WO2025259797A2 - Composite silk foam and fiber materials, and methods of making and using the same - Google Patents

Abstract

A printhead may include a co-extrusion nozzle configured to co-extrude a biopolymer-based cellular biomaterial with a continuous thread, the co-extrusion nozzle comprising: a first duct having a first diameter, the first duct comprising a fitting on a first end, a continuous thread duct having a continuous thread duct diameter, the continuous thread duct arranged at an incline with respect to the first duct, the continuous thread duct configured to receive and guide the continuous thread; and wherein a second end of the first duct is configured to receive the continuous thread exiting the continuous thread duct; and a co-flow tip arranged at the second end of the first duct and configured to receive a co-extruded material, the co-extruded material comprising the biopolymer-based cellular biomaterial and the continuous thread.

Description

COMPOSITE SILK FOAM AND FIBER MATERIALS, AND METHODS OF MAKING AND USING THE SAME

CLAIM TO PRIORITY

[0001] This application claims priority to and the benefit of U.S. Provisional Application No. 63/658,748 (T002832 PROV) filed in the U.S. Patent and Trademark Office on June 11, 2024, and U.S. Provisional Application No. 63/658,745 (T002833 PROV) filed on June 11, 2024. The foregoing patent applications are hereby incorporated by reference in their entireties for all purposes.

BACKGROUND

[0002] Presently, the fashion and textile industry generated a staggering 1.23 billion tons of waste in 2019 with projections estimating up to 2.23 billion in 2025. This includes silk-based waste, which accounts for 11 million tons, much of which remains unrecycled. Historically, the delicacy of printed silk foam structures has impeded their integration into fabric production despite their numerous advantages. A need exists for new materials overcoming these challenges.

SUMMARY

[0003] In some aspects, the techniques described herein relate to a printhead of a three-dimensional printer including: a co-extrusion nozzle configured to co-extrude a biopolymer-based cellular biomaterial with a continuous thread, the co-extrusion nozzle including: a first duct having a first diameter, the first duct including a fitting on a first end; a continuous thread duct having a continuous thread duct diameter, the continuous thread duct arranged at an incline with respect to the first duct, the continuous thread duct configured to receive and guide the continuous thread; and wherein a second end of the first duct is configured to receive the continuous thread exiting the continuous thread duct; and a co-flow tip arranged at the second end of the first duct and configured to receive a co-extruded material, the co-extruded material including the biopolymer-based cellular biomaterial and the continuous thread.

[0004] In some aspects, the techniques described herein relate to a method for generating a biopolymer-based cellular biomaterial and a suspended continuous thread, the method including: depositing at least one continuous thread from a printhead into a biopolymer-based cellular biomaterial foam bath to form an embedded filament article, the biopolymer-based cellular biomaterial including silk fibroin in an amount between 1% and 40%, glycerol monostearate in an amount between 1 % and 10%, and plasticizer in an amount between 20% and 75%.

[0005] In some aspects, the techniques described herein relate to a method for generating a coextruded biopolymer-based cellular biomaterial and continuous thread, including: flowing a biopolymer-based cellular biomaterial under pressure through a first duct of a co-extrusion nozzle of a printhead of a three-dimensional printer, the biopolymer-based cellular biomaterial including silk fibroin in an amount by weight of between 1% and 40%, a surfactant (e.g., glycerol monostearate) in an amount by weight of between 1% and 10%, and plasticizer in an amount by weight of between 20% and 75%; guiding a continuous thread within a continuous thread duct of the co-extrusion nozzle; and co-extruding the biopolymer-based cellular biomaterial and the continuous thread through a co-flow tip of the co-extrusion nozzle of the printhead to form a co-extruded material.

[0006] In some aspects, the techniques described herein relate to a method of making a biopolymerbased cellular biomaterial with a continuous thread, the method including: whipping a liquid composition for a predetermined whipping time at a predetermined speed to form a biopolymerbased cellular biomaterial, the liquid composition including silk fibroin, at least one polysaccharide, at least one surfactant (e.g., glycerol monostearate), and at least one plasticizer; pressurizing a volume of the biopolymer-based cellular biomaterial; and concurrently co-extruding the biopolymerbased cellular biomaterial with a continuous thread thereby forming a co-extruded material, and drying the co-extruded material.

[0007] These and other systems, methods, objects, features, and advantages of the present disclosure will be apparent to those skilled in the art from the following detailed description of the preferred embodiment and the drawings.

[0008] All documents mentioned herein are hereby incorporated in their entirety by reference. References to items in the singular should be understood to include items in the plural, and vice versa, unless explicitly stated otherwise or clear from the text. Grammatical conjunctions are intended to express any and all disjunctive and conjunctive combinations of conjoined clauses, sentences, words, and the like, unless otherwise stated or clear from the context.

BRIEF DESCRIPTION OF THE FIGURES

[0009] The disclosure and the following detailed description of certain embodiments thereof may be understood by reference to the following figures:

[0010] Fig. 1 depicts a biopolymer-based cellular biomaterial (scale bar = 300 pm) containing an embedded continuous silk thread.

[0011] Fig. 2 depicts an embodiment of the co-flow nozzle (scale bar = 5 mm).

[0012] Fig. 3A depicts a biopolymer-based cellular biomaterial with an open pattern.

[0013] Fig. 3B depicts a biopolymer-based cellular biomaterial with a closed or plain pattern.

[0014] Fig. 3C depicts extrusion of a biopolymer-based cellular biomaterial.

[0015] Fig. 3D depicts a biopolymer-based cellular biomaterial with a closed or plain pattern. [0016] Fig. 4A depicts a schematic of a co-flow printhead. [0017] Fig. 4B depicts a microscopic view of a silk continuous thread used in the co-extrusion process.

[0018] Fig. 4C depicts a microscopic view of a silk foam matrix material.

[0019] Fig. 5 is a schematic of an embodiment of the co-flow nozzle and its components.

[0020] Fig. 6 is a schematic of an embodiment of the co-flow nozzle and its components.

[0021] Fig. 7A shows the components of an embodiment of the thread feeding mechanism.

[0022] Fig. 7B shows a sensor of an embodiment of the thread feeding mechanism.

[0023] Fig. 8 A is a schematic of the co-extrusion printing process.

[0024] Fig. 8B shows a co-extruded article with an open pattern.

[0025] Fig. 8C shows the shear-thinning behavior of the foam-like material.

[0026] Fig. 9 shows the embedded filament printing process.

[0027] Fig. 10 shows the embedded filament printing process and further optional steps.

[0028] Fig. 11 shows a top down view (a) and perspective view (b) of the multiple feeder printhead for the embedded filament printing process.

[0029] Fig. 12 depicts a microscopic image of a composite cross-section. Depicted is the silk strand in the center surrounded by a silk foam matrix (post-compression).

DETAILED DESCRIPTION

[0030] Before the present disclosure is described in further detail, it is to be understood that the disclosure is not limited to the particular embodiments described. It is also understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. The scope of the present disclosure will be limited only by the claims. As used herein, the singular forms "a", "an", and "the" include plural embodiments unless the context clearly dictates otherwise.

[0031] In this application, unless otherwise clear from context, (i) the term “a” may be understood to mean “at least one”; (ii) the term “or” may be understood to mean “and/or”; (iii) the terms “comprising” and “including” may be understood to encompass itemized components or steps whether presented by themselves or together with one or more additional components or steps; and (iv) the terms “about” and “approximately” are used as equivalents and may be understood to permit standard variation as would be understood by those of ordinary skill in the art; and (v) where ranges are provided, endpoints are included.

[0032] Approximately: as used herein, the term “approximately” or “about,” as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0033] Composition: as used herein, may be used to refer to a discrete physical entity that comprises one or more specified components. In general, unless otherwise specified, a composition may be of any form - e.g., gas, gel, liquid, solid, etc. In some embodiments, “composition” may refer to a combination of two or more entities for use in a single embodiment or as part of the same article. It is not required in all embodiments that the combination of entities result in physical admixture, that is, combination as separate co-entities of each of the components of the composition is possible; however many practitioners in the field may find it advantageous to prepare a composition that is an admixture of two or more of the ingredients in a pharmaceutically acceptable carrier, diluent, or excipient, making it possible to administer the component ingredients of the combination at the same time.

[0034] Improve, increase, or reduce: as used herein or grammatical equivalents thereof, indicate values that are relative to a baseline measurement, such as a measurement in a similar composition made according to previously known methods.

[0035] Substantially: as used herein, the term “substantially” refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result. The term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0036] It should be apparent to those skilled in the art that many additional modifications beside those already described are possible without departing from the inventive concepts. In interpreting this disclosure, all terms should be interpreted in the broadest possible manner consistent with the context. Variations of the term "comprising" should be interpreted as referring to elements, components, or steps in a non-exclusive manner, so the referenced elements, components, or steps may be combined with other elements, components, or steps that are not expressly referenced. Embodiments referenced as "comprising" certain elements are also contemplated as "consisting essentially of" and "consisting of" those elements. When two or more ranges for a particular value are recited, this disclosure contemplates all combinations of the upper and lower bounds of those ranges that are not explicitly recited. For example, recitation of a value of between 1 and 10 or between 2 and 9 also contemplates a value of between 1 and 9 or between 2 and 10. When a value is given in an amount by weight, the value may be given in a dry solids basis, excluding the weight of water or other solvents within the composition.

[0037] As used herein, "silk fibroin" refers to silk fibroin protein whether produced by silkworm, spider, or other insect, or otherwise generated (Lucas et al., Adv. Protein Chem., 13: 107-242 (1958)). Any type of silk fibroin can be used in different embodiments described herein. Silk fibroin produced by silkworms, such as Bombyx mori, is the most common and represents an earth-friendly, renewable resource. For instance, silk fibroin used in a silk film may be attained by extracting sericin from the cocoons of B. mori. Organic silkworm cocoons are also commercially available. There are many different silks, however, including spider silk (e.g., obtained from Nephila clavipes), transgenic silks, genetically engineered silks, such as silks from bacteria, yeast, mammalian cells, transgenic animals, or transgenic plants, and variants thereof, that can be used. See, e.g., WO 97/08315 and U.S. Pat. No. 5,245,012, each of which is incorporated herein by reference in their entireties.

[0038] It should be appreciated that the term “foam” may be used in a different context elsewhere (e.g., where noted in the Examples), but the usage herein refers primarily to printing of a whipped silk cream as described in the Examples and in PCT International Application Number PCT/US2025/026472, filed April 25, 2025, which is incorporated by reference in its entirety herein for all purposes. A person of skill in the art would understand that where a foam is described herein, the foam may be the same as the whipped silk cream of PCT/US2025/026472 or a variation thereof. In some cases, the foam may have the same composition as the whipped silk cream. In some cases, the foam may have an adjusted composition. A skilled artisan would appreciate how to vary a whipped silk cream’ s composition to make it suitable for use with the claimed invention. For example, a skilled artisan, having the benefit of this disclosure and given a specific context, would understand that certain applications may require a different polysaccharide or combination of polysaccharides in the whipped silk cream composition depending on the ultimate material properties desired.

[0039] At a high level, this disclosure describes repurposing silk waste, among other materials, to develop innovative textiles. As used herein, silk waste may refer to any form of silk fibroin that is a byproduct or product of use, manufacturing, or reuse. Through this newly devised manufacturing method and the innovative component highlighted herein, a diverse range of textiles can now be crafted utilizing additive manufacturing techniques. The introduction of the co-flow nozzle described herein facilitates the simultaneous printing of silk foam and reinforcing thread. Consequently, the utilization of silk foam printing experiences significant enhancement, yielding composite materials with substantially augmented strength compared to foam alone. This breakthrough underscores the transformative potential of this technology in addressing the challenges posed by silk waste while advancing textile manufacturing practices towards sustainability.

[0040] Until now, various efforts have been made to recycle and repurpose silk waste; however, none have presented a comparable approach in tandem with additive manufacturing methodologies. This innovative approach represents a singular avenue for revitalizing silk into textile form, marking a significant advancement in waste utilization within the industry.

[0041] The co-flow nozzle described herein facilitates the fabrication of fiber-reinforced 3D printed bio-composites. An example of this material is shown in Fig. 1 . One embodiment of the co-flow nozzle entails a tailor-made nozzle designed for co-extrusion and simultaneous deposition of a biopolymer foam (e.g., silk foam) alongside a continuous thread (e.g., silk yarn) as shown in Fig. 2. The integration of this technology allows for the fabrication of silk composites with superior physical properties compared to conventional silk foam structures. This disclosure enables 3D printing of reinforced materials co-composed of a soft material and any kind of yarn, such as textiles. Notably, this method significantly enhances resistance to stretching and shear stress over traditional printing processes. The dimensions of the nozzle ensure precise material deposition. Additional functions can be implemented if reinforced with conductive thread/wire (e.g., cupronickel) or optical fibers such as electrical conductivity. Various shapes and patterns of articles and textiles can be produced as shown in Fig. 3A - Fig. 3D.

[0042] This innovative system opens avenues for designing a novel category of printed textiles based on reinforced silk foam. Furthermore, it empowers the creation of intricate 3D structures. The amalgamation of the foam’s insulating, elastic, and biocompatible properties with the yam's tensile strength has led to the conceptualization of a structural paradigm within this composite material, reminiscent of established textiles.

[0043] Historically, the delicacy of printed silk foam structures has impeded their integration into fabric production, despite their numerous advantages. However, the introduction of the co-flow nozzle marks a pivotal advancement, facilitating the simultaneous printing of silk foam and reinforcing thread. Consequently, the utilization of silk foam printing experiences significant enhancement, yielding composite materials with substantially augmented strength compared to foam alone. This breakthrough underscores the transformative potential of this technology in addressing the challenges posed by silk waste while advancing textile manufacturing practices towards sustainability.

[0044] Until now, various efforts have been made to recycle and repurpose silk waste; however, none have presented a comparable approach in tandem with additive manufacturing methodologies. This innovative approach represents a singular avenue for revitalizing silk into textile form, marking a significant advancement in waste utilization within the industry. While several experiments have been conducted previously, there has been a notable absence of a comparable component for fabricating silk-biocomposites. until now.

[0045] While the precise attributes of such silk biocomposites remain to be fully examined for textile industry applications, this material offers a viable alternative to conventional materials. Beyond its implications for textiles, the versatility of this disclosure extends to various other domains, including biomedical engineering and medical applications, promising widespread benefits across diverse sectors.

[0046] The present disclosure provides for a printhead. In some embodiments, the printhead includes a coaxial nozzle and a continuous thread dispenser. The printhead is configured to coextrude a biopolymer-based cellular biomaterial with a continuous thread. Fig. 4A shows a schematic of one embodiment of a co-flow printhead. The coaxial or co-flow nozzle includes two ducts: a first duct for a biomaterial (e.g., silk foam seen in Fig. 4C) extrusion, and a continuous thread duct for thread extrusion (e.g., silk yarn seen in Fig. 4B). The nozzle may be fabricated from biocompatible resins suitable for medical applications (e.g., BioMed Clear VI resin).

