Centrifugal molding articles and methods

Abstract

Centrifugal controlled biosolidification molding is disclosed. The method involves the use of aqueous solutions and centrifugal force in a unique processing method for producing highly uniform and useful articles from biopolymers previously formed by methods having significant drawbacks. In some cases, this technique can use a single biopolymer to produce highly uniform articles. In other cases, this technique can use different polymers to provide customizable and tunable properties, such as desired solubility. Articles produced by these methods eliminate significant disadvantages from current techniques, exhibiting the absence of external and internal lamination, as well as a uniform bulk material.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 511,572, filed on June 30, 2023, and is incorporated by reference in its entirety for all purposes.

[0002] Description of Government Support This invention was made with government support under Grant No. P41EB027062 awarded by the National Institutes of Health. The U.S. government has certain rights in this invention.

[0003] Sequence Listing The sequence listing is submitted with this application as an XML file of a sequence listing named "T002694 WO - 2095.0612.xml". This XML file is 32 kilobytes in size and was created on June 23, 2024. The sequence listing is submitted electronically with this application and is incorporated herein by reference in its entirety.

Background Art

[0004] Background Conventional silk - processing techniques can produce solid articles, but the current set of techniques has drawbacks that result in articles having one or more undesirable characteristics.

[0005] More generally, biopolymer and polymer - processing techniques currently lack a method for making biopolymer articles with material robustness closer to that of more conventional polymers.

[0006] Typical methods are limited by the diffusion of polymerization agents into the article. When the article reaches a certain size, non - uniform crystallization occurs due to diffusion.

[0007] One existing technique for making silk articles is the thermoforming and / or compression molding of silk powder. This technique has various drawbacks, including the lack of elastic and non - brittle mechanical characteristics.

[0008] Another existing technology is injection molding customized for silk fibroin. Unlike the conventional "melt and injection" process, the water-based injection molding process for silk fibroin traditionally produces materials with a high degree of internal voids that are not sufficiently uniform.

[0009] Another existing technology is three-dimensional printing. 3D printing using silk and biopolymers produces articles that have a layered nature and contain significant internal voids. In most cases, biopolymers are 3D printed using cylindrical extrusion of the biopolymer, and therefore there are large gaps between the cylindrical extrusions of the material.

[0010] New methods and products are needed to address one or more of the aforementioned shortcomings. [Overview of the Initiative] [Means for solving the problem]

[0011] overview A novel approach to biopolymer molding is disclosed herein, enabling the creation of high-resolution, complex geometric shapes and centimeter-scale objects using biopolymer solutions. This method is based on the alternating deposition of material into a mold, centrifugation to conform to the shape, and in-situ polymerization of the solution. This process is repeated multiple times until the mold is full, at which point the construct can be demolded and polymerization can be terminated.

[0012] In one embodiment, the present disclosure provides a centrifugal solidification-controlled biopolymer article.

[0013] In another embodiment, the present disclosure provides a centrifugal controlled biosolidification molding method. The method includes the steps of: A) introducing a first biopolymer aqueous solution into a mold and, if necessary, allowing the first biopolymer aqueous solution to settle in the mold; B) introducing a first curing aqueous solution onto the first biopolymer aqueous solution in the mold and, if necessary, allowing the first curing aqueous solution to settle in the mold, wherein the mold has a first orientation with respect to gravity during step B); and C) producing a first biosolid by subjecting the mold and its contents to a first centrifuge and stopping after a first centrifugation period, wherein the mold is within 45° of the first orientation with respect to the first centrifugation during step C). A first biopolymer article is produced from the first biosolid by waiting for a first final biosolidification period after step C).

[0014] In yet another embodiment, the Disclosure provides a method for producing a biopolymer article. The method comprises at least two iterative cycles of the following steps: X) repeatedly introducing a biopolymer aqueous solution into a mold and, if necessary, allowing the repeated biopolymer aqueous solution to settle in the mold; Y) repeatedly introducing a curing aqueous solution onto the repeated biopolymer aqueous solution in the mold and, if necessary, allowing the repeated curing aqueous solution to settle in the mold; and Z) repeatedly centrifugating the mold and its contents, stopping after the repeated centrifugation period, thereby repeatedly adding to the existing portion of the solidified body, wherein the repeated introduction of step X), the introduction of step Y), and the centrifugation of step Z) continues until a final biosolid is formed. The biopolymer article is produced by waiting for a final biosolidification period after the formation of the final biosolid.

[0015] In yet another aspect, the disclosure provides a method comprising the step of forming a first biosolidified body by centrifuging a first initial solidification aqueous solution of a biopolymer in a mold to a first late solidification stage.

[0016] Non-limiting embodiments of the present invention will be described by reference to the accompanying drawings, which are schematic diagrams and not intended to be drawn to an exact scale. In these drawings, each of the identical or nearly identical components shown is usually represented by a single number. For brevity, not all components are numbered in all drawings, and not all components of each embodiment of the invention are shown where it is not necessary to illustrate them for those skilled in the art to understand the invention. [Brief explanation of the drawing]

[0017] [Figure 1] Figure 1 is a schematic diagram of various embodiments of the centrifugal method disclosed herein.

[0018] [Figure 2] Figure 2 is a schematic diagram of one embodiment of loading a solution according to an aspect of this disclosure.

[0019] [Figure 3] Figure 3 is a pair of images showing tubular articles prepared by the disclosed method.

[0020] [Figure 4] Figure 4 illustrates the process for aqueous solvent-dependent centrifugal molding for the fabrication and enhancement of biopolymers. [Modes for carrying out the invention]

[0021] Detailed explanation Before the invention is described in further detail, it should be understood that the invention is not limited to the specific embodiments described herein. It should also be understood that the terminology used herein is intended to describe, and not to limit, specific embodiments. The scope of the invention is limited only by the claims. As used herein, the singular forms "a," "an," and "the" include multiple embodiments unless otherwise specified by the context.

[0022] Specific structures, devices, and methods related to forming surface patterns are disclosed. It should be understood by those skilled in the art that many additional changes are possible without departing from the concepts of the present invention in addition to what has already been described. When interpreting this disclosure, all terms should be interpreted as broadly as possible in context. Variations of the term "comprising" should be interpreted as non-exclusively referring to elements, components, or steps, so that the referenced elements, components, or steps may be combined with other elements, components, or steps not explicitly recited. Embodiments that are said to "comprise" certain elements may also be considered to "consist essentially of" and "consist of" those elements. If two or more ranges are stated for a particular value, this disclosure contemplates all combinations of upper and lower bounds of ranges not explicitly stated. For example, a description of values between 1 and 10 or between 2 and 9 also contemplates values between 1 and 9 or between 2 and 10.

