 0.245 ± 0.033 for a-actinin, 0.314 ± 0.052 for F-actin, mean ± s.e.m., n = 5). Consequently, the prestress generated by intra- and intercellular cytoskeletal structures determined the nuclear elongation and orientation. Nuclei were elongated on FIG scaffolds with a mean eccentricity ratio (the length of the major axis to the length of the minor axis) of 2.37 ± 0.17 compared to nuclei on the control Gel-Alg scaffolds, which remained relatively round (eccentricity ratio = 1.40 ± 0.02, mean ± s.e.m.; FIGS. 3D, 3E). The nuclear orientation of NRVMs on FIG scaffolds also aligned along the printing direction (0°) in contrast to those on the control Gel-Alg scaffolds, which showed no specific orientation (FIG. 3D-3F).

[0139] Conclusion

[0140] The examples demonstrated the ability to 3D print tissue-engineered ventricle models with hydrogel -based FIG ink, which simultaneously recapitulates the heart’s microstructural ECM architecture and macrostructural organ-level geometry. Here, infused gelatin fibers acted as a rheological modifier in our hydrogel inks, enabling the printing of complex 3D objects without the use of sacrificial baths. In addition, these fibers also provided biochemical and microstructural cues, which promoted cell adhesion and self-organization into a functional syncytium. Resulting in vitro ventricle models showed cyclic contractile motions of the heart.

[0141] Methods

[0142] Preparation of gelatin fiber filler

[0143] Gelatin fibers were produced by the focused rotary jet spinning method as previously described in more detail. See Chang, H. et al. Recreating the heart’s helical structure-function relationship with focused rotary jet spinning. Science 377, 180-185 (2022), which is incorporated by reference herein in its entirety. Gelatin (type A, gel strength -300 g Bloom, Sigma-Aldrich) was dissolved in 1,1,1,3,3,3-hexafuoro -2-propanol (HFIP; Oakwood Chemical, no. 003409) at 5% (w / v). The gelatin / HFIP solution was then injected into a customized spinneret at 1.8 ml min-1 using an automated syringe pump (Harvard Apparatus, part no. 703007). The spinneret was rotated at 10,000 r.p.m., extruding the gelatin solution out of three 400 pm holes into long fiber streams that were collected on a rotating collector (Heidolph Hei Torque Core) at 300 r.p.m. (Supplementary FIG. lE(i)). The distance from the spinneret to the collector was 15-20 cm, allowing enough time between the spinneret and collection for HFIP evaporation and fiber formation. Fibers were precut to about 1 mm using an array of razor blades and dispersed into anhydrous ethanol at 0.004 g ml-1. The precut fiber solution (40 ml total) was placed in an ice bath and underwent ultrasonication (FB-505, Fisher Scientific) to further fragment the gelatin fibers (Supplementary Fig. la(ii)). The length of the gelatin fibers can be controlled by the power amplitude and duration of the sonication. The 13 mg ml-1 of gelatin fibers were then crosslinked with a 5:2 molar ratio of N,N-(3-dimethylaminopropyl)-N'-ethyl- carbodiimide hydrochloride (EDC; Sigma-Aldrich) and N-hydroxysuccinimide (NHS; Sigma-Aldrich) in anhydrous ethanol for 3 hours while stirring at 260 r.p.m. (FIG. IE (iii)). After the gelatin fibers were washed with water and separated by centrifuge, the fibers were immersed in a solution containing fibronectin protein (50 pg ml-1 fibronectin, Corning), 2 pg ml-1 EDC (Thermo Fisher Scientific) and 5.5 pg ml-1 N-hydroxysulfosuccinimide (sulfo- NHS; Thermo Fisher Scientific) diluted in PBS and incubated overnight at 4 °C (FIG. lE(iv)). Fluorescent fibronectin (green fluorescent, HiLyte Fluor 488, Cytoskeleton) was used to confirm the fibronectin coating on the fibers. The fibers were then washed and centrifuged with deionized water and *2 concentrated PBS (x2 PBS). The x2 PBS was made of 16 mM NaH2PO4, 272 mM NaCl, 4 mM KH2PO4 and 5.2 mM KC1 in deionized water. A schematic illustration of the procedure to prepare the fiber fragmentation materials is depicted in FIG. IE. Fiber length distribution was measured from bright-field images taken by a x 10 EVOS FL microscope, and diameter distribution was measured from confocal microscope (Olympus ix83) images that were captured by a LUCPLFLN-PH x20 objective (Olympus). Fabrication of FIG inks and crosslinking process FIG inks were prepared by mechanically mixing 5-10 wt% prefabricated fibers into a 65 °C prewarmed solution consisting of 2.4 wt% gelatin (type A, gel strength -175 g Bloom, Sigma-Aldrich) and 2.4 wt% sodium alginate (Sigma-Aldrich) in x2 PBS (FIG. lE(vii)). After mixing thoroughly, the contents were transferred to a 3 ml syringe and centrifuged at 450 g for 2 min to remove air bubbles before printing FIG. lE(viii)). FIG inks were printed using a 3D bio-printer (Bio X, Cellink). The 2D FIG scaffolds were prepared with -100 pm thickness. After printing, FIG scaffolds were treated with a 1 wt% calcium chloride (Sigma) solution for 5 minutes to crosslink alginate and a 2-8 wt% microbial transglutaminase (Activa TI Transglutaminase, Ajinomoto) solution for 1 hour at room temperature to crosslink the printed FIG scaffolds. The printed scaffold was then treated with 0.1 M ethylenediaminetetraacetic acid disodium salt dihydrate (Sigma- Aldrich) solution for 15 min at room temperature. The Gel-Alg hydrogel inks with 4 wt% gelatin and 4 wt% alginate in the x2 PBS were used for printing the 3D-printed control models in FIGS. 1 A through 21, and Gel-Alg hydrogel inks with both 4 wt% gelatin and 4 wt% alginate, and 2.4 wt% gelatin and 2.4 wt% alginate, in the *2 PBS were used to compare line printability and cell alignment in FIGS. 3A-3F, respectively. All post procedures after printing were the same as with the FIG printed scaffolds.