[0047] A cutaway view of the co-flow nozzle is shown in Fig. 5. The first duct has a fitting on a first end. The fitting may be any type of fitting with a protruding component and a receiving component. The fitting may be a screw-type fitting (e.g., a Luer lock fitting). Biopolymer-based cellular biomaterial flows through as shown by the large arrow. The continuous thread duct, labeled as thread duct in Fig. 5, is arranged at an incline with respect to the first duct. The continuous thread duct is configured to receive and guide the continuous thread. A second end of the continuous thread duct is configured to receive the continuous thread exiting the continuous thread duct. The flow of the continuous thread is shown by the vertical arrow in Fig. 5. The printhead extrudes a co-extruded material which includes the biopolymer-based cellular biomaterial and the continuous thread. The biopolymer-based cellular biomaterial and the continuous thread may be co-extruded in a single path. A diameter of the biopolymer-based cellular biomaterial may be greater than a diameter of the continuous thread in the co-extruded material by between 5 times to 100 times, including at least 10 times, at least 30 times, or at least 50 times.

[0048] In some embodiments of the printhead, the printhead may comprise more than one nozzle for printing of more than one continuous thread simultaneously. Printheads may also be coupled together to print more than one continuous thread simultaneously. It should be understood that a person of skill in the art, having the benefit of this disclosure, would know when to couple printheads for certain applications, and further, if the coupled printheads should have the same or different features. It should be understood that coupled printheads may have the same or different features when compared to one another.

[0049] In some embodiments of the printhead, the printhead further includes a thread dispenser, which may also be referred to as a feeder, as shown in Fig. 6, Fig. 7 A, and Fig. 7B. The thread dispenser transforms the rotational movement of wheels (roller wheels in Fig. 7A) into a linear movement for the continuous thread. In some cases, the wheels of the thread dispenser may be made of at least one of thermoplastic polyurethane (TPU), silicon rubber, polyurethane, thermoplastic polyethylene, or any suitable material that may be extruded or molded.

[0050] The thread dispenser is a highly precise, custom-made mechanism with several unique features that minimize disruption to the coextrusion, with three features described herein, i) The continuous thread duct of the thread dispenser ensures deposition of the thread by propulsion, which is uncommon in thread deposition, ii) A precise pipe network prevents buckling effects by ensuring proper alignment of the continuous thread, iii) The thread dispenser is augmented with a sensor or sensor system that includes a sensor (seen in Fig. 7B) that detects any unwanted continuous thread behavior (e.g., poor positioning of the continuous thread or the continuous thread deviating from the desired trajectory). If the sensor detects any undesirable behavior of the continuous thread, the sensor may send a signal to the printer to pause printing or initiate a de-bottlenecking maneuver. The vertical alignment of the thread and the length of the continuous thread duct are also carefully selected to minimize friction and constraints in the thread extrusion method. The sensor may be an infrared (IR) sensor or an optical sensor. For example, an optical sensor may be coupled with a computer executing a machine learning algorithm to detect the presence/absence of the continuous thread.

[0051] In some embodiments, the continuous thread may be at least one of a silk yarn, a waste silk product, a filament, an electrode, a counter-electrode, an enzymatically-coated fiber, a non-spun thread, an electrochemical fiber, a conductive fiber, a copper material, a platinum material, a metallic material, a polyester material, a cotton material, a graphite fiber, or a nylon material. A diameter of the continuous thread may be between 35 pm and 80 pm or between 0.1 mm and 1 mm. A diameter of the continuous thread may be at least 35 pm, at least 60 pm, at least 70 pm, at least 0.2 mm, at least 0.3 mm, at least 0.5 mm, at least 0.7 mm, or at least 0.8 mm. A diameter of the continuous thread may be at most 1 mm, at most 0.6 mm, at most 0.4 mm, at most 80 pm, at most 65 pm, or at most 45 pm.

[0052] In some embodiments, the first duct of the printhead may include a pipe comprising copper and/or aluminum, or other suitable material. The first duct may lead the continuous thread through the wheels and have a diameter between 1 mm and 2 mm, including at least 1.2 mm, at least 1.4 mm, at least 1.6 mm, or at least 1.8 mm.

[0053] The continuous thread duct may have a funnel-shaped mouth with a diameter of between 2 mm and 5 mm. The diameter of the funnel-shaped mouth may be at least 2.5 mm, at least 3.5 mm, or at least 4.5 mm. The diameter of the funnel-shaped mouth may be at most 5 mm, at most 4 mm, or at most 3 mm. The continuous thread duct may have a diameter between 0. 1 mm and 1 mm including at least 0.2 mm, at least 0.3 mm, at least 0.5 mm, at least 0.7 mm, or at least 0.8 mm. The continuous thread duct may be between 5 mm and 100 mm in length, including but not limited to, between 10 mm and 80 mm in length. The continuous thread duct may be at least 10 mm in length, at least 30 mm in length, at least 50 mm in length, at least 70 mm in length, or at least 90 mm in length. The continuous thread duct may be at most 95 mm in length, at most 75 mm in length, at most 55 mm in length, at most 35 mm in length, or at most 15 mm in length. The continuous thread duct may have an angle of incline with respect to the first duct of between 5° and 45°. The angle of incline may be at least 5°, at least 15°, at least 25°, or at least 45°. The angle of incline may be at most 40°, at most 30°, at most 20°, or at most 10°.

[0054] In some embodiments, the printhead described herein may be configured to store the biopolymer-based cellular biomaterial under a storage pressure, which is optionally greater than atmospheric pressure. The storage pressure may be between 0.25 MPa and 25 MPa. The storage pressure may be at least 0.5 MPa, at least 1 MPa, at least 5 MPa, at least 10 MPa, or at least 20 MPa. The storage pressure may be at most 25 MPa, at most 15 MPa, at most 5 MPa, or at most 1 MPa. [0055] In some embodiments, the printhead described herein may further include at least one vessel for delivering the biopolymer-based cellular biomaterial to the coextrusion nozzle. The at least one vessel may be configured to connect to the coextrusion nozzle via the fitting. The at least one vessel may be at least one of a cartridge or a syringe that is at least one of removable, refillable, or replaceable. The vessel may be configured to store a volume of the biopolymer-based cellular biomaterial under the storage pressure. The volume may be between 100 mL and 400 mL. The volume may be at least 100 mL, at least 150 mL, at least 250 mL, or at least 350 mL. The volume may be at most 400 mL, at most 300 mL, at most 200 mL, or at most 100 mL.

[0056] In some embodiments, the printhead described herein may include a mechanical extrusion system. The mechanical extrusion system may include a bipolar stepper motor with a gearbox and a rear axle configured to operate at a torque of between 1 Nm and 4 Nm, including but not limited to, at least 1.5 Nm, at least 2 Nm, or at least 3 Nm.

[0057] In some embodiments, the printhead described herein may include a ventilation system positioned adjacent to the co-extrusion nozzle. Airflow from the ventilation system is directed to the co-extruded material that is being extruded from the co-extrusion nozzle. The ventilation system may include at least one fan, which may be directed towards the co-extruded material being extruded to enable layer-by-layer drying of the co-extruded material. The fan may be directed at an airflow angle with respect to a direction of co-extrusion of between 35° and 65°, including but not limited to, at least 40°, at least 42°, at least 45°, or at least 47°. The fan may have a speed between 3,000 bpm and 4,000 bpm, including but not limited to, at least 3100 bpm, at least 3200 bpm, at least 3500 bpm, or at least 3800 bpm.

[0058] The ventilation system may be configured to deliver airflow at a predetermined fluid velocity optionally between 4 m/s and 10 m/s, including but not limited to, at least 5 m/s, at least 7 m/s, or at least 9 m/s. The predetermined fluid velocity may be variable and may vary with a change in distance from the ventilation system to a surface of the co-extruded material, an extrusion rate from the extrusion nozzle, a layer height, a length of path, an extrusion width, and/or a composition of the biopolymer-based cellular biomaterial.

[0059] The ventilation system may be between 60 mm and 80 mm from a tip of the coextrusion needle, including but not limited to, at least 65 mm, at least 70 mm, or at least 75 mm. The ventilation system may be between 60 mm and 100 mm from the co-extruded material, including but not limited to, at least 65 mm, at least 80 mm, or at least 95 mm.

[0060] The printhead described herein may extrude the co-extruded material in accordance with a volumetric extrusion rate Q = OJ x h x v (where co = extrusion width, h = layer height, v = velocity of extrusion). A feed rate of the continuous thread may be equivalent to the extrusion rate. The extrusion rate may be further defined by a rate of volume change of the co-extruded material. The extrusion rate may vary by up to 45% of an original value of the extrusion rate, including but not limited to up to 75%, up to 65%, up to 60%, up to 55%, up to 48%, or up to 44%, The extrusion rate may be between 125 mm/min and 350 mm/min. The extrusion rate may be at least 265 mm/min, at least 290 mm/min, at least 310 mm/min, or at least 330 mm/min. The extrusion rate may be at most 310 mm/min, at most 250 mm/min, at most 200 mm/min, or at most 150 mm/min.

[0061] The co-extruded material extruded from the printhead described herein may have a firmness of between 100 N/m² and 400N/m², including but not limited to, at least 250 N/m². The co-extruded material may have a density of between 0.08 g/cm³ and 0.25 g/cm³, including but not limited to, at least 0.12 g/cm³.

[0062] The disclosure herein provides for a method of generating a co-extruded biopolymer-based cellular biomaterial and continuous thread. An overview of the method is shown in Fig. 8A and an example of the co-extruded material is shown in Fig. 8B. The co-extruded material exhibits shear thinning behavior (Fig. 8C). The method includes steps i-iii. The steps may be performed sequentially, simultaneously, or a combination thereof. A skilled artisan will recognize that the order of steps may have an impact on the outcome of the method.

[0063] Step i) includes flowing a biopolymer-based cellular biomaterial under pressure through a first duct of a co-extrusion nozzle of a printhead of a three-dimensional printer. The biopolymerbased cellular biomaterial includes 20% silk fibroin powder, 5% glycerol monostearate, and 60% plasticizer. Step ii) includes guiding a continuous thread within a continuous thread duct of the coextrusion nozzle. Step iii) includes co-extruding the biopolymer-based cellular biomaterial and the continuous thread through a co-flow tip of the co-extrusion nozzle of the printhead to form a coextruded material.

[0064] The method may further include drying the co-extruded material exiting the co-extrusion nozzle. The drying may include directing an airflow from a ventilation system adjacent to the co- extrusion nozzle towards the co-extruded material exiting the co-extrusion nozzle.

[0065] The coextrusion of the method may be in accordance with an extrusion rate Q = or x h x v (where co = extrusion width, h = layer height, v = velocity of extrusion). The biopolymer-based cellular biomaterial may further include 10% sodium alginate and 10% xanthan gum. The plasticizer may be glycerol.

[0066] The present disclosure provides a method of making a biopolymer-based cellular biomaterial with a continuous thread including steps i-iii. Step i) includes whipping a liquid composition including silk fibroin, at least one polysaccharide, and at least one plasticizer for a predetermined whipping time at a predetermined speed to form a biopolymer-based cellular biomaterial. Step ii) includes pressurizing a volume of the biopolymer-based cellular biomaterial. Step iii) includes concurrently co-extruding the biopolymer-based cellular biomaterial with a continuous thread thereby forming a co-extruded material and drying the coextruded material.

[0067] The predetermined whipping time may be at least 8.5 minutes or between 5 minutes and 30 minutes. The predetermined whipping time may be at least 5 minutes, at least 6 minutes, at least 7 minutes, at least 8 minutes, at least 9 minutes, or at least 10 minutes. The predetermined whipping time may be at most 30 minutes, at most 25 minutes, at most 20 minutes, at most 15 minutes, or at most 10 minutes.

[0068] The predetermined speed may be at least 185 rpm or between 160 rpm and 200 rpm. The predetermined speed may be at least 160 rpm, at least 170 rpm, at least 175 rpm, or at least 185 rpm. The predetermined speed may be at most 200 rpm, at most 190 rpm, or at most 180 rpm.

[0069] In some embodiments, the biopolymer-based cellular biomaterial may include silk fibroin, at least one plasticizer, at least one surfactant, and optionally at least one polysaccharide. The at least one plasticizer may be a di(ethylene glycol), a tri(ethylene glycol), a di(propylene glycol), a tri(propylene glycol), triethanolamine, a vegetable oil, and combinations thereof. The at least one polysaccharide may be xanthan gum, an alginate, a high molecular weight sugar, a cellulose derivative, or a combination thereof.

[0070] The inventors discovered that a surfactant may be used to fine tune the stability and flow behavior of the cream during extrusion, however the surfactant should be carefully selected. In some cases, the surfactant is a monoglyceride. In some cases, the surfactant is glycerol monostearate. The inventors unexpectedly discovered that some common surfactants, such as sodium dodecyl sulfate (SDS), do not generate stable foams. The absence of an adequate surfactant made the material more compressible which translates into the need to increase the pressure drastically during printing making the material difficult to control. In some cases, the biopolymer-based cellular biomaterial and/or the surfactant can be free of anionic surfactant, free of sodium sulfate surfactant, and/or free of sodium dodecyl sulfate.

[0071] The liquid composition may include silk fibroin, glycerol, sodium alginate, and xanthan gum. The alginate may be alginic acid sodium salt. The at least one plasticizer may include at least one -OH substituent. The at least one plasticizer may be present in the biopolymer-based cellular biomaterial in an amount by weight of between 20.0% and 75.0%. The at least one plasticizer may be present in at least 30%, at least 40%, at least 50%, or at least 60% by weight. The at least one plasticizer may be present in at most 70%, at most 65%, at most 55%, or at most 45% by weight.

[0072] In some cases, the biopolymer-based cellular biomaterial may include 20% silk fibroin, 5% glycerol monostearate, 55% glycerol, 10% sodium alginate, and 10% xanthan gum.

[0073] In some cases, silk fibroin is present in the biopolymer-based cellular biomaterial in an amount by weight of between 1% and 40%. Silk fibroin may be present in an amount by weight of at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, or at least 30% by weight. Silk fibroin may be present in an amount by weight of at most 40%, at most 35%, at most 30%, at most 25%, at most 20%, at most 15%, at most 10%, at most 9%, at most 8%, at most 7%, at most 6%, or at most 5%.

[0074] In some cases, the at least one polysaccharide may be present in the biopolymer-based cellular biomaterial in an amount by weight of between 0.1% and 30.0%. The polysaccharide may be present in an amount by weight of at least 0.1%, at least 0.5%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 10%, at least 15%, at least 20%, or at least 25% by weight. The polysaccharide may be present in an amount by weight of at most 30%, at most 25%, at most 20%, at most 15%, at mosl0%, at most 9%, at most 8%, at most 7%, at most 6%, or at most 5%. [0075] In some cases, the silk fibroin and the at least one polysaccharide (e.g., xanthan gum, sodium alginate) may be present in a weight ratio of between 1 :4 and 20: 1 or between 1 :2 and 10:1. The silk fibroin and the at least one polysaccharide may be present in at least 1:4, at least 1:3, or at least 1:2 by weight. The silk fibroin and the at least one polysaccharide may be present in at most 20: 1, al most 19: 1, al most 18: 1, at most 16: 1, at most 15: 1, at most 14: 1, at most 12: 1, at most 11 : 1, or at most 10:1 by weight.