[0023] As used herein, "film" refers to a laminated structure having size dimensions where two spatial dimensions are and the third spatial dimension is not limited, having a thickness between monolayer and 2 mm or less than 2 mm, including but not limited to a thickness of less than 1.5 mm, less than 1 mm, less than 0.5 mm, or less than 0.1 mm.

[0024] As used herein, "no cylindrical extrusion gap" refers to the absence of observable gaps within an article, which is specific to a three-dimensional printing process that utilizes extruded cylindrical polymers.

[0025] As used herein, “non-layered” refers to the absence of an observable layered structure within an article. Layers can be observed by imaging (e.g., scanning electron microscopy), spectroscopy (e.g., FTIR or fluorescence spectroscopy), or other analytical methods (e.g., differential scanning calorimetry). Examples of techniques that exhibit layering in the resulting products (i.e., these do not produce, to the best of our understanding, non-layered products) include immersion coating (where separate coating layers can be clearly identified) or three-dimensional printing (where the printing process is typically an identifiable layering process).

[0026] As used herein, “externally unlaminated” refers to an article that has no laminations visible from the outside.

[0027] As used herein, “absent internal lamination” refers to an article in which no lamination is observed on the inside of the article. In most cases, the absence of internal lamination can be observed by preparing a cross-sectional sample of the article and evaluating the newly exposed surface for evidence of lamination. For example, an article may have a cross-sectional profile that is uniform throughout the article. A uniform cross-sectional profile can be determined by at least one of the following: visual inspection, scanning electron microscopy, fluorescence microscopy, DSC, TEM, XRD, EPMA, or FTIR. The cross-sectional profile may relate to at least one of the following: density, absence of voids, consistent pattern, or chemical profile.

[0028] As used herein, “void-free” refers to an article that lacks internal voids having a maximum physical dimension between 50 μm and 1 mm or between 50 μm and 200 μm. It should be understood that, using certain porogenic approaches, it is possible to produce articles having pores intentionally placed within a solid structure that itself is void-free. In this example, a void-free article may be configured to have pores that fall within the size definition herein, but these pores are clearly distinguishable from voids unintentionally formed within the article and will be easily identifiable to those skilled in the art.

[0029] As used herein, “optically opaque” refers to a material having a visible light attenuation of 50% or more, including but not limited to 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more, for a given visible wavelength (or range of wavelengths).

[0030] As used herein, “random sampling” refers to any random or pseudo-random selection of a sampling site from an article. Techniques may be as close to true random as possible, while others may be pseudo-random. The randomness of sampling may be randomized per article, or randomized once for an entire series of articles. As a specific example, a script can be written for imaging software to randomly select sampling sites using a grid array and a random number generator. In some cases, random sampling involves more than one random selection (e.g., random selection of a cross-section of the article to be sampled, followed by random selection for analysis of a location on a newly exposed surface).

[0031] As used herein, “representative sampling” refers to a sampling method for evaluating an article for a given property, such that a person skilled in the art will be convinced with at least 90%, at least 95%, or at least 99% confidence that the sampling selection will yield accurate measurements of a given property over a given volume (the entire article or a specific area of ​​the article). Naturally, the confidence increases with the number of samples taken. The location of the samples is selected or randomly so as to ensure that the aforementioned confidence levels are adequately covered. In some cases, representative sampling involves more than one representative selection (e.g., a representative selection of the section of the article from which to sample, followed by a representative selection for analysis of the location on the newly exposed surface).

[0032] As used herein, “sacrificial” material is a material used within a mold during the molding process but which will ultimately not be present in the produced article. An example of sacrificial material is acrylonitrile butadiene styrene (ABS), which can be deposited within a portion of the mold, solidified, and subsequently dissolved in acetone. Other examples include, but are not limited to, polyvinyl alcohol (soluble in water), ABS (also soluble in alcohol), various materials soluble in specific solvents, or flexible materials that can be introduced and removed primarily by physical force, such as clay-like materials.

[0033] As used herein, “settling” refers to the process of prompting a viscous material to occupy the lowest center of gravity in a mold by the use of time, force, or physical manipulation, or a combination thereof. Settling may include tapping the mold surface to cause the viscous material to settle into the mold. Settling may include simply waiting for gravity to perform its function for a certain period of time. Settling may include initial centrifugal rotation at a given centrifugal force (e.g., the same as, or lower than, those used in more widespread methods) to prompt conformation to the mold.

[0034] As used herein, “uniform” means that the variability across the article is less than 10%, less than 5%, less than 1%, or less than 0.1%, as demonstrated by random sampling, representative sampling, or another sampling technique that has been shown to result in a statistically valid sampling.

[0035] The techniques disclosed herein make it possible to produce monoliths or integral bulk structures using materials that were previously only possible to make into films or 3D print. Since the inventors provide the ability to transform materials from 2D planes into 3D articles, the difference between the geometry produced using the inventors' novel molding process and that of films is evident to the observer. Compared to objects 3D printed using polymerization solutions, the inventors can create complex structures without using individual pattern formation of the extrusion path. 3D bioprints, due to the cylindrical extrusion shapes of filaments stacked on top of each other to form the shape, are inherently never completely bulk or have a filled volume: in bioprinting manufacturing methodologies, voids remain within the integral structure due to the gaps between the cylindrical extrudes.

[0036] One crucial achievement related to this disclosure is the ability to produce synthetic bone for mechanical testing, such as dog bone. Synthetic bone requires the formation of bulk material for accurate testing, and current methodologies for fabricating biopolymers (and other materials requiring aqueous assemblies) are unable to do so due to the lack of uniformity and pattern formation of the material within those products.

[0037] The techniques described herein also make it possible to create complex geometric shapes using a single material or a combination of multiple materials. By adding multi-material molds with sacrificial parts, the inventors can integrate combinations of materials in unique patterns. For example, the inventors can create a tracheal stent using a two-part mold: the outer mold may be a Boolean subtraction of the stent geometry and is removed after the materials have polymerized. The inventors can create multilayer structures simply by stacking them on top of each other in an iterative polymerization process. The inventors can create concentric multi-material structures by concentrically stacking different mold materials, which may have different sacrificial conditions, thereby allowing mixtures of different materials to iteratively replace each sacrificial mold portion. The same methodology for iteratively replacing materials in each sacrificial mold portion can also be applied to extremely unique geometric shapes where the sacrificial mold is the female mold of that geometry. (One example is a multi-material scaffold where the vascular structure was made of a different material from the surrounding scaffold.)