[0144] Rheological tests of FIG inks

[0145] The rheological properties of the FIG ink were highly influenced by fiber packing density in the polymer matrix. To investigate the effective fiber concentrations that enabled the printing of 3D scaffolds with accurate shape retention and preservation of the fiber alignment, the rheological properties of FIG inks consisting of a blended gelatin / alginate hydrogel (2.4 wt% gelatin and 2.4 wt% alginate) infused with either 0, 5, 6, 8, or 10 wt% of fibers were evaluated. At higher fiber concentrations, random movement of high aspect ratio fibers becomes reduced, as fiber-fiber contact interaction is increased. As the fibers are densely packed, the fiber-fiber contact interaction becomes dominant over fiber-fluid interaction (see FIG.2A), conferring high viscosity and solid-like behavior of FIG inks. Consequently, the FIG inks with a higher fiber concentration showed elastic properties at rest.

[0146] To assess these viscoelastic properties of FIG inks, we measured their dynamic modulus (Elastic storage modulus, G' and viscous loss modulus, G", representing elastic and viscous response, respectively) as a function of oscillation stress (FIG 2B). At 0 wt% fiber, the loss modulus was higher than the storage modulus within the tested range of applied shear stress (0.1 Pa-1000 Pa), indicative of a liquid-like behavior (FIG. 2B) as a result of low yield stress (FIG. 2D). In contrast, FIG inks show higher storage modulus than loss modulus, indicating solid-like behavior at rest (FIG. 2B). During printing, the FIG inks flow as the they experience shear stress above the yield stress corresponding to the crossover point of G’ and G” (FIG. 2B). While the FIG inks flow, fibers were aligned by shear stress at the nozzle, resulting in a decrease in viscosity (i.e. shear thinning behavior) (FIG. 2H). To quantitatively assess shear-thinning behavior, we used the power-law model, which is defined as i](y) = K(y), where / is viscosity, K is consistency index (defined as the viscosity at the shear rate of 1 s’1), y is the shear rate, and n is the shear-thinning index. When n is lower than 1, inks show shear-thinning behavior, and the ink with lower n has stronger shear-thinning behavior. The n indexes were derived from the viscosity-shear rate graph using the power-law equation FIG. 2H). The higher fiber concentration of the FIG inks showed a stronger shear thinning behavior (FIGS. 2H and 21). After FIGs inks were extruded out, they restored solid-like behavior quickly as demonstrated by the rapid response of crossover between storage and loss modulus under alternating small (1 %) or large (100 %) magnitudes of oscillation strains applied to the inks (FIG 2E). Quick and reversible gelation that occurs for the start / stop events of printing, allows for the preservation of shear-induced fiber alignment.