[0076] The co-extruded material of the printhead or method described herein may have at least one characteristic that is superior to a corresponding characteristic of an extruded material lacking the continuous thread. The characteristic may be resistance to stretching, and the co-extruded material may have a resistance to stretching that is at least 30%, at least 50%, at least 75%, at least 100%, or at least 150% higher than an extruded biopolymer-based material lacking the continuous thread. The characteristic may be resistance to shear stress, and the co-extruded material may have a resistance to shear stress that is at least 30%, at least 50%, at least 75%, at least 100%, or at least 150% higher than an extruded biopolymer-based material lacking the continuous thread.

[0077] The co-extruded material of the printhead or method described herein may have a porosity that is regular or irregular. The co-extruded material may have a firmness of at least 100 N/m², at least 200 N/m², at least 250 N/m², at least 300 N/m², or at least 400 N/m². The co-extruded material may have a density of at least 0.08 g/cm³, at least 0.1 g/cm³, at least 0.12 g/cm³, at least 0.2 g/cm³, or at least 0.25 g/cm³. The co-extruded material may include at least one of a sensing agent, a therapeutically active agent, a colorant, or an aroma-providing compound.

[0078] In some cases, it may be advantageous to produce articles by a process that does not include storing materials for use in the process under pressure. The present disclosure provides a method for generating a biopolymer-based cellular biomaterial, which optionally includes 20% silk fibroin, 5% glycerol monostearate, and 60% plasticizer, and a suspended continuous thread as shown in Fig. 9. A skilled artisan will appreciate that certain steps of this method may be substituted or combined with the previously described method to produce the desired articles.

[0079] In this method, the continuous thread is directly deposited into a bath of a biopolymer-based cellular biomaterial foam, which facilitates deposition of the continuous thread while preserving its initial layout. The continuous thread duct, labeled as ‘feeding pipe’ in Fig. 9, can move through the biopolymer-based cellular biomaterial foam bath without significantly altering the shape or properties of the bath. In some cases, the feeding duct may be no more than 1 mm, no more than 2 mm, no more than 2.5 mm, or no more than 3 mm in diameter. In some cases, the feeding duct may be circular in cross-section, and may comprise stainless steel (e.g., 316L). The distal end of the feeding pipe may either be in contact with the foam bath or submerged within the foam bath depending on the desired effect. If the distal end of the pipe is inside the foam bath, the printed thread will be integrated within the foam layer and covered on both sides. If the distal end is contacting the foam bath, only one side will be covered.

[0080] Consequently, the continuous thread can be deposited in one dimension, two dimensions, or three dimensions within the article, on a side of the article, or a combination of the two prior to an optional compressing step. Notably, this method uses the ‘support’ material as a base for the articles produced, meaning silk is used both as a matrix and a support, distinguishing this method from traditional suspended 3D printing processes.

[0081] Impressively, this method enables exponential scale-up of printing capacity due to the carefully selected steps and parameters. Scale-up can be a function of multiple continuous threads, larger bath sizes, larger and potentially more stable volumes of biomaterials, relative cost of a system without a pressurized component, or a combination thereof.

[0082] Fig. 10 provides an overview of the method (Steps 1 and 2) with optional further steps (Steps 3-5). Step 1 shows the biopolymer-based cellular biomaterial foam bath ready for the continuous thread. Step 2 includes depositing at least one continuous thread from a printhead into a biopolymer-based cellular biomaterial foam bath to form an embedded filament article. Optional Step 3 includes baking the embedded filament article to produce a baked embedded filament article. Optional Step 4 includes compression of the embedded filament article or the baked embedded filament article to produce a compressed embedded filament article or a compressed baked embedded filament article. Optional Step 5 shows recovery of the articles of Steps 2-4 which may be post-processed if desired, such as water vapor annealing, methanol treatment to increase beta sheet content, aesthetic enhancements or embellishments, or the like. In some cases, the articles may be redeposited into the foam bath such as for example to prepare a layered article by depositing additional continuous thread on at least a portion of the article. A skilled artisan will recognize that the optional compressing and baking steps may be performed sequentially or simultaneously to still provide the desired outcome. The inventors discovered that compressing before baking destabilizes the foam. The continuous thread is chemically and physically bonded by the foam matrix at the intersections. [0083] The printhead may include a continuous thread feeding mechanism. The biopolymer-based cellular biomaterial foam bath may include at least one layer of biopolymer-based cellular biomaterial foam. The printhead may include a feeding pipe with a proximal end contacting the printhead and a distal end contacting the biopolymer-based cellular biomaterial foam bath.

[0084] Compositionally, the compressed embedded filament article and/or the compressed baked embedded filament article, referred to hereinafter as ‘the compressed article(s)’, are generally the same as the embedded filament article or the baked embedded filament article, referred to hereinafter as the ‘uncompressed article(s)’. Structurally, the compressed articles have a reduced and/or compressed and/or damaged pore structure when compared with the uncompressed articles. Given that the compressed articles are downstream articles from the uncompressed articles, the compressed articles can include any component or feature of the uncompressed articles, unless the context clearly dictates otherwise (e.g., the porosity is reduced in the compressed articles). Similarly, as the uncompressed articles derive from upstream entities, the compressed articles can include any component or feature of the uncompressed articles, unless the context clearly dictates otherwise. [0085] In some cases, the present disclosure provides a method for making compressed articles. A method of making a compressed article can include compressing the uncompressed article with a force of between 0.25 MPa and 25 MPa for a length of time of between 15 minutes and 6 hours. In some cases, the compressing can be heat-compressing that is performed at an elevated temperature of between 80 °C and 200 °C. In some cases, the compressing can be performed with a calendar press. A method of making a compressed article can include compressing the uncompressed article with a force of between 0.25 MPa and 25 MPa for a length of time of between 5 minutes and 6 hours. A skilled artisan will recognize that the exact parameters of heat-compressing may vary outside of the specified parameters but still provide the desired outcome.

[0086] In some cases, the present disclosure provides a method for making baked embedded filament articles, compressed baked embedded filament articles, baked compressed embedded filament articles, or further baked compressed baked filament articles, hereinafter referred to as ‘baked articles’. A method for making a baked article can include baking the embedded filament article or the compressed embedded filament article, hereinafter referred to as ‘unbaked article(s)’ at a temperature of between 25 °C and 150 °C, between 30 °C and 120 °C, or between 50 °C and 80 °C for a length of time of between 2 hours and 24 hours to form the baked article. A compressed baked filament article may also be further baked to form a further baked compressed baked filament article. In an example, the baking is performed at a temperature of between 40 °C and 150 °C for a length of time of between 5 minutes and 24 hours.

[0087] In some cases, the present disclosure provides a method of making an article, such as baked embedded filament articles or compressed baked embedded filament articles. A method of making a baked article can include compressing the unbaked article or the uncompressed article with a force of between 0.25 MPa and 25 MPa for a length of time of between 15 minutes and 6 hours. In some cases, the compressing can be heat-compressing that is performed at an elevated temperature of between 80 °C and 200 °C. In some cases, the compressing can be performed with a calendar press. A method of making a baked article can include compressing the unbaked article with a force of between 0.25 MPa and 25 MPa for a length of time of between 5 minutes and 6 hours. [0088] The continuous thread may be deposited in one dimension, two dimensions, or three dimensions. A photograph of a continuous thread embedded in the silk foam is shown in Fig. 12. The continuous thread may be deposited in at least one layer, at least two layers, at least three layers, or more. The continuous thread may have at least one property of electrical conductance, magnetism, at least one sensor embedded therein, a thermal conductance, a fluorescence, a color, or a combination thereof. The at least one property may be constant or vary across the length of the continuous thread. The embedded filament article, baked embedded filament article, or the compressed baked embedded filament article may have a property that is the result of the at least one property of the continuous thread embedded therein.

[0089] The printhead may have one continuous thread (Fig. 9) or more than one continuous thread (Fig. 11 A,B) to be deposited, including but not limited to, at least two, at least three, or more continuous threads to be deposited. The multiple continuous threads may be deposited simultaneously or sequentially. The multiple continuous threads may have identical or different compositions.

[0090] The biopolymer-based cellular biomaterial foam bath of the method described herein may have an area of at least 1 cm², including but not limited to, at least 5 cm², at least 10 cm², at least 100 cm², at least 1 m², or greater. The bath is not limited to square or rectangular shapes, but could be rounded, triangular, hexagonal, octagonal, curved, or any desired shape. It should be understood that constraints on the foam bath size depend on processing capabilities and with proper procedure, a skilled artisan could scale the process up by orders of magnitude. A depth of the bath may be between 5 mm and 80 mm, including but not limited to, between 20 mm and 40 mm. A bath depth of between 20 mm and 40 mm enables more than 10 layers of embedded thread to be printed within. A skilled artisan will appreciate that the depth of the bath and number of layers of embedded thread may be scaled up in the same manner as the area as described herein.

[0091] The biopolymer-based cellular biomaterial foam bath may have more than one chamber. The chambers may have identical or different foam bath compositions to form articles with different compositions simultaneously.

[0092] A size or volume of the embedded filament article, the baked embedded filament article, or the compressed baked embedded filament article may be at least 75% of the size or volume of the biopolymer-based cellular biomaterial foam bath, including between 80% and 90% of bath size. The biomaterial-based cellular biomaterial foam bath may have at least one pattern or shape that is reflected in the embedded filament article, the baked embedded filament article, or the compressed baked embedded filament article. [0093] The embedded filament article, the baked embedded filament article, or the compressed baked embedded filament article produced by the method described herein may have a regular porosity or an irregular porosity. The embedded filament article, the baked embedded filament article, or the compressed baked embedded filament article may have a firmness of at least 100 N/m², at least 200 N/m², at least 250 N/m², at least 300 N/m², or at least 400 N/m². The embedded filament article, the baked embedded filament article, or the compressed baked embedded filament article may have a density of at least 0.08 g/cm³, at least 0.1 g/cm³, at least 0.12 g/cm³, at least 0.2 g/cm³, or at least 0.25 g/cm³. The embedded filament article, the baked embedded filament article, or the compressed baked embedded filament article may further include at least one of a sensing agent, a therapeutically active agent, a colorant, or an aroma-providing compound.

[0094] The mechanical properties and physical properties disclosed herein refer to the compressibility and density of the bulk foam in which the thread is embedded, under the assumption that these properties are not significantly affected by the presence of the filament. However, the materials obtained after baking and/or compression are expected to exhibit improved resistance, particularly under tensile stress. The enhancement of these mechanical properties strongly depends on the mechanical performance of the inserted thread. A skilled artisan will appreciate that the exact values of the properties may be dependent on the amount of foam within a sample or article.

[0095] Broadly, the articles with embedded filaments have similar properties and characteristics when compared to the coextruded articles. However, the coextruded articles may have hollow patterns that the embedded printing method cannot achieve. On the other hand, the embedded printing process offers a significant versatility in terms of matrix materials as the foam does not need to be extruded. Thus, a wider range of foam properties fit in the process. A skilled artisan will appreciate the different strengths of the methods and understand that in some cases, one method may be preferred over another.

[0096] The present disclosure provides for an article including the co-extruded material or the biopolymer-based cellular biomaterial described herein. The article may be at least one of a textile, a 3D structure, a structural material, a building material, an insulating material, a packaging material, a tool, a biomedical implant, a prosthetic, or an orthotic. The article may be subjected to at least one post-processing technique, including but not limited to, curing, compressing, molding, or aesthetically modifying the article.

[0097] According to various embodiments, a variety of functionalizing agents may be used with the silk-containing embodiments described herein (e.g., silk membrane, silk composition, silk articles, silk matrix, silk foam, silk microsphere, liquid composition, whipped silk cream, silk meringue, compressed silk meringue, hot-pressed silk meringue, silk leather, silk powder, silk toner, edible silk- based films, etc.). It should be understood that the examples herein may recite one or a few silkcontaining embodiments but are applicable to any silk-containing embodiment, as applicable. In some embodiments, a functionalizing agent may be any compound or molecule that facilitates the attachment to and/or development (e.g., growth) of one or more endothelial cells on a silk membrane. In some embodiments, a functionalizing agent may be any compound or molecule that facilitates the attachment and/or development (e.g., growth) of one or more megakaryocytes and/or hematopoietic progenitor cells on a silk matrix and/or silk membrane. In some embodiments, a functionalizing agent may be or comprise an agent suitable for facilitating the production of one or more of white blood cells and red blood cells.

[0098] In some embodiments, a functionalizing agent may be or comprise a cell attachment mediator and/or an extracellular matrix protein, for example: collagen (e.g., collagen type I, collagen type III, collagen type IV or collagen type VI), elastin, fibronectin, vitronectin, laminin, fibrinogen, von Willebrand factor, proteoglycans, decorin, perlecan, nidogen, hyaluronan, and/or peptides containing known integrin binding domains e.g. “RGD” integrin binding sequence, or variations thereof, that are known to affect cellular attachment.

[0099] In some embodiments, a functionalizing agent may be any soluble molecule produced by endothelial cells. Non- limiting examples include fibroblast growth factor- 1 (FGF1) and vascular endothelial growth factors (VEGF).

[0100] According to some embodiments, a plurality of functionalizing agents may be used. For example, in some embodiments wherein production of platelets is desired, provided compositions may comprise the use of laminin, fibronectin and/or fibrinogen, and type IV collagen in order to facilitate the attachment and growth of endothelial cells on a silk membrane (e.g., a porous silk membrane) and/or attachment of megakaryocytes to a silk matrix.

[0101] In some embodiments, a functionalizing agent may be embedded or otherwise associated with a silk membrane and/or silk matrix such that at least a portion of the functionalizing agent is surrounded by a silk membrane and/or silk matrix as contrasted to a functionalizing agent simply being positioned along the surface of a silk membrane and/or silk matrix. In some embodiments, a functionalizing agent is distributed along and/or incorporated in substantially the entire surface area of a silk membrane/silk wall. In some embodiments, a functionalizing agent is distributed and/or incorporated only at one or more discrete portions of a silk membrane/wall and/or silk matrix. In some embodiments, a functionalizing agent is distributed in and/or along at least one of the lumenfacing side of a silk wall and the matrix-facing side of a silk wall.

[0102] According to various embodiments, any application-appropriate amount of one or more functionalizing agents may be used. In some embodiments, the amount of an individual functionalizing agent may be between about 1 pg/ml and 1,000 pg/ml (e.g., between about 2 and 1,000, 5 and 1,000, 10 and 1,000, 10 and 500, 10 and 100 pg/ml). In some embodiments, the amount of an individual functionalizing agent may be at least 1 pg/ml (e.g., at least 5, 10, 15, 20 25, 50, 100, 200, 300 400, 500, 600, 700, 800, or 900 |ig/ml ). In some embodiments, the amount of an individual functionalizing agent is at most 1,000 pg/ml (e.g., 900, 800, 700, 600, 500, 400, 300 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, or 5 pg/ml).