[0038] This disclosure provides a method for producing biopolymer articles. The method overcomes significant shortcomings of existing technologies. For example, an alternative to the centrifugal molding process disclosed herein is 3D bioprinting. Indeed, 3D bioprinting is driving significant innovation in tissue engineering and the field of medical devices. Often, low-viscosity bioinks are extruded layer by layer into a polymerization bath, where they then cure into a desired geometry with unique properties (solvent-dependent molecular assembly). Various combinations of bioinks and bath liquids make it possible to customize constructs for desired material and mechanical properties. Solvent-dependent assembly of biopolymers has shown great success and potential in 3D bioprinting and is therefore considered promising. However, 3D printing also has its limitations, namely slow manufacturing times, difficulty in producing bulk materials, scalability, and the need for support baths to produce complex geometries. The disclosure herein addresses the shortcomings of 3D printing while leveraging the advantages and adaptability of solvent-driven biopolymer assembly. Novel, aqueous, solvent-dependent, centrifugal molding techniques for producing devices and bulk materials with tunable material properties from biopolymers are disclosed herein.

[0039] Many bioinks lack sufficient viscosity and have too high a loss modulus of elasticity to maintain their shape after extrusion. Often, the ink collapses, sagging occurs, resulting in impractical geometric shapes and overall print failure. Therefore, they are often extruded directly into a polymerization bath to induce crosslinking so that they can retain their intended structure or geometry. While different bioinks require different solvent bath properties to induce crosslinking, the overall concept of solidifying the extruded material remains the same. For example, chitosan dissolved in acetic acid is a common bioink used in regenerative medicine. It is often extruded into a bath solution consisting of sodium hydroxide to precipitate the chitosan and form a stable hydrogel. Silk is another bioink that can be extruded into a biomimetic salt bath, where the solvent polymerizes the silk through induced protein assembly, acidification, and dehydration processes. Ink lacking sufficient storage modulus of elasticity often requires a polymerization agent in the form of a bath solution.

[0040] Another alternative to the centrifugal molding process disclosed herein is conventional molding. Conventional molding involves depositing all the material into a mold, followed by polymerizing the material, but this technique has several limitations. For example, when working with larger structures, one limitation due to insufficient penetration into the depths of the polymerization bath is the maintenance of shape fidelity and polymerization. Centrifugal molding appears not to suffer from this limitation, even with larger structures. In another example, due to the viscous nature of the biopolymer solution used, it is not uncommon for air bubbles to enter the solution during casting. In conventional molding processes, these bubbles are eventually trapped in the solution, which can negatively affect the shape fidelity and mechanical properties of the resulting part. The centrifugal force applied to the solution removes the air bubbles, resulting in a flatter structure. In yet another example with complex geometries, liquid pockets may remain in certain areas of the mold, and the resulting impact on the shape fidelity and mechanism of the implant / device only becomes apparent after the part is removed from the mold. In this case as well, with the centrifugal molding approach, there are no remaining liquid pockets due to the centrifugal force applied to the solution.

[0041] Another alternative to the centrifugal molding process disclosed herein is thermoforming. Thermoforming involves filling a mold with freeze-dried biopolymer powder, and then applying heat and pressure to produce a part. Again, this technique presents limitations, which are addressed by the centrifugal molding approach. For example, the basic approach to thermoforming is to produce a blank larger than the desired object and obtain the resulting part using conventional manufacturing approaches such as milling or machining. This is inflexible, labor-intensive, and wasteful (subtractive manufacturing approaches result in far more waste material than additive manufacturing). The centrifugal molding strategy removes the constraints in terms of scalability and labor (many different parts can be molded simultaneously by a single operator) and the constraint in terms of waste (material is added only until the mold is full, and only a small amount of the resulting part is discarded as part of the post-processing). In another example, while it is possible to generate thermoforming molds that conform to complex geometries, obtaining the appropriate parts (three-part or more molds) and the correct tolerances to enable proper polymerization requires extensive research and expertise. On the other hand, using a centrifugal molding approach, molds can be easily generated using CAD software and 3D printed. In yet another example, modifying an existing mold is a very time-consuming and labor-intensive process, as it requires generating an entirely new mold. This is incompatible with incremental improvements to the design or mold. Using a centrifugal molding approach, it is possible to use a new biopolymer solution, make adjustments to the design, 3D print it, and verify it in a matter of hours. In the embodiments disclosed herein, techniques such as thermoforming, conventional molding, and / or machining can be used in conjunction with centrifugal molding techniques.

[0042] Centrifugal molding can be used in many different applications, such as in patients using implantable medical devices (e.g., middle ear ventilation tubes, nasal stents, nasal septum buttons, nerve capping devices, or Kirschner wires).

[0043] Medical devices or implants can be manufactured using biopolymers in aqueous solutions, while preserving one or more of the following: shape fidelity, reliability of the manufacturing process, scalability of manufacturing, simultaneous fabrication of multiple parts, generation of multi-material devices, generation of composite or multilayer structures, centimeter-scale devices, and high resolution of surface feature elements (at least tens of microns, possibly even lower feature element sizes, depending on the resolution of the 3D printer used to create the mold). In embodiments, the manufacturing technique may include robotic manufacturing, hybrid manufacturing, or modular manufacturing. For example, the mold and / or method may be robotically operated to produce the article. In one example, the mold may be adjusted during centrifugation to produce structural features. In another example, the method may be robotically programmed to produce an additive gradient in the article by modifying centrifugal parameters throughout the process.

[0044] Referring to Figures 1 and 2, schematic diagrams illustrating the principle of the method described herein are shown. Referring to Figure 3, an image of a tubular article produced by the disclosed method is shown.

[0045] Broadly speaking, this disclosure includes forming a first bio-solidified body by centrifuging an aqueous biopolymer solution in an initial solidification stage in a mold to a first late solidification stage. An article can be formed by further solidifying the first bio-solidified body. Alternatively, a second bio-solidified body containing the first bio-solidified body can be formed by adding a second aqueous biopolymer solution in an initial solidification stage to the first bio-solidified body to a second late solidification stage. To avoid misunderstanding, in many cases, sequentially added solutions do not need to have completed their bio-solidification before the introduction of the subsequent solution. In fact, for homogeneous materials, adding a subsequent solution before the previous layer has completed solidification is very advantageous because it allows the layers to become completely integrated with each other. If the second bio-solidification contains the first bio-solidified body (or any other), it does not mean that the first solidified body is distinguishable within the second solidified body. In many cases, the first bio-solidified material is seamlessly integrated with the second bio-solidified material.