[0147] The rheological properties were measured using a Discovery Hybrid 3 Rheometer (TA Instruments). A 0% fiber condition used as a control ink (without fiber) for rheology in FIG. ID and FIGS 2A-2D was prepared with 2.4 wt% gelatin and 2.4 wt% alginate in the *2 PBS. A 60 mm cone geometry for 0% Gel-Alg inks and a flat plane geometry for FIG inks were used. The plane geometry experimental set-ups used a 500 pm gap height. Viscosity was measured by a shear rate sweep from 0.1 to 1,000 s-1 (FIG. 2D). An oscillation amplitude sweep was performed by a shear stress sweep from 0.1 to 1,000 Pa at a frequency of 1 Hz (FIG. 2C), and the yield stress was evaluated and determined at the crossover point of the storage and loss moduli (FIG. 2E). The yield stress for the 0% fiber hydrogel inks could not be quantified using the oscillation amplitude sweep tests as the value ranges were too low. Therefore, the yield stress for the 0% fiber hydrogel inks in FIG. 2D was calculated using the stress-shear rate graphs (FIG. 2C), which showed a plateau region as the shear rate decreased. The cyclic strain test was measured with an oscillatory strain change between 1% and 100% every 1 minute at a frequency of 1 Hz and stress of 0.1 Pa (FIG. 2E). All measurements were performed at 25 °C. The temperature sweep test was performed from 10 °C to 40 °C with 2 °C steps at a frequency of 1 Hz and stress of 0.1 Pa (FIG. 2F).

[0148] 3D printing

[0149] G-code file, the computational language used to guide 3D printer nozzle pathways, was prepared by slicer software (slic3r), 3D bio-printer (Bio X, Cellink) or hand-script. Gel- Alg hydrogel and FIG scaffolds / pattems were printed onto Petri dishes or gelatin-layer- coated coverslips by a direct-ink-writing method. The gelatin-layer-coated coverslip was used to enhance adhesion between the printed scaffolds and coverslip. It was fabricated by printing a 14 mm by 14 mm mesh square onto coverslips and treating with ultraviolet-ozone for 5 min, using 4 wt% gelatin (type A, gel strength -175 g Bloom, Sigma- Aldrich) solution. The printing patterns were designed by hand-script G-code, MATLAB (MathWorks) scriptgenerated G-code or 3D bio-printer (Bio X, Cellink). The printing line distance for 2D scaffolds was set to 0.3 mm. STL files of 3D free-standing objects were processed and printed by the 3D bio-printer with a layer height of 0.2 mm for 23 gauge straight nozzles (inner diameter, 0.337 mm) and 0.15 mm for 27 gauge tapered nozzles (inner diameter, 0.21 mm). The G-code for the 3D cone-shaped ventricle chamber was prepared by 3D coordination of a cone shape derived from parabola equations (y = x2 / (4q); q, parameter for shape of parabola: mainly q = 0.5 was used) with a layer height of 0.2 mm using MATLAB. The G-codes were visualized with software (Repetier-Host). The printing condition for the scaffolds was set at a range of 50-100 kPa of printing pressure and 5-20 mm s-1 of printing speed with 23 gauge straight or 27 gauge tapered nozzles. The 3D free-standing objects were printed with 70-120 kPa of pressure and 5-10 mm s-1 of printing speed using 23 gauge straight nozzles and 27 gauge tapered nozzles at a 6 °C printing bed temperature.

[0150] Scaffold structural and biochemical imaging analysis

[0151] The gelatin fiber structure in the printed 3D FIG ventricle scaffold was captured by a spinning disc confocal microscope (Olympus ix83) with a UPLSAPO * 10 objective after staining the gelatin fibers using 1 pl ml-1 of NHS-Fluorescein (Thermo Fisher Scientific). Fiber alignments were analyzed by an ImageJ (National Institutes of Health) plug-in (Orientation!) with 2° bin size, and the images were colorized based on fiber angle. The colorized images were then processed using a custom MATLAB script to output an OOP value, falling on a normalized scale of 0 (perfectly random) to 1 (perfectly aligned), based on the distribution of reported angles from the OrientationJ-processed image. Scanning electron microscopy images (FESEM Ultra Plus, SmartSEM, ZEISS) and microcomputed tomography (Xradia Versa 620, ZEISS) images of 3D-printed scaffolds were taken after dehydration using a critical-point dryer (931 GL 2.5, Tousimis). Microcomputed tomography images of 3D structures were imaged by a Versa 620 X-ray microscope (ZEISS) using a microfocus X- ray source (tube voltage of 50 kV and current of 90 pA) and a *4 objective with wide-field mode at Harvard University’s Center for Nanoscale Systems. Some 4,501 projection images were captured per sample on a 16-bit, 2,048 x 2,048, 4 objective detector with achievable voxel resolutions of 3.19 pm. The 3D data visualization was performed using Dragonfly Pro software.