[0103] In some aspects, the composition comprises one or more sensing agents, such as a sensing dye. The sensing agents/sensing dyes are environmentally sensitive and produce a measurable response to one or more environmental factors. In some aspects, the environmentally- sensitive agent or dye may be present in the composition in an effective amount to alter the composition from a first chemical-physical state to a second chemical-physical state in response to an environmental parameter (e.g., a change in pH, light intensity or exposure, temperature, pressure or strain, voltage, physiological parameter of a subject, and/or concentration of chemical species in the surrounding environment) or an externally applied stimulus (e.g., optical interrogation, acoustic interrogation, and/or applied heat). In some cases, the sensing dye is present to provide one optical appearance under one given set of environmental conditions and a second, different optical appearance under a different given set of environmental conditions. Suitable concentrations for the sensing agents described herein can be the concentrations for the colorants and additives described elsewhere herein. A person having ordinary skill in the chemical sensing arts can determine a concentration that is appropriate for use in a sensing application of the inks described herein.

[0104] In some aspects, the first and second chemical-physical state may be a physical property of the composition, such as mechanical property, a chemical property, an acoustical property, an electrical property, a magnetic property, an optical property, a thermal property, a radiological property, or an organoleptic property. Exemplary sensing dyes or agents include, but are not limited to, a pH sensitive agent, a thermal sensitive agent, a pressure or strain sensitive agent, a light sensitive agent, or a potentiometric agent.

[0105] Exemplary pH sensitive dyes or agents include, but are not limited to, cresol red, methyl violet, crystal violet, ethyl violet, malachite green, methyl green, 2-(p- dimethylaminophenylazo)pyridine, paramethyl red, metanil yellow, 4-phenylazodiphenylamine, thymol blue, metacresol purple, orange IV, 4-o-Tolylazo-o-toluindine, quinaldine red, 2,4- dinitrophenol, erythrosine disodium salt, benzopurpurine 4B, N,N-dimethyl-p-(m-tolylazo) aniline, p-dimethylaminoazobenene, 4,4’-bis(2-amino-l-naphthylazo)-2,2’-stilbenedisulfonic acid, tetrabromophenolphthalein ethyl ester, bromophenol blue, Congo red, methyl orange, ethyl orange, 4-(4-dimethylamino-l-naphylazo)-3-methoxybenesulfonic acid, bromocresol green, resazurin, 4- phenylazo-l-napthylamine, ethyl red 2-(l-dimethylaminophenyazo) pyridine, 4-(p-ethoxyphenylazo)- m-phenylene-diamine monohydrochloride, resorcin blue, alizarin red S, methyl red, propyl red, bromocresol purple, chlorophenol red, p-nitrophenol, alizarin, 2-(2,4-dinitrophenylazo)-l-napthol-

3.6-disulfonic acid, bromothymol blue, 6, 8-dinitro-lH-quinazoline-2, 4-dione, brilliant yellow, phenol red, neutral red, m-nitrophenol, cresol red, turmeric, metacresol purple, 4,4’-bis(3-amino-l- naphthylazo)-2,2'-stilbenedisulfonic acid, thymol blue, p-naphtholbenzein, phenolphthalein, o- cresolphthalein, ethyl bis(2,4-dimethylphenyl) ethanoate, thymolphthalein, nitrazine yellow, alizarin yellow R, alizarin, p-(2,4-dihydroxyphenylazo) benzenesulfonic acid, 5,5'-indigodisulfonic acid,

2.4.6-trinitrotoluene, 1,3,5-trinitrobenezne, and clayton yellow.

[0106] Exemplary light responsive dyes or agents include, but are not limited to, photochromic compounds or agents, such as triarylmethanes, stilbenes, azastilbenes, nitrones, fulgides, spiropyrans, napthopyrans, spiro-oxazines, quinones, derivatives, and combinations thereof.

[0107] Exemplary potentiometric dyes include, but are not limited to, substituted amiononaphthylehenylpridinium (ANEP) dyes, such as di-4-ANEPPS, di-8-ANEPPS, and N-(4- Sulfobutyl)-4-(6-(4-(Dibutylamino)phenyl)hexatrienyl)Pyridinium (RH237).

[0108] Exemplary temperature sensitive dyes or agents include, but are not limited to, thermochromic compounds or agents, such as thermochromic liquid crystals, leuco dyes, fluoran dyes, octadecylphosphonic acid.

[0109] Exemplary pressure or strain sensitive dyes or agents include, but are not limited to, spiropyran compounds and agents.

[0110] Exemplary chemi-sensitive dyes or agents include, but are not limited to, antibodies such as immunoglobulin G (IgG) which may change color from blue to red in response to bacterial contamination.

[0111] In some aspects, the compositions comprise one or more additive, dopant, or biologically active agent suitable for a desired intended purpose. In some aspects, the additive or dopant may be present in the composition in an amount effective to impart an optical or organoleptic property to the composition. Exemplary additives or dopants that impart optical or organoleptic properties include, but are not limited to, dyes/pigments, flavorants, aroma compounds, granular or fibrous fillers.

[0112] Additionally or alternatively, the additive, dopant, or biologically active agent may be present in the composition in an amount effective to "functionalize" the composition to impart a desired mechanical property or added functionality to the composition. Exemplary additive, dopants, or biologically active agent that impart the desired mechanical property or added functionality include, but are not limited to: environmentally sensitive/sensing dyes; active biomolecules; conductive or metallic particles; micro and nanofibers (e.g., silk nanofibers for reinforcement, carbon nanofibers); nanotubes; inorganic particles (e.g., hydroxyapatite, tricalcium phosphate, bioglasses); drugs (e.g., antibiotics, small molecules or low molecular weight organic compounds); proteins and fragments or complexes thereof (e.g., enzymes, antigens, antibodies and antigen-binding fragments thereof); DNA/RNA (e.g., siRNA, miRNA, mRNA); cells and fractions thereof (viruses and viral particles; prokaryotic cells such as bacteria; eukaryotic cells such as mammalian cells and plant cells; fungi). [0113] In some aspects, the additive or dopant comprises a flavoring agent or flavorant.

[0114] Exemplary flavorants include ester flavorants, amino acid flavorants, nucleic acid flavorants, organic acid flavorants, and inorganic acid flavorants, such as, but not limited to, diacetyl, acetyl propionyl, acetoin, isoamyl acetate, benzaldehyde, cinnamaldehyde, ethyl propionate, methyl anthranilate, limonene, ethyl decadienoate, allyl hexanoate, ethyl maltol, ethylvanillin, methyl salicylate, manzanate, glutamic acid salts, glycine salts, guanylic acids salts, inosinic acid salts, acetic acid, ascorbic acid, citric acid, fumaric acid, lactic acid, malic acid, phosphoric acid, tartaric acid, derivatives, and mixtures thereof.

[0115] In some aspects, the additive or dopant comprises an aroma compound. Exemplary aroma compounds include ester aroma compounds, terpene aroma compounds, cyclic terpenes, and aromatic aroma compounds, such as, but not limited to, geranyl acetate, methyl formate, methyl acetate, methyl propionate, methyl butyrate, ethyl acetate, ethyl butyrate, isoamyl acetate, pentyl butyrate, pentyl pentanoate, octyl acetate, benzyl acetate, methyl anthranilate, myrcene, geraniol, nerol, citral, citronellal, cironellol, linalool, nerolidol, limonene, camphor, menthol, carone, terpineol, alpha-ionone, thujone, eucalyptol, benzaldehyde, eugenol, cinnamaldehyde, ethyl maltol, vanillin, anisole, anethole, estragole, thymol.

[0116] In some aspects, the additive or dopant comprises a colorant, such as a dye or pigment. In some aspects, the dye or pigment imparts a color or grayscale to the composition. The colorant can be different than the sensing agents and/or sensing dyes below. Any organic and/or inorganic pigments and dyes can be included in the inks. Exemplary pigments suitable for use in the present disclosure include International Color Index or C.I. Pigment Black Numbers 1 , 7, 1 1 and 31, C.I. Pigment Blue Numbers 15, 15:1, 15:2, 15:3, 15:4, 15:6, 16, 27, 29, 61 and 62, C.I. Pigment Green Numbers 7, 17, 18 and 36, C.I. Pigment Orange Numbers 5, 13, 16, 34 and 36, C.I. Pigment Violet Numbers 3, 19, 23 and 27, C.I. Pigment Red Numbers 3, 17, 22, 23, 48:1, 48:2, 57: 1, 81: 1, 81 :2, 81 :3, 81:5, 101, 114, 122, 144, 146, 170, 176, 179, 181, 185, 188, 202, 206, 207, 210 and 249, C.I. Pigment Yellow Numbers 1, 2, 3, 12, 13, 14, 17, 42, 65, 73, 74, 75, 83, 30, 93, 109, 1 10, 128, 138, 139, 147, 142, 151, 154 and 180, D&C Red No. 7, D&C Red No. 6 and D&C Red No. 34, carbon black pigment (such as Regal 330, Cabot Corporation), quinacridone pigments (Quinacridone Magenta (228-0122), available from Sun Chemical Corporation, Fort Lee, N.J.), diarylide yellow pigment (such as AAOT Yellow (274- 1788) available from Sun Chemical Corporation); and phthalocyanine blue pigment (such as Blue 15:3 (294-1298) available from Sun Chemical Corporation). The classes of dyes suitable for use in present invention can be selected from acid dyes, natural dyes, direct dyes (either cationic or anionic), basic dyes, and reactive dyes. The acid dyes, also regarded as anionic dyes, are soluble in water and mainly insoluble in organic solvents and are selected, from yellow acid dyes, orange acid dyes, red acid dyes, violet acid dyes, blue acid dyes, green acid dyes, and black acid dyes. European Patent 0745651, incorporated herein by reference, describes a number of acid dyes that are suitable for use in the present disclosure. Exemplary yellow acid dyes include Acid Yellow 1 International Color Index or C.I. 10316); Acid Yellow 7 (C.I. 56295); Acid Yellow 17 (C.I. 18965); Acid Yellow 23 (C.I. 19140); Acid Yellow 29 (C.I. 18900); Acid Yellow 36 (C.I. 13065); Acid Yellow 42 (C.I. 22910); Acid Yellow 73 (C.I. 45350); Acid Yellow 99 (C.I. 13908); Acid Yellow 194; and Food Yellow 3 (C.I. 15985). Exemplary orange acid dyes include Acid Orange 1 (C.I. 13090/1); Acid Orange 10 (C.I. 16230); Acid Orange 20 (C.I. 14603); Acid Orange 76 (C.I. 18870); Acid Orange 142; Food Orange 2 (C.I. 15980); and Orange B. [0117] Exemplary red acid dyes include Acid Red 1 (C.I. 18050); Acid Red 4 (C.I. 14710); Acid Red 18 (C.I. 16255); Acid Red 26 (C.I. 16150); Acid Red 27 (C.I. 16185); Acid Red 51 (C.I. 45430, available from BASF Corporation, Mt. Olive, N.J.); Acid Red 52 (C.I. 45100); Acid Red 73 (C.I. 27290); Acid Red 87 (C.I. 45380); Acid Red 94 (C.I. 45440) Acid Red 194; and Food Red 1 (C.I. 14700). Exemplary violet acid dyes include Acid Violet 7 (C.I. 18055); and Acid Violet 49 (C.I. 42640). Exemplary blue acid dyes include Acid Blue 1 (C.I. 42045); Acid Blue 9 (C.I. 42090); Acid Blue 22 (C.I. 42755); Acid Blue 74 (C.I. 73015); Acid Blue 93 (C.I. 42780); and Acid Blue 158A (C.I. 15050). Exemplary green acid dyes include Acid Green 1 (C.I. 10028); Acid Green 3 (C.I. 42085); Acid Green 5 (C.I. 42095); Acid Green 26 (C.I. 44025); and Food Green 3 (C.I. 42053). Exemplary black acid dyes include Acid Black 1 (C.I. 20470); Acid Black 194 (Basantol® X80, available from BASF Corporation, an azo/1 :2 CR-complex.

[0118] Exemplary direct dyes for use in the present disclosure include Direct Blue 86 (C.I. 74180); Direct Blue 199; Direct Black 168; Direct Red 253; and Direct Yellow 107/132 (C.I. Not Assigned). [0119] Exemplary natural dyes for use in the present disclosure include Alkanet (C.I. 75520,75530); Annatto (C.I. 75120); Carotene (C.I. 75130); Chestnut; Cochineal (C.I.75470); Cutch (C.I. 75250, 75260); Divi-Divi; Fustic (C.I. 75240); Brazilin (C.I. 75280); Logwood (C.I. 75200); Osage Orange (C.I. 75660); Paprika; Quercitron (C.I. 75720); Saffron (C.I. 75100) ; Sandal Wood (C.I. 75510, 75540, 75550, 75560); Sumac; and Turmeric (C.I. 75300). Exemplary reactive dyes for use in the present disclosure include Reactive Yellow 37 (monoazo dye); Reactive Black 31 (diazo dye); Reactive Blue 77 (phthalo cyanine dye) and Reactive Red 180 and Reactive Red 108 dyes. Suitable also are the colorants described in The Printing Ink Manual (5th ed., Leach et al. eds. (2007), pages 289-299). Other organic and inorganic pigments and dyes and combinations thereof can be used to achieve the colors desired.

[0120] In addition to or in place of visible colorants, compositions provided herein can contain ETV fluorophores that are excited in the ETV range and emit light at a higher wavelength (typically 400 nm and above). Examples of ETV fluorophores include but are not limited to materials from the coumarin, benzoxazole, rhodamine, napthalimide, perylene, benzanthrones, benzoxanthones or benzothia-xanthones families. The addition of a UV fluorophore (such as an optical brightener for instance) can help maintain maximum visible light transmission. The amount of colorant, when present, generally is between 0.05% to 5% or between 0.1% and 1% based on the weight of the composition.

[0121] For non-white compositions, the amount of pigmen t/dye generally is present in an amount of from at or about 0.1 wt% to at or about 20 wt% based on the weight of the composition. In some applications, a non-white ink can include 15 wt% or less pigment/dye, or 10 wt% or less pigment/dye or 5 wt% pigment/dye, or 1 wt% pigment/dye based on the weight of the composition. In some applications, a non-white ink can include 1 wt% to 10 wt%, or 5 wt% to 15 wt%, or 10 wt% to 20 wt% pigment/dye based on the weight of the composition. In some applications, a non-white ink can contain an amount of dye/pigment that is 1 wt%, 2 wt%, 3 wt%, 4 wt%, 5%, 6 wt%, 7 wt%, 8 wt%, 9 wt%, 10 wt%, 11 wt%, 12 wt%, 13 wt%, 14 wt%, 15%, 16 wt%, 17 wt%, 18 wt%, 19 wt% or 20 wt% based on the weight of the composition.