[0046] More specifically, the method of the present disclosure may include the following sequential steps: A) introducing a first biopolymer aqueous solution into a mold and, if necessary, allowing the first biopolymer aqueous solution to settle in the mold; B) introducing a first curing aqueous solution onto the first biopolymer aqueous solution in the mold and, if necessary, allowing the first curing aqueous solution to settle in the mold, wherein the mold has a first orientation with respect to gravity during step B); and C) producing a first biosolidified body by subjecting the mold and its contents to a first centrifuge and stopping after a first centrifugation period, wherein the mold is within 45° of the first orientation with respect to the first centrifuge during step C. A first biopolymer article is produced from the first biosolidified body by waiting for a first final biosolidification period after step C. In some cases, the method includes the step of forming a first biopolymer article from a first biosolidified body by waiting for a first biosolidification period.

[0047] In some cases, the sedimentation in step A) is performed, but not as necessary. Sedimentation can be achieved in various ways, but centrifugal sedimentation is obviously conceivable. Centrifugal sedimentation disclosed elsewhere in this specification, or other methods understood by those skilled in the art to be useful for sedimentation, may be used. Manual sedimentation (e.g., tapping the surface) is also conceivable. The mold can be oriented during sedimentation to within 45° of the first orientation relative to gravity.

[0048] After steps A to C), the method may further include the following sequential steps: D) introducing a second biopolymer aqueous solution onto the first biosolid in a mold and, if necessary, allowing the second biopolymer aqueous solution to settle in the mold; E) introducing a second curing aqueous solution onto the second biopolymer aqueous solution in a mold and, if necessary, allowing the second curing aqueous solution to settle in the mold, wherein the mold has a second orientation with respect to gravity during step E); and F) producing a second biosolid containing the first biosolid by subjecting the mold and its contents to a second centrifugation and stopping after the second centrifugation period, wherein the mold is within 45° of the second orientation with respect to the second centrifugation during step F). After step F), a second final biosolidification period is awaited to produce a second biopolymer article from the second biosolid. In some cases, the method further includes a step of producing a second biopolymer article from a second biosolidified body by waiting for a second biosolidification period after step F).

[0049] In some cases, the sedimentation in step D) is performed, but not as necessary. Sedimentation can be achieved in various ways, but centrifugal sedimentation is obviously conceivable. Centrifugal sedimentation disclosed elsewhere in this specification, or other methods understood by those skilled in the art to be useful for sedimentation, may be used. Manual sedimentation (e.g., tapping the surface) is also conceivable. The mold can be oriented during sedimentation to within 45° of the second orientation relative to gravity.

[0050] After steps A to F), the method may further include the following sequential steps: G) introducing a third biopolymer aqueous solution onto the second biosolid in a mold and, if necessary, allowing the third biopolymer aqueous solution to settle in the mold; H) introducing a third curing aqueous solution onto the third biopolymer aqueous solution in a mold and, if necessary, allowing the third curing aqueous solution to settle in the mold, wherein the mold has a third orientation with respect to gravity during step H); and I) producing a third biosolid containing the first and second biosolids by subjecting the mold and its contents to a third centrifuge and stopping after a third centrifugation period, wherein the mold is within 45° of the third orientation with respect to the third centrifuge during step I). A third biopolymer article is produced from the third biosolid by waiting for a third final biosolidification period after step I). In some cases, the method further includes a step of producing a third biopolymer article from a third biosolidified body by waiting for a third biosolidification period after step I).

[0051] In some cases, the sedimentation in step G) is performed, but not as necessary. Sedimentation can be achieved in various ways, but centrifugal sedimentation is obviously conceivable. Centrifugal sedimentation disclosed elsewhere in this specification, or other methods understood by those skilled in the art to be useful for sedimentation, may be used. Manual sedimentation (e.g., tapping the surface) is also conceivable. The mold can be oriented during sedimentation to within 45° of a third orientation relative to gravity.

[0052] After steps A to I), the method may further include the following sequential steps: J) introducing a fourth biopolymer aqueous solution onto the third biosolid in a mold and, if necessary, allowing the fourth biopolymer aqueous solution to settle in the mold; K) introducing a fourth curing aqueous solution onto the fourth biopolymer aqueous solution in a mold and, if necessary, allowing the fourth curing aqueous solution to settle in the mold, wherein the mold has a fourth orientation with respect to gravity during step J); and L) producing a fourth biosolid containing the first, second and third biosolids by subjecting the mold and its contents to a fourth centrifuge and stopping after a fourth centrifugation period, wherein the mold is within 45° of the fourth orientation with respect to the fourth centrifuge during step L). After step L), a fourth final biosolidification period is awaited to produce a fourth biopolymer article from the fourth biosolid. In some cases, the method further includes a step of producing a fourth biopolymer article from a fourth biosolidified body by waiting for a fourth biosolidification period after step L).

[0053] In some cases, the sedimentation in step J) is performed, but not as necessary. Sedimentation can be achieved in various ways, but centrifugal sedimentation is obviously conceivable. Centrifugal sedimentation disclosed elsewhere in this specification, or other methods understood by those skilled in the art to be useful for sedimentation, can be utilized. Manual sedimentation (e.g., tapping the surface) is also conceivable. The mold can be oriented during sedimentation to within 45° of a third orientation relative to gravity.

[0054] In some cases, the first and second orientations are the same, but in other cases, they are different. In some cases, for example, when forming alternating layers, it may be important that the orientations are the same. In other cases, for example, when forming intricate lateral feature elements in an article, it may be important to use different orientations. Similarly, the third orientation may be the same as or different from the first and second orientations, and the fourth orientation may be the same as or different from the first, second, and third orientations. The same is true for the fifth, sixth, and up to n orientations for each iteration of these method steps.