[0152] NRVM and hiPSC-CM cell cultures

[0153] NRVMs. The animal protocol numbered 24-01-2 was approved by the Harvard Animal Care and Use Committee and is based on previously published methods. Ventricular tissue was removed from two-day-old Sprague Dawley rats. The tissue was minced by scissors, rinsed in Hank’s Balanced Salt Solution (HBSS) and digested in 1 mg ml-1 trypsin in HBSS solution at 4 °C for 14 hours overnight. The next day, the tissue was washed in customized media (details in the following) at 37 °C for 4 minutes. The tissue was then further dissociated using 1 mg ml-1 collagenas in HBSS solution at 37 °C for 2 minutes. Cells dissociated from the tissue were collected in a prechilled tube containing fresh HBSS without collagenase to quench the digestion. The collagenase step was repeated a total of four times until most of the tissue was digested. The dissociated cells were centrifuged at 250g for 8 minutes, resuspended in chilled HBSS and filtered through a 40 pm strainer to remove any remaining bulk tissue. The cells were then centrifuged again at 250g for 8 minutes and plated in a T 175 flask for 2 hours and 15 minutes to remove the fast-adhering fibroblasts. The nonadhering cells were collected as the primary NRVMs and plated on tissue-engineered scaffolds. Culture media for NRVM were Medium 199 supplemented with 0.01 M HEPES buffer solution, 1% MEM non-essential amino acids, 20 mM glucose, 2 mM 1-glutamine, 1.5 pM vitamin B 12 and 50 U ml-1 penicillin. Some 10% heat-inactivated fetal bovine serum was supplemented to the media for the first two days, and 2% fetal bovine serum supplement was used starting on the third day and changed every other day onward.

[0154] The hiPSC-CM culture. The hiPSCs (WTC-11, Coriell Institute, no. GM25256) were seeded onto Geltrex-coated (LDEV-free matrix, Thermo Fisher Scientific) six-well plates and maintained in Essential 8 (E8) medium. At -60-70% confluency, hiPSCs were passaged using Versene (Life Technologies) into six-well plates for maintenance and twelvewell plates for hiPSC cardiomyocyte differentiation. E8 medium with 5 pM Y-27632 (R&D) was used on the first day of passage. The differentiation process for hiPSC-CMs was conducted for 15 days following published protocols. Differentiation started (day 0) at -60- 70% confluence using 6 pM CHIR909921 (STEMCELL Technologies) with a differentiation medium (RPMI / B27(-)) consisting of RPMI-1640 (Life Technologies) and a B-27 minus insulin supplement after a short rinse with PBS. On day 2, the medium was replaced with the RPMI / B27(-) for 24 hours and changed to the RPMI / B27(-) with 5 pM IWR-1 (STEMCELL Technologies; day 3). After 48 hours of incubation and a quick PBS rinse, the medium was replaced with RPMI / B27(-). On day 7, the medium was replaced with RPMI / B27(-) again. On day 9, cardiomyocyte selection was conducted using 0.4 mM lactate (no. L7022, Sigma- Aldrich) added to the differentiation medium for 48 hours, and 0.4 mM lactate added to RPMI-1680 for the next 48 hours. After cardiomyocyte selection, cells were cultured in the differentiation medium until hiPSC-CM isolation on days 15-17. After visual confirmation of cardiomyocyte contractility, hiPSC-CMs were isolated after incubating in Accutase (STEMCELL Technologies) for 30 min. Isolated hiPSC-CMs were seeded using STEMdiff Cardiomyocyte Support Medium (STEMCELL Technologies). The culture medium was changed to RPMI / B27(-) every other day.

[0155] Cell culture on the printed FIG scaffolds

[0156] Before cell seeding, printed scaffolds were sterilized with a 70% ethanol wash for 5 min and ultraviolet-ozone exposure for 4 min. Subsequently, scaffolds were incubated with 50 pg ml-1 solution of fibronectin (Corning) in PBS (Gibco, Thermo Fisher Scientific) at 37 °C for 1 h. After the fibronectin solution was washed with PBS, the 2D scaffolds were seeded with 2 M NRVMs and hiPSC-CMs in twelve-well plates. For 3D-printed ventricle chamber seeding, we inserted a printed ventricle scaffold into a polydimethylsiloxane (PDMS) mould made by casting the same 3D-printed ventricle plastic model (VEROCLEAR, Object30, Stratasys). Considering the cells are less likely to be evenly distributed in the 3D structure, NRVMs and hiPSC-CMs were seeded for 3D-printed ventricle models with 5 M and 8 M, and we turned the ventricles upside down after 1 h and 0.5 h, respectively.