[0122] For white compositions, the amount of white pigment generally is present in an amount of from at or about 1 wt% to at or about 60 wt% based on the weight of the composition. In some applications, greater than 60 wt% white pigment can be present. Preferred white pigments include titanium dioxide (anatase and rutile), zinc oxide, lithopone (calcined coprecipitate of barium sulfate and zinc sulfide), zinc sulfide, blanc fixe and alumina hydrate, and combinations thereof, although any of these can be combined with calcium carbonate. In some applications, a white ink can include 60 wt% or less white pigment, 55 wt% or less white pigment, 50 wt% white pigment, 45 wt% white pigment, 40 wt% white pigment, 35 wt% white pigment, 30 wt% white pigment, 25 wt% white pigment, 20 wt% white pigment, 15 wt% white pigment, or 10 wt% white pigment, based on the weight of the composition. In some applications, a white ink can include 5 wt% to 60 wt%, 5 wt% to 55 wt%, 10 wt% to 50 wt%, 10 wt% to 25 wt%, 25 wt% to 50 wt%, 5 wt% to 15 wt%, or 40 wt% to 60 wt% white pigment based on the weight of the composition. In some applications, a non-white ink can an amount of dye/pigment that is 5%, 6 wt%, 7 wt%, 8 wt%, 9 wt%, 10 wt%, 11 wt%, 12 wt%, 13 wt%, 14 wt%, 15%, 16 wt%, 17 wt%, 18 wt%, 19 wt%, 20 wt%, 21 wt%, 22 wt%, 23 wt%, 24 wt%, 25%, 26 wt%, 27 wt%, 28 wt%, 29 wt%, 30 wt%, 31 wt%, 32 wt%, 33 wt%, 34 wt%, 35%, 36 wt%, 37 wt%, 38 wt%, 39 wt%, 40 wt%, 41 wt%, 42 wt%, 43 wt%, 44 wt%, 45%, 46 wt%, 47 wt%, 48 wt%, 49 wt%, 50 wt%, 51 wt%, 52 wt%, 53 wt%, 54 wt%, 55%, 56 wt%, 57 wt%, 58 wt%, 59 wt% or 60 wt% based on the weight of the composition.

[0123] In some aspects, the additive or dopant comprises a conductive additive. Exemplary conductive additives include, but are not limited to graphite, graphite powder, carbon nanotubes, and metallic particles or nanoparticles, such as gold nanoparticles. In some aspects, the conductive additive is biocompatible and non-toxic.

[0124] In some aspects, the additive is a biologically active agent. The term “biologically active agent” as used herein refers to any molecule which exerts at least one biological effect in vivo. For example, the biologically active agent can be a therapeutic agent to treat or prevent a disease state or condition in a subject. Biologically active agents include, without limitation, organic molecules, inorganic materials, proteins, peptides, nucleic acids (e.g., genes, gene fragments, gene regulatory sequences, and antisense molecules), nucleoproteins, polysaccharides, glycoproteins, and lipoproteins. Classes of biologically active compounds that can be incorporated into the composition provided herein include, without limitation, anticancer agents, antibiotics, analgesics, antiinflammatory agents, immunosuppressants, enzyme inhibitors, antihistamines, anti-convulsants, hormones, muscle relaxants, antispasmodics, ophthalmic agents, prostaglandins, anti-depressants, anti-psychotic substances, trophic factors, osteoinductive proteins, growth factors, and vaccines. [0125] The term “active agent” may also be used herein to refer to a biological sample (e.g., a sample of tissue or fluid, such as for instance blood) or a component thereof, and/or to a biologically active entity or compound, and/or to a structurally or functionally labile entity.

[0126] Exemplary active agents include, but are not limited to, therapeutic agents, diagnostic agents (e.g., contrast agents), and any combinations thereof. In some embodiments, the active agent present in a silk matrix (e.g., a silk microsphere), composition, or the like can include a labile active agent, e.g., an agent that can undergo chemical, physical, or biological change, degradation and/or deactivation after exposure to a specified condition, e.g., high temperatures, high humidity, light exposure, and any combinations thereof. In some embodiments, the active agent present in the silk matrix (e.g., a silk microsphere), composition, or the like can include a temperature-sensitive active agent, e.g., an active agent that will lose at least about 30% or more, of its original activity or bioactivity, upon exposure to a temperature of at least about 10 °C. or above, including at least about 15 °C. or above, at least about room temperature or above, or at least about body temperature (e.g., about 37 °C.) or above. [0127] The active agent can be generally present in the silk matrix (e.g., a silk microsphere), composition, or the like in an amount of about 0.01% (w/w) to about 70% (w/w), about 0.1% (w/w) to about 50% (w/w), or about 1% (w/w) to about 30% (w/w). The active agent can be present on a surface of the silk matrix (e.g., a silk microsphere), composition, or the like and/or encapsulated and dispersed in the silk matrix (e.g., a silk microsphere), composition, or the like homogeneously, heterogeneously, or in a gradient. In some embodiments, the active agent can be added into the silk solution, which is then subjected to the methods described herein for preparing a silk matrix (e.g., a silk microsphere), composition, or the like. In some embodiments, the active agent can be coated on a surface of the silk matrix (e.g., a silk microsphere), composition, or the like. In some embodiments, the active agent can be loaded in a silk matrix (e.g., a silk microsphere), composition, or the like by incubating the silk microsphere in a solution of the active agent for a period of time, during which an amount of the active agent can diffuse into the silk matrix (e.g., a silk microsphere), composition, or the like, and thus distribute within the silk matrix (e.g., a silk microsphere), composition, or the like. [0128] In some aspects, the additive is a therapeutic agent. As used herein, the term “therapeutic agent” means a molecule, group of molecules, complex or substance administered to an organism for diagnostic, therapeutic, preventative medical, or veterinary purposes. As used herein, the term “therapeutic agent” includes a “drug” or a “vaccine.” This term includes externally and internally administered topical, localized and systemic human and animal pharmaceuticals, treatments, remedies, nutraceuticals, cosmeceuticals, biologicals, devices, diagnostics and contraceptives, including preparations useful in clinical and veterinary screening, prevention, prophylaxis, healing, wellness, detection, imaging, diagnosis, therapy, surgery, monitoring, cosmetics, prosthetics, forensics and the like. This term can also be used in reference to agriceutical, workplace, military, industrial and environmental therapeutics or remedies comprising selected molecules or selected nucleic acid sequences capable of recognizing cellular receptors, membrane receptors, hormone receptors, therapeutic receptors, microbes, viruses or selected targets comprising or capable of contacting plants, animals and/or humans. This term can also specifically include nucleic acids and compounds comprising nucleic acids that produce a therapeutic effect, for example deoxyribonucleic acid (DNA), ribonucleic acid (RNA), nucleic acid analogues (e.g., locked nucleic acid (LNA), peptide nucleic acid (PNA), xeno nucleic acid (XNA)), or mixtures or combinations thereof, including, for example, DNA nanoplexes, siRNA, microRNA, shRNA, aptamers, ribozymes, decoy nucleic acids, antisense nucleic acids, RNA activators, and the like. Generally, any therapeutic agent can be included in the composition provided herein.

[0129] The term “therapeutic agent” also includes an agent that is capable of providing a local or systemic biological, physiological, or therapeutic effect in the biological system to which it is applied. For example, the therapeutic agent can act to control infection or inflammation, enhance cell growth and tissue regeneration, control tumor growth, act as an analgesic, promote anti-cell attachment, and enhance bone growth, among other functions. Other suitable therapeutic agents can include anti-viral agents, hormones, antibodies, or therapeutic proteins. Other therapeutic agents include prodrugs, which are agents that are not biologically active when administered but upon administration to a subject are converted to biologically active agents through metabolism or some other mechanism. Additionally, a silk-based drug delivery composition can contain one therapeutic agent or combinations of two or more therapeutic agents.

[0130] A therapeutic agent can include a wide variety of different compounds, including chemical compounds and mixtures of chemical compounds, e.g., small organic or inorganic molecules; saccharides; oligosaccharides; polysaccharides; biological macromolecules, e.g., peptides, proteins, and peptide analogs and derivatives; peptidomimetics; antibodies and antigen binding fragments thereof; nucleic acids; nucleic acid analogs and derivatives; an extract made from biological materials such as bacteria, plants, fungi, or animal cells; animal tissues; naturally occurring or synthetic compositions; and any combinations thereof. In some aspects, the therapeutic agent is a small molecule.

[0131] The term “bioactivity,” as used herein in reference to an active agent, generally refers to the ability of an active agent to interact with a biological target and/or to produce an effect on a biological target. For example, bioactivity can include, without limitation, elicitation of a stimulatory, inhibitory, regulatory, toxic or lethal response in a biological target. The biological target can be a molecule or a cell. For example, a bioactivity can refer to the ability of an active agent to modulate the effect/activity of an enzyme, block a receptor, stimulate a receptor, modulate the expression level of one or more genes, modulate cell proliferation, modulate cell division, modulate cell morphology, or any combination thereof. In some instances, a bioactivity can refer to the ability of a compound to produce a toxic effect in a cell. Exemplary cellular responses include, but are not limited to, lysis, apoptosis, growth inhibition, and growth promotion; production, secretion, and surface expression of a protein or other molecule of interest by the cell; membrane surface molecule activation including receptor activation; transmembrane ion transports; transcriptional regulations; changes in viability of the cell; changes in cell morphology; changes in presence or expression of an intracellular component of the cell; changes in gene expression or transcripts; changes in the activity of an enzyme produced within the cell; and changes in the presence or expression of a ligand and/or receptor (e.g., protein expression and/or binding activity). Methods for assaying different cellular responses are well known to one of skill in the art, e.g., western blot for determining changes in presence or expression of an endogenous protein of the cell, or microscopy for monitoring the cell morphology in response to the active agent, or FISH and/or qPCR for the detection and quantification of changes in nucleic acids. Bioactivity can be determined in some embodiments, for example, by assaying a cellular response.

[0132] In reference to an antibody, the term “bioactivity” includes, but is not limited to, epitope or antigen binding affinity, the in vivo and/or in vitro stability of the antibody, the immunogenic properties of the antibody, e.g., when administered to a human subject, and/or the ability to neutralize or antagonize the bioactivity of a target molecule in vivo or in vitro. The aforementioned properties or characteristics can be observed or measured using art-recognized techniques including, but not limited to, scintillation proximity assays, ELISA, ORIGEN immunoassay (IGEN), fluorescence quenching, fluorescence ELISA, competitive ELISA, SPR analysis including, but not limited to, SPR analysis using a BIAcore biosensor, in vitro and in vivo neutralization assays (see, for example, International Publication No. WO 2006/062685), receptor binding, and immunohistochemistry with tissue sections from different sources including human, primate, or any other source as needed. In reference to an immunogen, the “bioactivity” includes immunogenicity, the definition of which is discussed in detail later. In reference to a virus, the “bioactivity” includes infectivity, the definition of which is discussed in detail later. In reference to a contrast agent, e.g., a dye, the “bioactivity” refers to the ability of a contrast agent when administered to a subject to enhance the contrast of structures or fluids within the subject's body. The bioactivity of a contrast agent also includes, but is not limited to, its ability to interact with a biological environment and/or influence the response of another molecule under certain conditions.

[0133] As used herein, the term “small molecule” can refer to compounds that are “natural productlike,” however, the term “small molecule” is not limited to “natural product-like” compounds. Rather, a small molecule is typically characterized in that it contains several carbon — carbon bonds and has a molecular weight of less than 5000 Daltons (5 kDa), preferably less than 3 kDa, still more preferably less than 2 kDa, and most preferably less than 1 kDa. In some cases, it is preferred that a small molecule has a molecular weight equal to or less than 700 Daltons.

[0134] Exemplary therapeutic agents include, but are not limited to, those found in Harrison’ s Principles of Internal Medicine, 13th Edition, Eds. T.R. Harrison et al. McGraw-Hill N.Y., NY; Physicians’ Desk Reference, 50th Edition, 1997, Oradell New Jersey, Medical Economics Co.; Pharmacological Basis of Therapeutics, 8th Edition, Goodman and Gilman, 1990; United States Pharmacopeia, The National Formulary, ETSP XII NF XVII, 1990, the complete contents of all of which are incorporated herein by reference.

[0135] Therapeutic agents include the herein disclosed categories and specific examples. It is not intended that the category be limited by the specific examples. Those of ordinary skill in the art will recognize also numerous other compounds that fall within the categories and that are useful according to the present disclosure. Examples include a radiosensitizer, a steroid, a xanthine, a beta- 2-agonist bronchodilator, an anti-inflammatory agent, an analgesic agent, a calcium antagonist, an angiotensin-converting enzyme inhibitors, a beta-blocker, a centrally active alpha- agonist, an alpha- 1 -antagonist, an anticholinergic/antispasmodic agent, a vasopressin analogue, an antiarrhythmic agent, an antiparkinsonian agent, an antiangina/antihypertensive agent, an anticoagulant agent, an antiplatelet agent, a sedative, an anxiolytic agent, a peptidic agent, a biopolymeric agent, an antineoplastic agent, a laxative, an antidiarrheal agent, an antimicrobial agent, an antifungal agent, a vaccine, a protein, or a nucleic acid. In a further aspect, the pharmaceutically active agent can be coumarin, albumin, steroids such as betamethasone, dexamethasone, methylprednisolone, prednisolone, prednisone, triamcinolone, budesonide, hydrocortisone, and pharmaceutically acceptable hydrocortisone derivatives; xanthines such as theophylline and doxophylline; beta-2- agonist bronchodilators such as salbutamol, fenterol, clenbuterol, bambuterol, and salmeterol; antiinflammatory agents, including antiasthmatic anti-inflammatory agents, antiarthritis antiinflammatory agents, and non-steroidal anti-inflammatory agents, examples of which include but are not limited to sulfides, mesalamine, budesonide, salazopyrin, diclofenac, pharmaceutically acceptable diclofenac salts, nimesulide, naproxen, acetaminophen, ibuprofen, ketoprofen and piroxicam; analgesic agents such as salicylates; calcium channel blockers such as nifedipine, amlodipine, and nicardipine; angiotensin converting enzyme inhibitors such as captopril, benazepril hydrochloride, fosinopril sodium, trandolapril, ramipril, lisinopril, enalapril, quinapril hydrochloride, and moexipril hydrochloride; beta-blockers (i.e., beta adrenergic blocking agents) such as sotalol hydrochloride, timolol maleate, esmolol hydrochloride, carteolol, propanolol hydrochloride, betaxolol hydrochloride, penbutolol sulfate, metoprolol tartrate, metoprolol succinate, acebutolol hydrochloride, atenolol, pindolol, and bisoprolol fumarate; centrally active alpha-2-agonists such as clonidine; alpha- 1 -antagonists such as doxazosin and prazosin; anticholinergic/antispasmodic agents such as dicyclomine hydrochloride, scopolamine hydrobromide, glycopyrrolate, clidinium bromide, flavoxate, and oxybutynin; vasopressin analogues such as vasopressin and desmopressin; antiarrhythmic agents such as quinidine, lidocaine, tocainide hydrochloride, mexiletine hydrochloride, digoxin, verapamil hydrochloride, propafenone hydrochloride, flecainide acetate, procainamide hydrochloride, moricizine hydrochloride, and disopyramide phosphate; antiparkinsonian agents, such as dopamine, L-Dopa/Carbidopa, selegiline, dihydroergocryptine, pergolide, lisuride, apomorphine, and bromocriptine; antiangina agents and antihypertensive agents such as isosorbide mononitrate, isosorbide dinitrate, propranolol, atenolol and verapamil; anticoagulant and antiplatelet agents such as coumadin, warfarin, acetylsalicylic acid, and ticlopidine; sedatives such as benzodiazepines and barbiturates; anxiolytic agents such as lorazepam, bromazepam, and diazepam; peptidic and biopolymeric agents such as calcitonin, leuprolide and other LHRH agonists, hirudin, cyclosporin, insulin, somatostatin, protirelin, interferon, desmopressin, somatotropin, thymopentin, pidotimod, erythropoietin, interleukins, melatonin, granulocyte/macrophage-CSF, and heparin; antineoplastic agents such as etoposide, etoposide phosphate, cyclophosphamide, methotrexate, 5 -fluorouracil, vincristine, doxorubicin, cisplatin, hydroxyurea, leucovorin calcium, tamoxifen, flutamide, asparaginase, altretamine, mitotane, and procarbazine hydrochloride; laxatives such as senna concentrate, casanthranol, bisacodyl, and sodium picosulphate; antidiarrheal agents such as difenoxin hydrochloride, loperamide hydrochloride, furazolidone, diphenoxylate hydrochloride, and microorganisms; vaccines such as bacterial and viral vaccines; antimicrobial agents such as penicillins, cephalosporins, and macrolides, antifungal agents such as imidazolic and triazolic derivatives; and nucleic acids such as DNA sequences encoding for biological proteins, and antisense oligonucleotides.