[0055] As a general principle, the method may include the following sequential steps after steps A-C), A-F), A-I), or A-L): X) introducing an additional biopolymer aqueous solution onto an existing portion of the biosolid, and optionally allowing the additional biopolymer aqueous solution to settle in the mold, wherein the existing portion of the biosolid includes a first solid and optionally includes second, third, and fourth biosolids; Y) introducing an additional curing aqueous solution onto the additional biopolymer aqueous solution in the mold, and optionally allowing the additional curing aqueous solution to settle in the mold; and Z) centrifugating the mold and its contents for an additional period, stopping after the additional centrifugation period, thereby producing an additional biosolid containing the existing portion of the biosolid. Additional biopolymer articles are produced from the additional biosolid by waiting for an additional final biosolidification period after step Z. In some cases, the method includes waiting for a final biosolidification period.

[0056] As another general principle, the method may include at least two iterative cycles of the following steps: X) repeatedly introducing an aqueous biopolymer solution into a mold and, if necessary, allowing the aqueous biopolymer solution to settle in the mold; Y) repeatedly introducing a curing solution onto the aqueous biopolymer solution in the mold and, if necessary, allowing the curing solution to settle in the mold; and Z) repeatedly centrifugating the mold and its contents, stopping after the repeated centrifugation period to repeatedly add to the existing portion of the solidified body, wherein the repeated introduction of step X), the introduction of step Y), and the centrifugation of step Z) continues until the final biosolid is formed. The biopolymer article is produced by waiting for a final biosolidification period after the formation of the final biosolid. In some cases, the method includes waiting for a final biosolidification period.

[0057] In some cases, the sedimentation in step X) is performed, but not as necessary. Sedimentation can be achieved in various ways, but centrifugal sedimentation is obviously conceivable. Centrifugal sedimentation disclosed elsewhere in this specification, or other methods understood by those skilled in the art to be useful for sedimentation, may be used. Manual sedimentation (e.g., tapping on the surface) is also conceivable. The mold can be oriented during sedimentation to within 45° of a third orientation relative to gravity.

[0058] In some cases, residual aqueous solutions may be aspirated from the mold as needed, taking care not to disturb the deposited polymer. In some cases, rinsing may also be required. Specifically, in cases where alternating layers of materials are desired, if the curing conditions for one of the materials impair the others (i.e., if the acidic curing solution for one biopolymer decomposes the other biopolymer), the mold is aspirated and rinsed before adding the other biopolymer to the mold.

[0059] The mold size and the volume of liquid added in these methods are not intended to be limiting. While there is no physical lower limit to the size of articles produced by these methods, it is acknowledged that very small 1D or 2D articles (e.g., fibers or films) may be produced using existing methods to produce articles having one or more of the characteristic centrifugal properties described above. In other words, there may be some minimum size threshold for these methods to produce materials qualitatively different from previously produced materials. There are also other size thresholds above which the results achieved by the invention described herein may become even more particularly surprising. For example, if an article has an interior point at at least 0.05 mm, at least 0.1 mm, at least 0.2 mm, at least 0.3 mm, at least 0.4 mm, at least 0.5 mm, at least 1.0 mm, or at least 1.5 mm from the surface of the article, no prior method has been shown to produce the material properties and uniformity achieved by the disclosed method.

[0060] The centrifugation described throughout these methods differs somewhat from the conventional use of centrifugation, as the process is not necessarily intended to separate components as described elsewhere in this specification, but rather to facilitate the removal of bubbles and the maintenance of physical conformability. Consequently, centrifugation techniques do not offer much guidance on using centrifuges in this manner. Surprisingly, the inventors have discovered many advantages to processing materials in the manner described herein.

[0061] The centrifugal force used in the example was 2000 × g, but other centrifugal forces, including those between 1000 × g and 5000 × g, are possible. The centrifugation period can also be adjusted as desired. The centrifugation period may be between 30 seconds and 10 minutes or between 1 minute and 1 hour. Longer centrifugation times may retain additional advantages, but these will likely be offset by increased time and energy consumption.

[0062] Each aqueous solution described herein may be degassed before use.

[0063] Each biopolymer aqueous solution contains the respective biopolymer. The biopolymers can be selected from the group consisting of silk fibroin, alginate, fibrinogen, chitosan, collagen, and combinations thereof. These biopolymers can be paired with their respective curing aqueous solutions.

[0064] One pair consists of silk fibroin as a biopolymer and a curing solution that initiates beta-sheet crystallization and / or hydrogel formation.