[0157] Tissue immunostaining and structural analysis

[0158] For live / dead cell staining, we cultured NRVMs on fibronectin-coated glass coverslips and FIG scaffolds with 0.5 M cm-2 for 96 h. Live / dead-cell-stained images were captured by a confocal microscope (Olympus ix83) with a LUCPLFLNPh *20 objective after incubating the cells for 5 minutes in 1 pg ml-1 Hoechst 33342 in media for live cell staining, and using the ReadyProbes Cell Viability Imaging Kit, Green Tissue structure analysis was conducted using confocal microscope immunostaining images of NRVM tissue cultured on the scaffold for 12 days and ventricle for 7-12 days. Washed samples were fixed with 4% (v / v) paraformaldehyde in PBS for 15 min and permeabilized with 0.1% (v / v) Triton-X 100 solution in PBS for 15 min at room temperature. The samples were then incubated in 5% (w / v) bovine serum albumin (BSA) solution in PBS for 30 min at room temperature. Samples were then incubated overnight with 1 : 100 diluted monoclonal sarcomeric a-actinin (clone EA-53; abeam) or 1 : 100 Anti-Connexin-43 antibody produced in rabbit (Sigma-Aldrich, no. C-6219) primary antibody in 1 wt% BSA solution at 4 °C. After being washed three times in 0.5 wt% BSA solution for 5 min, the samples were counterstained with 1 :200 diluted Alexa Fluor 546-conjugated anti-mouse secondary antibody (Invitrogen, no. A-11003), 1 : 1,000 diluted Alexa Fluor 633 -conjugated phalloidin (Life Technologies, no. A22284) and 1 :500 diluted DAPI (Invitrogen) in 1 wt% BSA solution for 2 h at room temperature. After the samples were washed three times with 0.5 wt% BSA solution, they were mounted on the glass to observe immunostaining images using a spinning disc confocal microscope (Olympus ix83). Images were captured by LUCPLFLNPh *20 andx40 objectives on a Hamamatsu Orca Flash 4.0 Cl 1440 at 16-bit depth. Background subtraction and Z-stack maximum projection were applied for displayed confocal microscope images in figures using Imaged software. We used *40 confocal microscope images with 1,024 x 1,024 pixels to quantify nucleus alignment, eccentricity ratio, and sarcomere and F-actin alignments after rotating those images to position the print orientation horizontally. We analyzed the angle and length of nuclei in the major and minor axes using an Imaged plug-in (Analyze particle). We subtracted 90 degrees from the angle results to ensure that the print direction was oriented towards 0 degrees. The eccentricity ratio was calculated by dividing the length of the major axis by the length of the minor axis. Sarcomere and F-actin images were processed with an Imaged plug-in (Orientationd) to get color-coded maps based on cytoskeletal fiber orientation. Based on the distribution of angular orientations, we calculated OOP values that range from zero (random organization) to one (perfect alignment) using custom-made MATLAB (MATHWORKS) code.