[0136] Anti-cancer agents include alkylating agents, platinum agents, antimetabolites, topoisomerase inhibitors, antitumor antibiotics, antimitotic agents, aromatase inhibitors, thymidylate synthase inhibitors, DNA antagonists, farnesyltransferase inhibitors, pump inhibitors, histone acetyltransferase inhibitors, metalloproteinase inhibitors, ribonucleoside reductase inhibitors, TNF alpha agonists/antagonists, endothelin A receptor antagonists, retinoic acid receptor agonists, immunomodulators, hormonal and antihormonal agents, photodynamic agents, and tyrosine kinase inhibitors. [0137] Antibiotics include aminoglycosides (e.g., gentamicin, tobramycin, netilmicin, streptomycin, amikacin, neomycin), bacitracin, carbapenems (e.g., imipenem/cilastatin), cephalosporins, colistin, methenamine, monobactams (e.g., aztreonam), penicillins (e.g., penicillin G, penicillin V, methicillin, nafcillin, oxacillin, cloxacillin, dicloxacillin, ampicillin, amoxicillin, carbenicillin, ticarcillin, piperacillin, mezlocillin, azlocillin), polymyxin B, quinolones, and vancomycin; and bacteriostatic agents such as chloramphenicol, clindamycin, macrolides (e.g., erythromycin, azithromycin, clarithromycin), lincomycin, nitrofurantoin, sulfonamides, tetracyclines (e.g., tetracycline, doxycycline, minocycline, demeclocy cline), and trimethoprim. Also included are metronidazole, fluoroquinolones, and rifampin.

[0138] Enzyme inhibitors are substances which inhibit an enzymatic reaction. Examples of enzyme inhibitors include edrophonium chloride, N-methylphysostigmine, neostigmine bromide, physostigmine sulfate, tacrine, 1 -hydroxymaleate, iodotubercidin, p-bromotetranisole, 10-(alpha- diethylaminopropionyl)-phenothiazine hydrochloride, calmidazolium chloride, hemicholinium-3,3,5- dinitrocatechol, diacylglycerol kinase inhibitor I, diacylglycerol kinase inhibitor II, 3- phenylpropargylamine, N°-monomethyl-L-arginine acetate, carbidopa, 3-hydroxybenzylhydrazine, hydralazine, clorgyline, deprenyl, hydroxylamine, iproniazid phosphate, 6-MeO-tetrahydro-9H- pyrido-indole, nialamide, pargyline, quinacrine, semicarbazide, tranylcypromine, N,N- diethylaminoethyl-2,2-diphenylvalerate hydrochloride, 3-isobutyl-l-methylxanthine, papaverine, indomethacin, 2-cyclooctyl-2-hydroxyethylamine hydrochloride, 2,3-dichloro-a-methylbenzylamine (DCMB), 8,9-dichloro-2,3,4,5-tetrahydro-lH-2-benzazepine hydrochloride, p-aminoglutethimide, p- aminoglutethimide tartrate, 3-iodotyrosine, alpha-methyltyrosine, acetazolamide, dichlorphenamide, 6-hydroxy-2-benzothiazolesulfonamide, and allopurinol.

[0139] Antihistamines include pyrilamine, chlorpheniramine, and tetrahydrozoline, among others. [0140] Anti-inflammatory agents include corticosteroids, nonsteroidal anti-inflammatory drugs (e.g., aspirin, phenylbutazone, indomethacin, sulindac, tolmetin, ibuprofen, piroxicam, and fenamates), acetaminophen, phenacetin, gold salts, chloroquine, D-Penicillamine, methotrexate colchicine, allopurinol, probenecid, and sulfinpyrazone.

[0141] Muscle relaxants include mephenesin, methocarbamol, cyclobenzaprine hydrochloride, trihexyphenidyl hydrochloride, levodopa/carbidopa, and biperiden.

[0142] Anti-spasmodics include atropine, scopolamine, oxyphenonium, and papaverine.

[0143] Analgesics include aspirin, phenylbutazone, indomethacin, sulindac, tolmetin, ibuprofen, piroxicam, fenamates, acetaminophen, phenacetin, morphine sulfate, codeine sulfate, meperidine, nalorphine, opioids (e.g., codeine sulfate, fentanyl citrate, hydrocodone bitartrate, loperamide, morphine sulfate, noscapine, norcodeine, normorphine, thebaine, nor-binaltorphimine, buprenorphine, chlomaltrexamine, funaltrexamine, nalbuphine, nalorphine, naloxone, naloxonazine, naltrexone, and naltrindole), procaine, lidocaine, tetracaine and dibucaine. Ophthalmic agents include sodium fluorescein, rose bengal, methacholine, adrenaline, cocaine, atropine, alphachymotrypsin, hyaluronidase, betaxolol, pilocarpine, timolol, timolol salts, and combinations thereof. [0144] Prostaglandins are art recognized and are a class of naturally occurring chemically related long-chain hydroxy fatty acids that have a variety of biological effects.

[0145] Anti-depressants are substances capable of preventing or relieving depression.

[0146] Examples of anti-depressants include imipramine, amitriptyline, nortriptyline, protriptyline, desipramine, amoxapine, doxepin, maprotiline, tranylcypromine, phenelzine, and isocarboxazid. [0147] Trophic factors are factors whose continued presence improves the viability or longevity of a cell trophic factors include, without limitation, platelet-derived growth factor (PDGP), neutrophilactivating protein, monocyte chemoattractant protein, macrophage- inflammatory protein, platelet factor, platelet basic protein, and melanoma growth stimulating activity; epidermal growth factor, transforming growth factor (alpha), fibroblast growth factor, platelet- derived endothelial cell growth factor, insulin-like growth factor, glial derived growth neurotrophic factor, ciliary neurotrophic factor, nerve growth factor, bone growth/cartilage-inducing factor (alpha and beta), bone morphogenetic proteins, interleukins (e.g., interleukin inhibitors or interleukin receptors, including interleukin 1 through interleukin 10), interferons (e.g., interferon alpha, beta and gamma), hematopoietic factors, including erythropoietin, granulocyte colony stimulating factor, macrophage colony stimulating factor and granulocyte-macrophage colony stimulating factor; tumor necrosis factors, and transforming growth factors (beta), including beta-1, beta-2, beta-3, inhibin, and activin. [0148] Hormones include estrogens (e.g., estradiol, estrone, estriol, diethylstilbestrol, quinestrol, chlorotrianisene, ethinyl estradiol, mestranol), anti-estrogens (e.g., clomiphene, tamoxifen), progestins (e.g., medroxyprogesterone, norethindrone, hydroxyprogesterone, norgestrel), antiprogestin (mifepristone), androgens (e.g., testosterone cypionate, fluoxymesterone, danazol, testolactone), anti- androgens (e.g., cyproterone acetate, flutamide), thyroid hormones (e.g., triiodothyronne, thyroxine, propylthiouracil, methimazole, and iodixode), and pituitary hormones (e.g., corticotropin, somatotropin, oxytocin, and vasopressin). Hormones are commonly employed in hormone replacement therapy and/or for purposes of birth control. Steroid hormones, such as prednisone, are also used as immunosuppressants and anti-inflammatories. In some aspects, the additive is an agent that stimulates tissue formation, and/or healing and regrowth of natural tissues, and any combinations thereof. Agents that increase formation of new tissues and/or stimulates healing or regrowth of native tissue at the site of injection can include, but are not limited to, fibroblast growth factor (FGF), transforming growth factor- beta (TGF-beta, platelet-derived growth factor (PDGF), epidermal growth factors (EGFs), connective tissue activated peptides (CTAPs), osteogenic factors including bone morphogenic proteins, heparin, angiotensin II (A-II) and fragments thereof, insulin-like growth factors, tumor necrosis factors, interleukins, colony stimulating factors, erythropoietin, nerve growth factors, interferons, biologically active analogs, fragments, and derivatives of such growth factors, and any combinations thereof.

[0149] In some aspects, the silk composition can further comprise at least one additional material for soft tissue augmentation, e.g., dermal filler materials, including, but not limited to, poly(methyl methacrylate) microspheres, hydroxyapatite, poly(L-lactic acid), collagen, elastin, and glycosaminoglycans, hyaluronic acid, commercial dermal filler products such as BOTOX® (from Allergan), DYSPORT®, COSMODERM®, EVOLENCE®, RADIESSE®,RESTYLANE®, IUVEDERM® (from Allergan), SCULPTRA®, PERLANE®, and CAPTIQEIE®, and any combinations thereof.

[0150] In some aspects, the additive is a wound healing agent. As used herein, a “wound healing agent" is a compound or composition that actively promotes wound healing process. [0151] Exemplary wound healing agents include, but are not limited to dexpanthenol; growth factors; enzymes; hormones; povidon-iodide; fatty acids; anti-inflammatory agents; antibiotics; antimicrobials; antiseptics; cytokines; thrombin; analgesics; opioids; aminoxyls; furoxans; nitrosothiols; nitrates and anthocyanins; nucleosides, such as adenosine; and nucleotides, such as adenosine diphosphate (ADP) and adenosine triphosphate (ATP); neurotransmitter/neuromodulators, such as acetylcholine and 5 -hydroxy tryptamine (serotonin/5-HT); histamine and catecholamines, such as adrenalin and noradrenalin; lipid molecules, such as 5 -sphingosine- 1 -phosphate and lysophosphatidic acid; amino acids, such as arginine and lysine; peptides such as the bradykinins, substance P and calcium gene-related peptide (CGRP); nitric oxide; and any combinations thereof. [0152] In certain aspects, the active agents provided herein are immunogens. In one aspect, the immunogen is a vaccine. Most vaccines are sensitive to environmental conditions under which they are stored and/or transported. For example, freezing may increase reactogenicity (e.g., capability of causing an immunological reaction) and/or loss of potency for some vaccines (e.g., HepB, and DTaP/IPV/FQB), or cause hairline cracks in the container, leading to contamination. Further, some vaccines (e.g., BCG, Varicella, and MMR) are sensitive to heat. Many vaccines (e.g., BCG, MMR, Varicella, Meningococcal C Conjugate, and most DTaP-containing vaccines) are light sensitive. See, e.g., Galazka et al., Thermostability of vaccines, in Global Programme for Vaccines & Immunization (World Health Organization, Geneva, 1998); Peetermans et al., Stability of freeze-dried rubella virus vaccine (Cendehill strain) at various temperatures, J. Biological Standardization 179 (1973). Thus, the compositions and methods provided herein also provide for stabilization of vaccines regardless of the cold chain and/or other environmental conditions.

[0153] In some aspects, the additive is a cell, e.g., a biological cell. Cells useful for incorporation into the composition can come from any source, e.g., mammalian, insect, plant, etc. In some aspects, the cell can be a human, rat or mouse cell. In general, cells to be used with the compositions provided herein can be any types of cells. In general, the cells should be viable when encapsulated within compositions. In some aspects, cells that can be used with the composition include, but are not limited to, mammalian cells (e.g. human cells, primate cells, mammalian cells, rodent cells, etc.), avian cells, fish cells, insect cells, plant cells, fungal cells, spore cells, bacterial cells, and hybrid cells. In some aspects, exemplary cells that can be used with the compositions include platelets, activated platelets, stem cells, totipotent cells, pluripotent cells, and/or embryonic stem cells. In some aspects, exemplary cells that can be encapsulated within compositions include, but are not limited to, primary cells and/or cell lines from any tissue. For example, cardiomyocytes, myocytes, hepatocytes, keratinocytes, melanocytes, neurons, astrocytes, embryonic stem cells, adult stem cells, hematopoietic stem cells, hematopoietic cells (e.g. monocytes, neutrophils, macrophages, etc.), ameloblasts, fibroblasts, chondrocytes, osteoblasts, osteoclasts, neurons, sperm cells, egg cells, liver cells, epithelial cells from lung, epithelial cells from gut, epithelial cells from intestine, liver, epithelial cells from skin, etc., and/or hybrids thereof, can be included in the silk/platelet compositions disclosed herein. Those skilled in the art will recognize that the cells listed herein represent an exemplary, not comprehensive, list of cells. Cells can be obtained from donors (allogenic) or from recipients (autologous). Cells can be obtained, as a non-limiting example, by biopsy or other surgical means known to those skilled in the art.

[0154] In some aspects, the cell can be a genetically modified cell. A cell can be genetically modified to express and secrete a desired compound, e.g. a bioactive agent, a growth factor, differentiation factor, cytokines, and the like. Methods of genetically modifying cells for expressing and secreting compounds of interest are known in the art and easily adaptable by one of skill in the art.

[0155] Differentiated cells that have been reprogrammed into stem cells can also be used.

[0156] For example, human skin cells reprogrammed into embryonic stem cells by the transduction of Oct3/4, Sox2, c-Myc and Klf4 (Junying Yu, et. ah, Science, 2007, 318, 1917-1920 and Takahashi K. et. al, Cell, 2007, 131, 1-12).

[0157] Unless otherwise specified or indicated by context, the terms “a”, “an”, and “the” mean “one or more.” For example, “a molecule” should be interpreted to mean “one or more molecules”.

[0158] As used herein, “about”, “approximately”, “substantially”, and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” and “approximately” will mean plus or minus <10% of the particular term and “substantially” and “significantly” will mean plus or minus > 10% of the particular term.

[0159] As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.” The terms “comprise” and “comprising” should be interpreted as being “open” transitional terms that permit the inclusion of additional components further to those components recited in the claims. The terms “consist” and “consisting of’ should be interpreted as being “closed” transitional terms that do not permit the inclusion of additional components other than the components recited in the claims. The term “consisting essentially of’ should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter.