[0065] Fibroin: As used herein, the term “fibroin” includes silkworm silk fibroin or insect or spider silk protein (Lucas et al, Adv. Protein Chem 13: 107-242 (1958)). Any type of silk fibroin can be used according to embodiments of the present invention. There are many different types of silk produced by a wide variety of species, including, but not limited to, Antheraea mylitta; Antheraea pemyi; Antheraea yamamai; Galleria mellonella; Bombyx mori; Bombyx mandarina; Galleria mellonella; Nephila clavipes; Nephila senegalensis; Gasteracantha mammosa; Argiope aurantia; Araneus diadematus; Latrodectus geometricus; Araneus bicentenarius; Tetragnatha versicolor; Araneus ventricosus; Dolomedes tenebrosus; Euagrus chisoseus; Plectreurys tristis; Argiope trifasciata; and Nephila madagascariensis. In some embodiments, fibroin is obtained from a solution containing dissolved silkworm silk or spider silk. Silkworm silk proteins are obtained, for example, from Bombyx mori, and spider silk is obtained from Nephila clavipes. Other silks include transgenic silk, genetically modified silk (recombinant silk), for example, silk from bacteria, yeast, mammalian cells, transgenic animals or transgenic plants, and variants thereof. See, for example, WO97 / 08315 and U.S. Patent No. 5,245,012, the contents of both of which are incorporated herein by reference in their entirety. In some embodiments, silk fibroin may be derived from other sources, for example, spiders, other silkworms, bees, synthetic silk-like peptides, and variants thereof made by biotechnology.In some embodiments, silk fibroin may be extracted from the glands of silkworms or transgenic silkworms. See, for example, WO2007 / 098951, the entire contents of which are incorporated herein by reference. Different species of silk-producing organisms and different types of silk have different amino acid compositions, but various fibroin proteins share certain structural features. A common tendency of silk fibroin structures is an amino acid sequence usually characterized by alternating glycine and alanine or alanine alone. Such configurations allow fibroin molecules to self-assemble into beta-sheet higher-order structures. These “Ala-rich” and “Gly-rich” hydrophobic blocks are typically separated by amino acid segments having bulky side groups (e.g., hydrophilic spacers). In some embodiments, the core repeat sequence of the hydrophobic block of fibroin is the following amino acid sequence and / or formula: (GAGAGS)5-15 (SEQ ID NO: 1); (GX)5-15 (X=V, I, A) (SEQ ID NO: 2); GAAS (SEQ ID NO: 3); (S1-2A11-13) (SEQ ID NO: 4); GX1-4 GGX (SEQ ID NO: 5); GGGX (X=A, S, Y, R, DV, W, R, D) (SEQ ID NO: 6); (Sl-2Al-4)l-2 (SEQ ID NO: 7); GLGGLG (SEQ ID NO: 8); GXGGXG (X=L, I, V, P) (SEQ ID NO: 9); GPX (X=L, Y, I); (GP(GGX)l-4 Y)n (X=Y, V, S, A) (SEQ ID NO: 10); GRGGAn (SEQ ID NO: 11); GGXn (X=A, T, V, S); GAG(A)6-7GGA (SEQ ID NO: 12); and GGX GX It may be represented by GXX(X=Q, Y, L, A, S, R) (Sequence ID 13). In some embodiments, the fibroin peptide may contain multiple hydrophobic blocks, e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 hydrophobic blocks within the peptide. In some embodiments, the fibroin peptide may contain hydrophobic blocks between 4 and 17. In some embodiments of the present invention, the fibroin peptide includes at least one hydrophilic spacer sequence ("hydrophilic block") having a length of about 4 to 50 amino acids.Non-limiting examples of hydrophilic spacer sequences include TGSSGFGPYVNGGYSG (SEQ ID NO: 14); YEYAWSSE (SEQ ID NO: 15); SDFGTGS (SEQ ID NO: 16); RRAGYDR (SEQ ID NO: 17); EVIVIDDR (SEQ ID NO: 18); TTHEDLDITIDGADGPI (SEQ ID NO: 19); and TISEELTI (SEQ ID NO: 20). In certain embodiments, the fibroin peptide may contain a hydrophilic spacer sequence that is a derivative of any one of the spacer sequences listed above. Such a derivative is at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to any one of the hydrophilic spacer sequences. In some embodiments, the fibroin peptide suitable for the present invention does not contain a spacer. Silk is generally a fibrous protein characterized by modular units linked together to form high molecular weight, highly repeatable proteins. These modular units or domains, each having a specific amino acid sequence and chemical properties, are thought to provide specific functions. For example, arrangement motifs such as poly-alanine (Poly-A) and poly-alanine-glycine (Poly-AG) tend to be beta-sheet forming; GXX motifs contribute to 31-helix formation; GXG motifs provide rigidity; and GPGXX (SEQ ID NO: 22) contributes to beta-helical formation. These are examples of different components in various silk structures where arrangement and sequence are linked to the final material properties of silk-based materials (outlined in Omenetto and Kaplan (2010) Science 329: 528-531). See also WO2011 / 130335 (PCT / US2011 / 032195), the contents of which are incorporated herein by reference for all purposes.

[0066] One pair consists of alginate as a biopolymer and a curing solution, which is a calcium ion solution.

[0067] One pair consists of fibrinogen as a biopolymer and a curing solution, which is a thrombin solution that cleaves and polymerizes the fibrinogen into fibrin.

[0068] One pair consists of chitosan as a biopolymer, which is acidic, and a curing solution, which is a neutralizing agent that neutralizes the acid and solidifies the chitosan. Chitosan, a naturally occurring polysaccharide biopolymer, and its derivatives are non-toxic, biocompatible, and biodegradable, and possess certain important biological properties, such as inherent antimicrobial properties, mucosal adhesion, and permeability-enhancing properties, which can be imparted to articles containing chitosan or its derivatives. Other biological activities include antifungal, antitumor, anticancer, antidiabetic, wound healing, and antioxidant activity. Depending on the degree of deacetylation, modification, and / or molecular weight (MW) of chitosan, certain properties (e.g., biological activity, biocompatibility, and biodegradability) may be present, absent, enhanced, or less pronounced. Examples of curing solutions used with chitosan include sodium hydroxide, a mixture of sodium hydroxide and ethanol, ethanol, urea (carbamide), carbonic acid, or sodium tripolyphosphate.

[0069] One pair consists of collagen as a biopolymer and a curing solution, which is a pH adjuster or a series of buffer solutions that directs the collagen to self-assemble.

[0070] When multiple different polymers and solutions are used, some degree of compatibility between the polymers is required, as will be understood by those skilled in the art. One exemplary combination is silk fibroin and chitosan, where the alternating layers are quite strong, perhaps due to the electrostatic interaction between the negatively charged silk layer and the positively charged chitosan layer.

[0071] In some cases, the first, second, third, fourth and / or repeated biopolymer aqueous solutions, and / or the first, second, third, fourth and / or repeated curing aqueous solutions, may further contain one or more additives. In some cases, the additives may be introduced into the mold before introducing one of the aqueous solutions. In these cases, the solution fills around the additive, solidifies, and does not dissolve and / or dissolve / suspend the soluble or suspendable additive.

[0072] Additives may include nanoparticles, microparticles, structural modification materials, and / or bioactive materials. Nanoparticles / microparticles may include silk, metals, semiconductor nanoparticles, ceramics, magnetic particles, contrast agents, and / or radiopaque particles. Structural modification materials include, but are not limited to, fibers (including microfibers / nanofibers), fabrics, scaffolds, foams, plasticizers, carbon nanotubes, and / or pologens. Bioactive materials include cells and / or growth factors or other biopharmaceuticals. Other examples of bioactive materials include, but are not limited to, enzymes, antibiotics, anti-inflammatory drugs, analgesics, chemotherapeutic agents, miRNA, mRNA, or siRNA.

[0073] When additives are affected by gravity, their position can be important. For example, adding bulky additives too early in the process can cause them to accumulate entirely at the "bottom" of the mold, while adding them too late can result in them remaining at the "top" of the mold. When adding additives into a biopolymer solution, care is taken to ensure that the additive is simply incorporated into the solution and that the centrifugal force or centrifugation time (or a combination of force and time) is controlled to ensure that the additive does not precipitate from the structure. When adding additives via a polymerization agent solution, it is crucial to characterize the g-force and centrifugation time required for the additive to penetrate and move / permeate through the polymer solution. If the g-force is too high or the centrifugation time is too long, the additive will migrate to the bottom of the layer and possibly even penetrate into previously deposited layers. If the g-force is too low or the centrifugation time is too short, the additive will not penetrate the polymer solution and will exist locally on the polymer solution as its own independent layer (which can also be considered another characteristic element). It is also important to consider the effect of centrifugation on previously deposited layers and additives. Viscosity, polymerization time, material density, additive density, and layer height are all confounding factors that affect centrifugal force and time. If the additives respond to gravity, it should be understood that the centrifugal force and centrifugation time can be adjusted to produce a predetermined gradient for the additives.