[0159] Optical mapping experiments of cardiac tissues

[0160] After observing the spontaneous beating of NRVM tissue at day 3 and iPSC-CM tissue at day 5, we observed Ca2+ activities of cardiac tissues on the 3D-printed scaffolds using an optical mapping system at day 5 and day 7, respectively. For hiPSC-CM tissues on the 3D-printed ventricle scaffolds, we cultured for 14 days before conducting the optical mapping experiment to ensure the structural and functional coupling of the cells in tissues due to the higher number of cells in 3D structures. The optical mapping system includes a modified tandem-lens macroscope (Scimedia) equipped with a high-speed camera (MiCAM Ultima, Scimedia), a Plan APO x0.63 or x l objective (Zeiss), a collimator (Lumencor), a 200 mW mercury lamp (X-Cite exacte, Lumen Dynamics), a high-spatial-resolution scientific complementary metal-oxide semiconductor camera (pco.edge, PCO AG) and 880 nm dark- field light-emitting diode (LED) light. For Ca2+ imaging, we used an excitation filter with 580 / 14 nm, a dichroic mirror with a 593 nm cut-off and an emission filter with 641 / 75 nm (Semrock). For dark-field imaging, we added a dichroic mirror with a 685 nm cut-off and long-pass emission filter with a 664 nm cut-off filter (Semrock). Cultured tissues were incubated with 2 pM X-Rhod-1 (Invitrogen) for 60 min at 37 °C, rinsed and incubated in dye- free media for an additional 15 min at 37 °C before recording. The recording was conducted in Tyrode’s solution (1.8 mM CaC12, 5 mM glucose, 5 mM HEPES, 1 mM MgC12, 5.4 mM KC1, 135 mM NaCl and 0.33 mM NaH2PO4 in deionized water, pH 7.4, at 37 °C) for NRVM tissue and fresh RPMI / B27(-) for hiPSC-CM tissue. Point stimulation was applied to in vitro cardiac tissues using two platinum electrodes (Sigma-Aldrich) with 1 mm spacing and with 10 V amplitude and 10 ms duration. The platinum electrodes were located at 1.0 mm from the corner of the tissue samples (lonOptix MyoPacer). For each recording, Ca2+ and dark-field images were acquired with frame rates of 400 frames per second (f. p.s.) for 10 s. The recording of the top view was performed after the 3D-printed in vitro ventricles moved to the PDMS mould without external stimulation. Post-processing of data was conducted with custom software written in MATLAB (MathWorks) and MiCAM imaging software (MiCAM Ultima, Scimedia). A spatial filter with 3 x 3 pixels was applied to improve the signal-to- noise ratio. The activation time of each pixel was calculated at the average maximum upstroke slope of multiple pulses of X-Rhod-1 signals over a 10 second recording window. Velocity was derived from the difference in activation time that occurred between 1 mm and 5 mm.

[0161] PIV analysis

[0162] The hiPSC-CMs were cultured for 15 days in printed 3D FIG ventricle chamber scaffolds. The in vitro model of the heart chamber was moved to the 35 mm Petri dish filled with RPMI / B27(-) with 0.1 pg ml-1 of fluorescent beads (green fluorescent microspheres, Cospheric). A 1 Hz field electrical stimulation (lonOptix MyoPacer) was applied with 10-20 V using two parallel platinum electrodes positioned ~30 cm apart. Bead movements were recorded using a stereoscopic microscope (ZEISS Discovery. V12 stereomicroscope) with a HBO 100 fluorescent light source at a x 14 magnification with frame rates of 30 f.p.s. Bright- field images were recorded with a Basler electric ACA2500-14UC USB 3.0 camera at an *8.5 magnification and 30 f.p.s. with and without 1 Hz field electrical stimulation

[0163] (spontaneous contraction). Fluorescent image sequences were prepared for analysis using a rolling ball background subtraction (ImageJ) and were analyzed by custom software based on the open-source package OpenPIV to reconstruct velocity maps. This resulted in velocityfield profiles as a function of time. These time-dependent velocity profiles were then phase- averaged over the entire cycle to smooth out local fluctuations in performance. For quantitative metrics of cardiac performance, we examined the total mass flux (cardiac output) and ejection fraction resulting from the 3D-printed ventricles as previously reported. Briefly, here we summed the velocity profiles over the basal opening and normalized this value by the fluid density to obtain a 2D instantaneous mass flux with units of grams per meter. Further integrating this 2D value over the basal opening resulted in a 3D estimate of the total mass flux occurring during systole. Finally, knowing this mass flux value, the density of the fluid and the initial starting volume of the ventricle, we could estimate the fraction of fluid ejected during contraction. Based on printing dimensions, a width of 10 mm, height of 10 mm and depth of 11 mm were used to estimate the starting volume, assuming the shape of a half- oblate ellipsoid.

[0164] Statistics and reproducibility

[0165] Statistical analysis was conducted to compare cell viability, tissue alignment, nucleus aspect ratio, nucleus angle distribution, tissue contractility and the Ca2+ conduction anisotropy ratio in the longitudinal direction versus the transverse direction between tissues on the printed Gel-Alg hydrogel and the FIG scaffolds. All error bars are given as the standard error of the mean (s.e.m.) unless otherwise noted. Statistical analyses were performed with a two-tailed unpaired Student’s t-test, assuming unequal variance, a one-way analysis of variance with Tukey post-hoc test within the groups and a two-sample Kolmogorov- Smirnov test, and were conducted unless otherwise noted. P values of <0.05 were considered statistically significant. Sample sizes are given for each experimental condition in the figure legends. For representative data and images given in the figures, at least three or more independent experiments were conducted, showing similar results.

[0166] The contents of the following article are incorporated by reference herein in their entirety, Choi et al, “Fiber-infused gel scaffolds guide cardiomyocyte alignment in 3D- printed ventricles,” Nature Materials volume 22, 1039-1046 (2023).