[0160] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0161] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0162] Preferred aspects of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred aspects may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect a person having ordinary skill in the art to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0163] While the invention has been illustrated and described in detail in the foregoing drawings and description, the same is to be considered as illustrative and not restrictive in character, it being understood that only illustrative embodiments thereof have been shown and described and that all changes and modifications that come within the spirit of the invention are desired to be protected. For example, any of the features or functions of any of the embodiments disclosed herein may be incorporated into any of the other embodiments disclosed herein.

[0164] The following examples illustrate some embodiments and aspects of the invention. It will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like can be performed without altering the spirit or scope of the invention, and such modifications and variations are encompassed within the scope of the invention as defined in the claims which follow. The following examples do not in any way limit the invention.

[0165] EXAMPLES

[0166] Example 1

[0167] A printhead and method as described herein was used to print fiber-reinforced 3D printed bio-composites as shown in Fig. 1. Fig. 1 shows a bottom view of a coextruded sample using the coextrusion method described herein. A silk cord was used as the continuous thread. Two perpendicular layers were printed to fabricate the article.

[0168] EQUIVALENTS AND SCOPE [0169] The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combinations (or subcombinations) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions 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 of the invention described herein.

[0170] In addition to the features described above and elsewhere herein, the present disclosure also includes the following clauses:

1. A printhead of a three-dimensional printer comprising: a co-extrusion nozzle configured to co-extrude a biopolymer-based cellular biomaterial with a continuous thread, the co-extrusion nozzle comprising: a first duct having a first diameter, the first duct comprising a fitting on a first end; a continuous thread duct having a continuous thread duct diameter, the continuous thread duct arranged at an incline with respect to the first duct, the continuous thread duct configured to receive and guide the continuous thread; and wherein a second end of the first duct is configured to receive the continuous thread exiting the continuous thread duct; and a co-flow tip arranged at the second end of the first duct and configured to receive a co-extruded material, the co-extruded material comprising the biopolymer-based cellular biomaterial and the continuous thread.

2. The printhead of clause 1 , wherein the first diameter is between 1 mm and 2 mm including at least 1.2 mm, at least 1.4 mm, at least 1.6 mm, or at least 1.8 mm.

3. The printhead of clause 1 , wherein the continuous thread duct diameter is between 0.1 mm and 1 mm, including at least 0.2 mm, at least 0.3 mm, at least 0.5 mm, at least 0.7 mm, or at least 0.8 mm.

4. The printhead of clause 1 , wherein the co-extrusion nozzle has an overall width of at least 18 mm at a widest point and an overall length of 25 mm at a longest point.

5. The printhead of clause 1 , wherein an angle of incline of the continuous thread duct with respect to the first duct is between 5° and 45°, including at least 10°, at least 20°, at least 35°, or at least 45°.

6. The printhead of clause 1, wherein the fitting is a Luer Lock screw-type fitting.

7. The printhead of clause 1, wherein the printhead is configured to store the biopolymer-based cellular biomaterial under a storage pressure.

8. The printhead of clause 7, wherein the storage pressure is greater than atmospheric pressure. 9. The printhead of clause 7, wherein the storage pressure is between 0.25 MPa and 25 MPa, including at least 0.5 MPa, at least 1 MPa, at least 5 MPa, at least 10 MPa, and at least 20 MPa.

10. The printhead of clause 1, further comprising at least one vessel for delivering the biopolymerbased cellular biomaterial to the co-extrusion nozzle, the at least one vessel configured to connect to the co-extrusion nozzle via the fitting.

11. The printhead of clause 10, wherein the at least one vessel comprises a syringe that is at least one of removable, refillable, or replaceable.

12. The printhead of clause 10, wherein the at least one vessel is configured to store a volume of the biopolymer-based cellular biomaterial under a storage pressure.

13. The printhead of clause 12, wherein the volume is between 100 mb and 400 mL, including at least 150 mL, at least 250 mL, or at least 300 mL.

14. The printhead of clause 1, wherein the printhead comprises a mechanical extrusion system.

15. The printhead of clause 14, wherein the mechanical extrusion system comprises a bipolar stepper motor with a gearbox and a rear axle configured to operate at a torque of between 1 N-m and 4 N-m, including at least 1.5 N-m, at least 2 N-m, or at least 3 N-m.

16. The printhead of clause 15, wherein the bipolar stepper motor enables both clockwise and counter-clockwise rotation of a belt and a threaded rod, guiding a piston and facilitating the biopolymer-based cellular biomaterial extrusion.

17. The printhead of clause 1, further comprising a ventilation system, the ventilation system positioned adjacent to the co-extrusion nozzle, wherein an airflow from the ventilation system is directed to the co-extruded material extruded from the co-extrusion nozzle.

18. The printhead of clause 17, wherein the ventilation system comprises at least one fan.

19. The printhead of clause 18, wherein the at least one fan is directed towards the co-extruded material exiting the co-extrusion nozzle to enable layer-by-layer drying of the co-extruded material.

20. The printhead of clause 18, wherein an airflow from the at least one fan is directed at an airflow angle with respect to a direction of co-extrusion.

21. The printhead of clause 20, wherein the airflow angle is between 35° and 65°, including at least 40°, at least 42°, at least 45°, or at least 47°.

22. The printhead of clause 18, wherein a fan speed of the at least one fan is between 3000 bpm and 4000 bpm, including at least 3100 bpm, at least 3200 bpm, at least 3500 bpm, or at least 3800 bpm.

23. The printhead of clause 17, wherein the ventilation system is configured to deliver the airflow at a predetermined fluid velocity. 24. The printhead of clause 23, wherein the predetermined fluid velocity is between 4 m/s and 10 m/s, including at least 5 m/s, at least 7 m/s, or at least 9 m/s.

25. The printhead of clause 17, wherein the ventilation system is configured to deliver an airflow at a variable fluid velocity.

26. The printhead of clause 25, wherein the variable fluid velocity varies with at least one of a change in distance from the ventilation system to a surface of the co-extruded material, an extrusion rate from the co-extrusion nozzle, a layer height, a length of path, an extrusion width, or a composition of the biopolymer-based cellular biomaterial.

27. The printhead of any one of clauses 17 to the immediately preceding clause, wherein the ventilation system is arranged at a distance of between 60 mm and 80 mm from a tip of the co-extrusion nozzle, including at least 65 mm, at least 70 mm, or at least 75 mm.

28. The printhead of any one of clauses 17 to the immediately preceding clause, wherein the ventilation system is arranged at a distance of between 60 mm and 100 mm from the co-extruded material, including at least 65 mm, at least 80 mm, or at least 95 mm.

29. The printhead of clause 1, wherein the printhead extrudes the co-extruded material in accordance with an extrusion rate defined by Q= 0) x h x v (where 0) = extrusion width, h = layer height, v = extrusion velocity).

30. The printhead of clause 29, wherein the extrusion rate is between 125 mm/min and 350 mm/min, including at least 265 mm/min, at least 290 mm/min, at least 310 mm/min, and at least 330 mm/min, or at most 310 mm/min, at most 250 mm/min, at most 200 mm/min, or at most 150 mm/min.

31 . The printhead of clause 29, wherein the extrusion rate is further defined by a rate of volume change of the co-extruded material.

32. The printhead of clause 29, wherein the extrusion rate varies by up to 45% of an original value of the extrusion rate, including up to 75%, up to 65%, up to 60%, up to 55%, up to 48%, or up to 44%.

33. The printhead of clause 1, wherein a feed rate of the continuous thread is equivalent to a rate of co-extrusion.

34. The printhead of clause 1, wherein the co-extruded material has a firmness of between 100 N/m² and 400 N/m², including at least 250 N/m², and a density of between 0.08 g/cm³ and 0.25 g/cm³, including at least 0.12 g/cm³.

35. The printhead of clause 1, wherein the continuous thread duct is further configured to extrude the continuous thread via propulsion.

36. The printhead of clause 1, further comprising a sensor system to detect an undesired behavior of the continuous thread (e.g., poor positioning of the continuous thread or the thread moving out of a proper trajectory). 37. The printhead of the immediately preceding clause, wherein detection of the undesired behavior causes a sensor of the sensor system to at least one of pause printing or correct the undesired behavior.

38. The printhead of any one of the two immediately preceding clauses, wherein the sensor system includes at least one of an infrared sensor or an optical sensor.

39. A method for generating a co-extruded biopolymer-based cellular biomaterial and continuous thread, comprising: flowing a biopolymer-based cellular biomaterial under pressure through a first duct of a coextrusion nozzle of a printhead of a three-dimensional printer, the biopolymer-based cellular biomaterial comprising silk fibroin in an amount by weight of between 1% and 40%, a surfactant (e.g., glycerol monostearate) in an amount by weight of between 1 % and 10%, and plasticizer in an amount by weight of between 20% and 75%; guiding a continuous thread within a continuous thread duct of the co-extrusion nozzle; and co-extruding the biopolymer-based cellular biomaterial and the continuous thread through a co-flow tip of the co-extrusion nozzle of the printhead to form a co-extruded material.

40. The method of clause 39, wherein the co-extruding is in accordance with an extrusion rate defined by Q= co x h x v (where co = extrusion width, h = layer height, v = extrusion velocity).

41. The method of clause 39, further comprising drying the co-extruded material exiting the co- extrusion nozzle.

42. The method of the immediately preceding clause, wherein drying comprises directing an airflow from a ventilation system adjacent to the co-extrusion nozzle towards the co-extruded material exiting the extrusion nozzle.

43. The method of clause 39, wherein the biopolymer-based cellular biomaterial further comprises 10% sodium alginate and 10% xanthan gum.

44. The method of clause 39, wherein the plasticizer is glycerol.

45. A method of making a biopolymer-based cellular biomaterial with a continuous thread, the method comprising: whipping a liquid composition for a predetermined whipping time at a predetermined speed to form a biopolymer-based cellular biomaterial, the liquid composition comprising silk fibroin, at least one polysaccharide, at least one surfactant (e.g., glycerol monostearate), and at least one plasticizer; pressurizing a volume of the biopolymer-based cellular biomaterial; and concurrently co-extruding the biopolymer-based cellular biomaterial with a continuous thread thereby forming a co-extruded material, and drying the co-extruded material.

46. The method of clause 45, wherein the predetermined whipping time is at least 8.5 minutes. 47. The method of clause 45, wherein the predetermined speed is at least 185 rpm.

48. The method of clause 45, wherein the predetermined whipping time is between 5 minutes and 30 minutes, including but not limited to, at least 5 minutes, at least 6 minutes, at least 7 minutes, at least 8 minutes, at least 9 minutes, or at least 10 minutes and at most 30 minutes, at most 25 minutes, at most 20 minutes, at most 15 minutes, or at most 10 minutes.

49. The method of clause 45, wherein the predetermined speed is between 160 rpm and 200 rpm, including but not limited to, at least 160 rpm, at least 170 rpm, at least 175 rpm, or at least 185 rpm, and at most 200 rpm, at most 190 rpm, or at most 180 rpm.

50. The method of clause 45, wherein the at least one plasticizer is selected from the group consisting of a di(ethylene glycol), a tri(ethylene glycol), a di(propylene glycol), a tri(propylene glycol), triethanolamine, a vegetable oil, and combinations thereof.

51. The method of clause 45, wherein the at least one polysaccharide is selected from the group consisting of xanthan gum, an alginate, a high molecular weight sugar, a cellulose derivative, or a combination thereof.

52. The method of clause 45, wherein the liquid composition comprises silk fibroin, glycerol, sodium alginate, and xanthan gum.

53. The printhead or method of any one of the preceding clauses wherein the co-extruded material possesses at least one characteristic that is superior to a corresponding characteristic of an extruded material lacking the continuous thread.

54. The printhead or method of clause 53, wherein the characteristic is a resistance to stretching, and the co-extruded material has a resistance to stretching that is at least 30%, at least 50%, at least 75%, at least 100%, or at least 150% higher than an extruded biopolymer-based material lacking the continuous thread.

55. The printhead or method of clause 53, wherein the characteristic is a resistance to shear stress, and the co-extruded material has a resistance to shear stress that is at least 30%, at least 50%, at least 75%, at least 100%, or at least 150% higher than an extruded biopolymer-based material lacking the continuous thread.

56. The printhead or method of any one of the preceding clauses, wherein the co-extruded material has an irregular porosity.

57. The printhead or method of any of one of clauses 1 to 55, wherein the co-extruded material has a regular porosity.

58. The printhead or method of any one of the preceding clauses, wherein the co-extruded material has a firmness of at least 100 N/m², at least 200 N/m², at least 250 N/m², at least 300 N/m², or at least 400 N/m². 59. The printhead or method of any one of the preceding clauses, wherein the co-extruded material has a density of at least 0.08 g/cm³, at least 0. 1 g/cm³, at least 0.12 g/cm³, at least 0.2 g/cm³, or at least 0.25 g/cm³.

60. The printhead or method of any one of the preceding clauses, wherein the co-extruded material further comprises at least one of a sensing agent, a therapeutically active agent, a colorant, or an aromaproviding compound.

61. A method for generating a biopolymer-based cellular biomaterial and a suspended continuous thread, the method comprising: depositing at least one continuous thread from a printhead into a biopolymer-based cellular biomaterial foam bath to form an embedded filament article, the biopolymer-based cellular biomaterial comprising silk fibroin in an amount between 1% and 40%, glycerol monostearate in an amount between 1% and 10%, and plasticizer in an amount between 20% and 75%.

62. The method of the immediately preceding clause, wherein the printhead comprises a continuous thread feeding mechanism.

63. The method of any one of the two preceding clauses, wherein the process further comprises baking the embedded filament article to produce a baked embedded filament article.

64. The method of any one of clauses 61 to the immediately preceding clause, wherein the biopolymer-based cellular biomaterial foam bath comprises at least one layer of biopolymer-based cellular biomaterial foam.

65. The method of any one of clauses 61 to the immediately preceding clause, wherein the method further comprises compressing the embedded filament article or the baked embedded filament article to produce a compressed embedded filament article or a compressed baked embedded filament article.

66. The method of any one of clauses 61 to the immediately preceding clause, wherein the method further comprises baking the compressed embedded filament article or further baking the compressed baked filament article to form a baked compressed embedded filament article or a further baked compressed baked filament article.

67. The method of any one of clauses 61 to the immediately preceding clause, wherein the printhead further comprises a feeding pipe with a proximal end contacting the printhead and a distal end contacting the biopolymer-based cellular biomaterial foam bath.

68. The method of any one of clauses 61 to the immediately preceding clause, wherein the continuous thread is deposited in one dimension, two dimensions, or three dimensions.

69. The method of any one of clauses 61 to the immediately preceding clause, wherein the continuous thread is deposited in at least one layer, at least two layers, at least three layers, or more. 70. The method of any one of clauses 61 to the immediately preceding clause, wherein the at least one continuous thread has at least one property of an electrical conductance, a magnetism, at least one sensor embedded therein, a thermal conductance, a fluorescence, a color, or a combination thereof.

71. The method of the immediately preceding clause, wherein the at least one property is constant across a length of the at least one continuous thread.

72. The method of clause 70, wherein the at least one property varies across a length of the at least one continuous thread.

73. The method of any one of clauses 70 to the immediately preceding clause, wherein the continuous thread has at least one thread property, thereby providing at least one article property of the embedded filament article, the baked embedded filament article, or the compressed baked embedded filament article.