[0074] The molds used herein may have a variety of feature elements suitable for use in this disclosure. In some cases, the mold may have intricate internal feature elements and may produce article feature elements that cannot be produced by any other means. In some cases, the mold may have mounting supports or other mounting feature elements that allow the mold to be forced to hold at a desired angle during centrifugation. In other words, the mold may have fixing points that produce a predictable orientation under gravity.

[0075] The mold itself can be 3D printed. The methods described herein may include 3D printing. If the shape of the desired article is known, a person skilled in the art can customize and produce a mold from 3D printing techniques known to the art. The molds used herein may be made from 3D printing materials, graphite, ceramic, stainless steel, iron, steel, aluminum, nickel, copper, acrylic, polylactic acid, polyamide, nylon, or a combination thereof.

[0076] In some cases, the mold may have multiple parts. In one specific embodiment, the mold has an upper mold portion, which is located above the solution and molds the top surface of the bio-solidified article and / or the final article.

[0077] Various sacrificial materials can be used in these techniques to create more complex geometric shapes. For example, the sacrificial material can be introduced into a portion of the mold, remain during the process, and then removed, with the process performed in the location previously occupied by the sacrificial material. One example of a sacrificial material is acrylonitrile butadiene styrene (ABS), which can be removed using acetone or alcohol. Other sacrificial materials include, but are not limited to, polyvinyl alcohol and inert clay.

[0078] Articles produced by the methods described herein may take various forms, and the specific forms mentioned herein are not intended to be limiting. Articles may be medical devices. Illustrative articles include, but are not limited to, catheters, stents, tympanostomy tubes, nasal septum buttons, nerve cap devices, Kirschner wires, artificial lenses, dental implants, bone scaffolds, spinal implants, plates, screws, pins, rods, tissue scaffolds, collagen structures, or tendon scaffolds.

[0079] In some cases, the article may be a microfluidic device or a microfluidic chip. One particular approach for forming a microfluidic element involves physical etching, which is a design attribute of this process. The mold used herein can be designed to have a convex topography of the microfluidic device, which will be imprinted on the resulting article during formation by the centrifugal method described herein. These feature elements are transferred with high reliability so that forces ensure strong contact between the article and the mold.

[0080] The bio-solidified materials and articles described herein can be processed in a variety of ways, including but not limited to chemical crosslinking, physical crosslinking, chemical etching, chemical smoothing, chain extension and / or branching of polymers, and / or chemical functionalization.

[0081] The articles described herein can be machined into various useful shapes.

[0082] Although this disclosure is described in particular in relation to biopolymers, the method is thought to be more broadly applicable to other aqueous processing methods and / or other aqueous polymer systems.

[0083] Centrifugal solidification-controlled biopolymer articles are disclosed herein. Centrifugal solidification-controlled biopolymer articles are not films. At least a portion of the article includes at least one characteristic centrifugal property selected from the group consisting of uniform density, optical opacity, absence of external lamination, absence of internal lamination, absence of internal voids, absence of cylindrical extrusion gaps, and absence of lamination. In an example, the centrifugal solidification-controlled biopolymer article includes at least two of the at least one characteristic centrifugal property disclosed. In an example, the centrifugal solidification-controlled biopolymer article includes at least three of the at least one characteristic centrifugal property disclosed. In an example, the centrifugal solidification-controlled biopolymer article includes at least four of the at least one characteristic centrifugal property disclosed. In an example, the centrifugal solidification-controlled biopolymer article includes at least five of the at least one characteristic centrifugal property disclosed. In an example, the centrifugal solidification-controlled biopolymer article includes at least six of the at least one characteristic centrifugal property disclosed. In the example, the centrifugal solidification-controlled biopolymer article includes at least seven of the at least one characteristic centrifugal properties disclosed.

[0084] A centrifugal controlled biosolidification molding method is disclosed herein. The method comprises the following sequential steps: A) introducing a first biopolymer aqueous solution into a mold and, if necessary, allowing the first biopolymer aqueous solution to settle in the mold; B) introducing a first curing aqueous solution onto the first biopolymer aqueous solution in the mold and, if necessary, allowing the first curing aqueous solution to settle in the mold, wherein the mold has a first orientation with respect to gravity during step B); and C) producing a first biosolid by subjecting the mold and its contents to a first centrifuge and stopping after a first centrifugation period, wherein the mold is within 45° of the first orientation with respect to the first centrifuge during step C). A first biopolymer article is produced from the first biosolid by waiting for a first final biosolidification period after step C).

[0085] In some examples, step A) includes the step of settling a first biopolymer aqueous solution into a mold. In an example, the settling step includes subjecting the mold and its contents to centrifugal sedimentation and stopping the centrifugation after the centrifugation period, thereby causing the first biopolymer solution to conform to the geometry of the mold, with the mold being within 45° of the first orientation to the first centrifuge in step B) during centrifugal sedimentation. In an example, the settling step includes causing the first biopolymer solution to conform to the geometry of the mold by tapping the mold and its contents on the surface, with the mold being within 45° of the first orientation to the first centrifuge in step B) during tapping.

[0086] In some examples, the method further includes a step of producing a first biopolymer article by waiting for a first final biosolidification period after step C).

[0087] In some examples, the first biopolymer aqueous solution is a silk aqueous solution, an alginate aqueous solution, a fibrinogen aqueous solution, a chitosan aqueous solution, a collagen aqueous solution, or a combination thereof. In one example, the first biopolymer aqueous solution is a silk aqueous solution, and the first curing aqueous solution is a silk crosslinking and / or hydrogel initiation aqueous solution. In another example, the first biopolymer aqueous solution is an alginate aqueous solution, and the first curing aqueous solution is an alginate crosslinking aqueous solution. In yet another example, the first biopolymer aqueous solution is a fibrinogen aqueous solution, and the first curing aqueous solution is a fibrinogen curing aqueous solution. In yet another example, the first biopolymer aqueous solution is a collagen aqueous solution, and the first curing aqueous solution is a collagen curing aqueous solution.