[0167] The contents of U.S. Patent Application No. 16 / 756,214 entitled “Methods of Forming Three-Dimensional Tissues Scaffolding Using Biological Fiber Inks and Methods of Use Thereof,” which published as U.S. Patent Application Publication US 2020 / 0330644A1, are incorporated by reference herein in its entirety.

[0168] VIII. Equivalents

[0169] In describing embodiments of the invention, specific terminology is used for the sake of clarity. For purposes of description, each specific term is intended to at least include all technical and functional equivalents that operate in a similar manner to accomplish a similar purpose. Additionally, in some instances where a particular embodiment of the invention includes a plurality of system elements or method steps, those elements or steps may be replaced with a single element or step; likewise, a single element or step may be replaced with a plurality of elements or steps that serve the same purpose. Further, where parameters for various properties are specified herein for embodiments of the invention, those parameters can be adjusted up or down by l / 20th, 1 / 1 Oth, Uth, ’Ard, %, etc., or by rounded-off approximations thereof, unless otherwise specified. Moreover, while this invention has been shown and described with references to particular embodiments thereof, those skilled in the art will understand that various substitutions and alterations in form and details may be made therein without departing from the scope of the invention; further still, other aspects, functions and advantages are also within the scope of the invention. The contents of all references, including patents and patent applications, cited throughout this application are hereby incorporated by reference in their entirety. The appropriate components and methods of those references may be selected for the invention and embodiments thereof. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments and methods described herein. Such equivalents are intended to be encompassed by the scope of the following claims.

Claims

Claims1. A method of forming a three-dimensional, self-supporting structure of a fiberhydrogel composite material by extrusion, the method comprising: providing or obtaining a fiber-hydrogel composite material that is solid prior to extrusion, the fiber-hydrogel composite material comprising: a plurality of polymeric fibers, an average diameter of the plurality of polymer fibers falling in a range of 3-200 microns; and a hydrogel; extruding the fiber-hydrogel composite material through at least one aperture onto a support to form the three-dimensional self-supporting structure, wherein shear stress on the fiber-hydrogel composite material during extrusion liquefies the fiber-hydrogel composite material before the fiber-hydrogel composite material exits the at least one aperture and the fiber-hydrogel composite material returns to a solid state after extrusion, and wherein no support bath, no coagulating bath and no cross-linking bath are used during extrusion of the fiber-hydrogel composite material onto the support to form the three- dimensional self-supporting structure.

2. The method of claim 1, wherein the three-dimensional, self-supporting structure is a three-dimensional, multilayer structure.

3. The method of claim 1, wherein polymeric fibers in the plurality of polymeric fibers are at least partially aligned with an extrusion direction during extrusion resulting in shear thinning that liquefies the fiber-hydrogel composite material.

4. The method of claim 3, wherein more than 50% of the polymeric fibers in the plurality of polymeric fibers show anisotropic alignment along an extrusion direction in the formed three-dimensional self-supporting structure.

5. The method of claim 1, wherein the fibers of plurality of polymeric fibers of the fiberhydrogel composite material have an aspect ratio of average length to average diameter that falls in a range of 5: 1 to 100: 1.

6. The method of claim 1, wherein the fibers of plurality of polymeric fibers of the fiberhydrogel composite material have an aspect ratio of average length to average diameter that falls in a range of 5: 1 to 50: 1.

7. The method of claim 1, wherein the fibers of the plurality of polymeric fibers of the fiber-hydrogel composite material have an aspect ratio of length to diameter that falls in a range of 10: 1 to 30:

18. The method of claim 1, wherein a weight percent of the plurality of polymeric fibers in the fiber-hydrogel composite material is in a range of 8% to 40%.

10. The method of claim 1, wherein a weight percent of the plurality of polymeric fibers in the fiber-hydrogel composite material is in a range of 8% to 15%.

11. The method of claim 1, wherein the plurality of polymeric fibers comprises one or more proteins, one or more polysaccharides, or both.

12. The method of claim 11, wherein the plurality of polymeric fibers comprises gelatin and the one or more proteins are one or more gelatin proteins.

13. The method of claim 11, wherein the plurality of polymeric fibers comprises: gelatin and the one or more proteins are gelatin proteins, alginate and the one or more polysaccharides are alginate polysaccharides, or both.

14. The method of claim 11, wherein the plurality of polymeric fibers comprises hyaluronic acid (HA) and the one or more polysaccharides include HA.