74. The method of any one of clauses 61 to the immediately preceding clause, wherein the printhead comprises at least two continuous threads to be deposited, at least three continuous threads to be deposited, or more.

75. The method of the immediately preceding clause, wherein the at least two continuous threads to be deposited are deposited simultaneously.

76. The method of any one of the two preceding clauses, wherein the at least two continuous threads have identical compositions.

77. The method of any one of clause 74 or 75, wherein the at least two continuous threads have different compositions.

78. The method of any one of clauses 61 to the immediately preceding clause, wherein the biopolymer-based cellular biomaterial foam bath has an area of at least 1 cm², including but not limited to, at least 5 cm², at least 10 cm², at least 100 cm², at least 1 m², or greater.

79. The method of any one of clauses 61 to the immediately preceding clause, wherein a size or volume of the embedded filament article, the baked embedded filament article, the compressed baked embedded filament article, the baked compressed embedded filament article, or the further baked compressed baked filament article is at least 75% of the size or volume of the biopolymer-based cellular biomaterial foam bath.

80. The method of any one of clauses 61 to the immediately preceding clause, wherein a shape of the embedded filament article, the baked embedded filament article, the compressed baked embedded filament article, the baked compressed embedded filament article, or the further baked compressed baked filament article is the same shape as a shape of the biopolymer-based cellular biomaterial foam bath. 81. The method of any one of clauses 61 to the immediately preceding clause, wherein the embedded filament article, the baked embedded filament article, the compressed baked embedded filament article, the baked compressed embedded filament article, or the further baked compressed baked filament article has an irregular porosity.

82. The method of any one of clauses 61 to the immediately preceding clause, wherein the embedded filament article, the baked embedded filament article, the compressed baked embedded filament article, the baked compressed embedded filament article, or the further baked compressed baked filament article has a regular porosity.

83. The method of any one of clauses 61 to the immediately preceding clause, wherein the embedded filament article, the baked embedded filament article, the compressed baked embedded filament article, the baked compressed embedded filament article, or the further baked compressed baked filament article has a firmness of at least 100 N/m², at least 200 N/m², at least 250 N/m², at least 300 N/m², or at least 400 N/m².

84. The method of any one of clauses 61 to the immediately preceding clause, wherein the embedded filament article, the baked embedded filament article, the compressed baked embedded filament article, the baked compressed embedded filament article, or the further baked compressed baked filament article has a density of at least 0.08 g/cm³, at least 0.1 g/cm³, at least 0.12 g/cm³, at least 0.2 g/cm³, or at least 0.25 g/cm³.

85. The method of any one of clauses 61 to the immediately preceding clause, wherein the embedded filament article, the baked embedded filament article, the compressed baked embedded filament article, the baked compressed embedded filament article, or the further baked compressed baked filament article further comprises at least one of a sensing agent, a therapeutically active agent, a colorant, or an aroma-providing compound.

86. The printhead or method of any one of the preceding clauses, wherein the continuous thread or at least one continuous thread is at least one of a silk yam, a waste silk product, a filament, an electrode, a counter-electrode, an enzymatically-coated fiber, a non-spun thread, an electrochemical fiber, a conductive fiber, a copper material, a platinum material, a metallic material, a polyester material, a cotton material, a graphite fiber, or a nylon material.

87. The printhead or method of any one of the preceding clauses, wherein a diameter of the continuous thread or at least one continuous thread is between 35 pm and 80 pm, including but not limited to, at least 35 pm, at least 60 pm, or at least 70 pm, and at most 80 pm, at most 65 pm, or at most 45 pm. 88. The printhead or method of any one of the preceding clauses, wherein a diameter of the continuous thread is between 0. 1 mm and 1 mm including at least 0.2 mm, at least 0.3 mm, at least 0.5 mm, at least 0.7 mm, or at least 0.8 mm.

89. The printhead or method of any one of the preceding clauses wherein the biopolymer-based cellular biomaterial comprises silk fibroin.

90. The printhead or method of any one of the preceding clauses, wherein the biopolymer-based cellular biomaterial comprises silk fibroin, at least one polysaccharide, at least one plasticizer, and a surfactant.

91. The printhead or method of clause 90, wherein the at least one polysaccharide is xanthan gum, an alginate, a high molecular weight sugar, a cellulose derivative, or a combination thereof.

92. The printhead or method of the immediately preceding clause, wherein the at least one polysaccharide is the alginate, wherein the alginate is alginic acid sodium salt.

93. The printhead or method of any one of clauses 90 to the immediately preceding clause, wherein the at least one plasticizer is selected from the group consisting of a glycerol, a di(ethylene glycol), a tri(ethylene glycol), a di(propylene glycol), a tri(propylene glycol), triethanolamine, a vegetable oil, and combinations thereof.

94. The printhead or method of any one of clauses 90 to the immediately preceding clause, wherein the at least one plasticizer comprises at least one -OH substituent.

95. The printhead or method of any one of clauses 90 to the immediately preceding clause, wherein the at least one plasticizer is present in the biopolymer-based cellular biomaterial in an amount by weight of between 20.0% and 75.0%, including but not limited to at least 30%, at least 40%, at least 50%, or at least 60%, or at most 70%, at most 65%, at most 55%, or at most 45%.

96. The printhead or method of any one of the preceding clauses, wherein the biopolymer-based cellular biomaterial comprises between 15% and 25% silk fibroin, between 3% and 7% glycerol monostearate, between 50% and 65% glycerol, between 5% and 15% sodium alginate, and between 5% and 15% xanthan gum.

97. The printhead or method of any one of the preceding clauses, wherein the silk fibroin is present in the biopolymer-based cellular biomaterial in an amount by weight of between 1 % and 30% , between 15% and 25%, or between 3% and 7%, including but not limited to, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, or at least 20% and at most 30%, at most 25%, at most 22%, at most 20%, at most 15%, at most 10%, at most 7%, or at most 5%.

98. The printhead or method of any one of clauses 90 to the immediately preceding clause, wherein the at least one polysaccharide is present in the biopolymer-based cellular biomaterial in an amount by weight of between 0.1% and 30%, including but not limited to, at least 0.1%, at least 0.5%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, or at least 12% and at most 30%, at most 25%, at most 23%, at most 20%, at most 17%, at most 15%, at most 12%, at most 10%, or at most 8%.

99. The printhead or method of any one of clauses 90 to the immediately preceding clause, wherein the silk fibroin and the at least one polysaccharide are present in the biopolymer-based cellular biomaterial in a weight ratio of between 1 : 4 and 20 : 1 or between 1 : 2 and 10:1, including but not limited to, at least 1:4, at least 1:3, or at least 1:2, and at most 20:1, at most 19:1, at most 18:1, at most 16:1, at most 15:1, at most 14:1, at most 12:1 , at most 11 :1 , or at most 10:1.

100. The printhead or method of any one of clauses 90 to the immediately preceding clause, wherein the surfactant is present in an amount by weight of between 1% and 10%, including but not limited to, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, or at least 6% and at most 10%, at most 9%, at most 8%, at most 7%, at most 6%, at most 5%, or at most 4%.

101. The printhead or method of any one of clauses 90 to the immediately preceding clause, wherein the surfactant is a monoglyceride.

102. The printhead or method of any one of clauses 90 to the immediately preceding clause, wherein the surfactant is glycerol monostearate.

103. The printhead or method of any one of the preceding clauses, wherein the biopolymer-based cellular biomaterial and/or the surfactant is free of anionic surfactant, free of sodium sulfate surfactant, and/or free of sodium dodecyl sulfate.

104. The printhead or method of any one of the preceding clauses, wherein the biopolymer-based cellular biomaterial and continuous thread are co-extruded in a single path.

105. An article, comprising, consisting essentially of, or consisting of the co-extruded biopolymerbased cellular biomaterial or the biopolymer-based cellular biomaterial and continuous thread of or made by the method of any one of clauses 45 to 103.

106. The article of clause 105, wherein the article is at least one of a textile, a 3D structure, a structural material, a building material, an insulating material, a packaging material, a tool, a biomedical implant, a prosthetic, or an orthotic.

107. The article of clause 105 wherein at least one post-processing technique is employed on the article.

108. The article of clause 107, wherein the post-processing technique is at least one of curing, compressing, molding, or aesthetically modifying.

109. The article of clause 105, wherein a diameter of the biopolymer-based cellular biomaterial is greater than a diameter of the continuous thread in the co-extruded material by between 5 times to 100 times, including at least 10 times, at least 30 times, or at least 50 times. 110. An article, comprising, consisting essentially of, or consisting of the co-extruded material of or made by the method of any one of clauses 39 to 44 or 53 to 103.

111. The article of clause 110, wherein the article is at least one of a textile, a 3D structure, a structural material, a building material, an insulating material, a packaging material, a tool, a biomedical implant, a prosthetic, or an orthotic.

112. The article of clause 110, further comprising, employing at least one post-processing technique on the article.

113. The article of clause 112, wherein the post-processing technique is at least one of curing, compressing, molding, or aesthetically modifying.

114. The article of clause 110, wherein a diameter of the biopolymer-based cellular biomaterial is greater than a diameter of the continuous thread in the co-extruded material by between 5 times to 100 times, including at least 10 times, at least 30 times, or at least 50 times.

[0171] The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the following claims:

Claims

CLAIMS What is claimed is:

1. A printhead of a three-dimensional printer comprising: a co-extrusion nozzle configured to co-extrude a biopolymer-based cellular biomaterial with a continuous thread, the co-extrusion nozzle comprising: a first duct having a first diameter, the first duct comprising a fitting on a first end; a continuous thread duct having a continuous thread duct diameter, the continuous thread duct arranged at an incline with respect to the first duct, the continuous thread duct configured to receive and guide the continuous thread; and wherein a second end of the first duct is configured to receive the continuous thread exiting the continuous thread duct; and a co-flow tip arranged at the second end of the first duct and configured to receive a coextruded material, the co-extruded material comprising the biopolymer- based cellular biomaterial and the continuous thread.

2. The printhead of claim 1 , wherein the biopolymer-based cellular biomaterial comprises silk fibroin, at least one polysaccharide, at least one plasticizer, and a surfactant.

3. The printhead of claim 2, wherein the at least one plasticizer is present in the biopolymerbased cellular biomaterial in an amount by weight of between 20.0% and 75.0%, including but not limited to at least 30%, at least 40%, at least 50%, or at least 60%, or at most 70%, at most 65%, at most 55%, or at most 45%.

4. The printhead of claim 2, wherein the silk fibroin is present in the biopolymer-based cellular biomaterial in an amount by weight of between 1% and 30%, between 15% and 25%, or between 3% and 7%, including but not limited to, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, or at least 20% and at most 30%, at most 25%, at most 22%, at most 20%, at most 15%, at most 10%, at most 7%, or at most 5%.

5. The printhead of claim 2, wherein the at least one polysaccharide is present in the biopolymer-based cellular biomaterial in an amount by weight of between 0.1 % and 30%, including but not limited to, at least 0.1%, at least 0.5%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, or at least 12% and at most 30%, at most 25%, at most 23%, at most 20%, at most 17%, at most 15%, at most 12%, at most 10%, or at most 8%.

6. The printhead of claim 2, wherein the surfactant is present in an amount by weight of between 1% and 10%, including but not limited to, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, or at least 6% and at most 10%, at most 9%, at most 8%, at most 7%, at most 6%, at most 5%, or at most 4%.

7. The printhead of claim 6, wherein the surfactant is a monoglyceride.

8. The printhead of claim 7, wherein the surfactant is glycerol monostearate.

9. The printhead of claim 1 , wherein the biopolymer-based cellular biomaterial and/or the surfactant is free of anionic surfactant, free of sodium sulfate surfactant, and/or free of sodium dodecyl sulfate.

10. The printhead of claim 1 , wherein the first diameter is between 1 mm and 2 mm including at least 1.2 mm, at least 1.4 mm, at least 1.6 mm, or at least 1.8 mm, wherein the continuous thread duct diameter is between 0.1 mm and 1 mm, including at least 0.2 mm, at least 0.3 mm, at least 0.5 mm, at least 0.7 mm, or at least 0.8 mm, and/or wherein the co-extrusion nozzle has an overall width of at least 18 mm at a widest point and an overall length of 25 mm at a longest point.

11. The printhead of claim 1 , wherein the printhead is configured to store the biopolymer-based cellular biomaterial under a storage pressure, wherein the storage pressure is greater than atmospheric pressure.

12. The printhead of claim 1 , wherein the printhead comprises a mechanical extrusion system.

13. The printhead of claim 1 , further comprising a ventilation system, the ventilation system positioned adjacent to the co-extrusion nozzle, wherein an airflow from the ventilation system is directed to the co-extruded material extruded from the co-extrusion nozzle.

14. The printhead of claim 13, wherein the ventilation system is configured to deliver the airflow at a predetermined fluid velocity.

15. The printhead of claim 13, wherein the ventilation system is arranged at a distance of between 60 mm and 80 mm from a tip of the co-extrusion nozzle, including at least 65 mm, at least 70 mm, or at least 75 mm.

16. The printhead of claim 1 , wherein the printhead extrudes the co-extruded material in accordance with an extrusion rate defined by Q= m x h x v, where m = extrusion width, h = layer height, and v = extrusion velocity.

17. The printhead of claim 1 , further comprising a sensor system to detect an undesired behavior of the continuous thread (e.g., poor positioning of the continuous thread or the thread moving out of a proper trajectory), wherein the sensor system includes an infrared sensor or an optical sensor.

18. A method for generating a biopolymer-based cellular biomaterial and a suspended continuous thread, the method comprising: depositing at least one continuous thread from a printhead into a biopolymer-based cellular biomaterial foam bath to form an embedded filament article, the biopolymer-based cellular biomaterial comprising silk fibroin in an amount between 1% and 40%, glycerol monostearate in an amount between 1% and 10%, and plasticizer in an amount between 20% and 75%.

19. A method for generating a co-extruded biopolymer-based cellular biomaterial and continuous thread, comprising: flowing a biopolymer-based cellular biomaterial under pressure through a first duct of a coextrusion nozzle of a printhead of a three-dimensional printer, the biopolymer-based cellular biomaterial comprising silk fibroin in an amount by weight of between 1% and 40%, a surfactant (e.g., glycerol monostearate) in an amount by weight of between 1% and 10%, and plasticizer in an amount by weight of between 20% and 75%; guiding a continuous thread within a continuous thread duct of the co-extrusion nozzle; and co-extruding the biopolymer-based cellular biomaterial and the continuous thread through a co-flow tip of the co-extrusion nozzle of the printhead to form a co-extruded material.

20. A method of making a biopolymer-based cellular biomaterial with a continuous thread, the method comprising: whipping a liquid composition for a predetermined whipping time at a predetermined speed to form a biopolymer-based cellular biomaterial, the liquid composition comprising silk fibroin, at least one polysaccharide, at least one surfactant (e.g., glycerol monostearate), and at least one plasticizer; pressurizing a volume of the biopolymer-based cellular biomaterial; and concurrently co-extruding the biopolymer-based cellular biomaterial with a continuous thread thereby forming a co-extruded material, and drying the co-extruded material.