15. The method of claim 1, wherein the plurality of polymeric fibers comprises a synthetic biodegradable polymer.

16. The method of claim 1, wherein the synthetic biodegradable polymer is selecting from the group consisting of polylactic acid (PCL), poly(L-lactide-co-s-caprolactone) PLCL, and polyacrylamide (PAA).

17. The method of claim 1, wherein a standard distribution of lengths for the plurality of polymeric fibers is within a range of 10% of the average length.

18. The method of claim 1, wherein a standard distribution of lengths for the plurality of polymeric fibers is within a range of 6% of the average length.

19. The method of claim 1, wherein the hydrogel comprises one or more proteins, one or more polysaccharides, or both.

20. The method of claim 19, wherein the hydrogel comprises: gelatin and the one or more proteins are proteins of gelatin, alginate and the one or more polysaccharides are alginate polysaccharides, or both.

21. The method of any one of claims 1-20, wherein the at least one aperture is defined by an orifice of a three-dimensional printing system or an additive manufacturing system.

22. The method of any one of claims 1-20, further comprising moving the at least one aperture relative to the support, moving the support relative to the at least one aperture, or both, during extrusion using a three-dimensional printing system or the additive manufacturing system to control relative movement of the at least one aperture and the support.

23. The method of any one of claims 1-20, wherein the at least one aperture is defined by an extruder.

24. The method of any one of claims 1-20, wherein the at least one aperture is defined by a die of a die former, a depositer, or a screw extruder.

25. The method of any one of claims 1-13 and 17-20, wherein the fiber-hydrogel composite material is edible.

26. The method of any one of claims 1-20, wherein the fiber-hydrogel composite material further comprises one or more additional agents for cell programming.

27. The method of claim 26, wherein the one or more additional agents for cell programming are disposed on or in the plurality of polymeric fibers.

28. The method of any one of claims 1-20, wherein the fiber-hydrogel composite material further comprises one or more biologically active agents.

29. The method of claim 28, wherein the one or more biologically active agents are disposed on or in the plurality of polymeric fibers.

30. The method of any one of claims 1-20, wherein the fiber-hydrogel composite material further comprises one or more pharmaceutically active agents.

31. The method of claim 20, wherein the one or more pharmaceutically active agents are disposed on or in the plurality of polymeric fibers.

32. The method of any one of claims 1-20, wherein the fiber-hydrogel composite material further comprises cells.

33. The method of any one of claims 1-20, wherein the fiber-hydrogel composite material further comprises fluorescent molecules.

34. The method of claim 33, wherein the fluorescent molecules are disposed on or in the plurality of polymeric fibers.

43. The method of any one of claims 1-20, wherein the fiber-hydrogel composite material further comprises nanoparticles.

44. A method of forming an engineered food product comprising: providing a three-dimensional self-supporting structure that is a tissue scaffold using the method of any one of claims 1-13 and 17-20; seeding the scaffold with muscle cells; and culturing the muscle cells under suitable conditions to form a muscle tissue, thereby forming a three-dimensional engineered food product.

45. A method of forming a three-dimensional engineered tissue comprising: providing a three-dimensional self-supporting structure that is a tissue scaffold using the method of any one of claims 1-20; seeding the tissue scaffold with cells; and culturing the cells under suitable conditions to form a tissue, thereby forming a three- dimensional engineered tissue.

46. A fiber-hydrogel composite material for forming a three-dimensional self-supporting structure by extrusion, the fiber-hydrogel composite material comprising: a plurality of polymeric fibers, an average diameter of the plurality of polymer fibers falling in a range of 5-200 microns, the fibers of the plurality of polymeric fibers having an aspect ratio of average length to average diameter that falls in a range of 5: 1 to 50: 1, and a hydrogel comprising one or more proteins, one or more polysaccharides, or both, the fiber-hydrogel composite material being solid at room temperature prior to extrusion, and the fiber-hydrogel composite material configured to liquefy due to shear stress during extrusion and configured to return to a solid state after extrusion.

47. The fiber-hydrogel composite material of claim 46, wherein the hydrogel comprises: gelatin and the one or more proteins are gelatin proteins, alginate and the one or more polysaccharides are alginate polysaccharides, or both.

48. The fiber-hydrogel composite material of claim 46, wherein the plurality of polymeric fibers includes gelatin and the one or more proteins are gelatin proteins.

49. The fiber-hydrogel composite material of claim 46, wherein a weight percentage of the polymeric fibers in the fiber-hydrogel composite material is in a range of 8% to 15%.

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