[0088] In some examples, the method further includes the following sequential steps: D) introducing a second biopolymer aqueous solution onto a first biosolid in a mold and, if necessary, allowing the second biopolymer aqueous solution to settle in the mold; E) introducing a second curing aqueous solution onto the second biopolymer aqueous solution in a mold and, if necessary, allowing the second curing aqueous solution to settle in the mold, wherein the mold has a second orientation with respect to gravity during step E); and F) producing a second biosolid containing the first biosolid by subjecting the mold and its contents to a second centrifuge and stopping after the second centrifugation period, wherein the mold is within 45° of the second orientation with respect to the second centrifuge during step F). A second biopolymer article is produced from the second biosolid by waiting for a second final biosolidification period after step F). In the example, the method further includes a step of producing a second biopolymer article by waiting for a second final biosolidification period after step F). The second orientation may be the same as the first orientation or different from the first orientation. For example, the method comprises the following steps: G) introducing a third biopolymer aqueous solution onto a second biosolid in a mold and, if necessary, allowing the third biopolymer aqueous solution to settle in the mold; H) introducing a third curing aqueous solution onto the third biopolymer aqueous solution in a mold and, if necessary, allowing the third curing aqueous solution to settle in the mold, wherein the mold has a third orientation with respect to gravity during step H); and I) subjecting the mold and its contents to a third centrifuge and stopping after a third centrifugation period to produce a third biosolid containing the first and second biosolids, wherein the mold is within 45° of the third orientation with respect to the third centrifugation during step I), and a third biopolymer article is produced from the third biosolid by waiting for a third final biosolidification period after step I). In the example, the method may further include a step of producing a third biopolymer article by waiting for a third final solidification period after step I).In this example, the method comprises the following sequential steps: J) introducing a fourth biopolymer aqueous solution onto a third biosolid in a mold and, if necessary, allowing the fourth biopolymer aqueous solution to settle in the mold; K) introducing a fourth curing aqueous solution onto the fourth biopolymer aqueous solution in a mold and, if necessary, allowing the fourth curing aqueous solution to settle in the mold, wherein the mold has a fourth orientation with respect to gravity during step J); and L) subjecting the mold and its contents to a fourth centrifuge and stopping after a fourth centrifugation period to produce a fourth biosolid containing the first, second, and third biosolids, wherein the mold is within 45° of the fourth orientation with respect to the fourth centrifugation during step L), and a fourth biopolymer article is produced from the fourth biosolid by waiting for a fourth final biosolidification period after step L). In this example, the method may further include a step of producing a third biopolymer article by waiting for a third final solidification period after step L).

[0089] In some examples, the method further comprises the following sequential steps: X) introducing an additional biopolymer aqueous solution onto an existing portion of the biosolid, and optionally allowing the additional biopolymer aqueous solution to settle in the mold, wherein the existing portion of the biosolid includes a first solid and optionally includes second, third, and fourth biosolids; Y) introducing an additional curing aqueous solution onto the additional biopolymer aqueous solution in the mold, and optionally allowing the additional curing aqueous solution to settle in the mold; and Z) producing an additional biosolid containing the existing portion of the biosolid by subjecting the mold and its contents to additional centrifugation and stopping after an additional centrifugation period, wherein an additional biopolymer article is produced from the additional biosolid by waiting for an additional final biosolidification period after step Z). In one example, the method further comprises producing an additional biopolymer article by waiting for an additional final solidification period after step Z).

[0090] A method for producing a biopolymer article is disclosed herein, comprising at least two iterative cycles of the following steps: X) repeatedly introducing a biopolymer aqueous solution into a mold and, if necessary, allowing the repeated biopolymer aqueous solution to settle in the mold; Y) repeatedly introducing a curing aqueous solution onto the repeated biopolymer aqueous solution in the mold and, if necessary, allowing the repeated curing aqueous solution to settle in the mold; and Z) repeatedly centrifugating the mold and its contents and stopping the centrifugation after the repeated centrifugation period to repeatedly add to the existing portion of the solidified body, wherein the repeated introduction of step X), introduction of step Y), and subjecting to step Z) continues until a final biosolid is formed, and after the formation of the final biosolid, a final biosolidification period is observed to produce the biopolymer article. The method may further include a step of producing the biopolymer article by waiting for a final biosolidification period after step Z).

[0091] A method is disclosed herein that includes the step of forming a first biosolid by centrifuging a first initial solidification aqueous solution of a biopolymer in a mold to a first late solidification stage. In one example, the method further includes the step of forming a second biosolid containing the first biosolid by centrifuging a second initial solidification aqueous solution of a biopolymer over the first biosolid in a mold to a second late solidification stage.

[0092] In any of the methods disclosed herein, the mold is three-dimensionally printed. In any of the methods disclosed herein, the method includes the step of three-dimensionally printing the mold before step A).

[0093] In any of the methods disclosed herein, a sacrificial molding is used in the process. For example, the sacrificial molding is acrylonitrile butadiene styrene. In one example, the method includes a step of removing the sacrificial molding using acetone or alcohol.

[0094] In any of the methods disclosed herein, the presence of an upper mold during one or more centrifugal steps introduces additional molding of one or more biosolidified articles.

[0095] In any of the methods disclosed herein, the first biopolymer in the first biopolymer aqueous solution, the second biopolymer in the second biopolymer solution, the third biopolymer in the third biopolymer solution, the fourth biopolymer in the fourth biopolymer solution, and / or the repeating biopolymer in the repeating biopolymer solution are selected from the group consisting of silk fibroin, alginate, fibrinogen, chitosan, collagen, and combinations thereof. In one example, the first, second, third, fourth, and / or repeating biopolymers are silk fibroin. In one example, the first, second, third, fourth, and / or repeating curing solutions are beta-sheet initiators or hydrogel initiators. In one example, the first, second, third, fourth, and / or repeating biopolymers are alginate. In this example, the first, second, third, fourth, and / or repeating curing solutions are calcium ion solutions. In one example, the first, second, third, fourth and / or iterative biopolymer is fibrinogen. In this example, the first, second, third, fourth and / or iterative curing solution is a thrombin solution that cleaves and polymerizes the fibrinogen into fibrin. In another example, the first, second, third, fourth and / or iterative biopolymer is chitosan. In this example, the chitosan is acidic, and the first, second, third, fourth and / or iterative curing solution is a neutralizing agent that neutralizes the acid and solidifies the chitosan. In yet another example, the first, second, third, fourth and / or iterative biopolymer is collagen. In this example, the collagen is acidic, and the first, second, third, fourth and / or iterative curing solution is a pH adjuster that directs the self-assembly of the collagen.

[0096] Any of the methods disclosed herein further comprises a step of chemically crosslinking the first, second, third, fourth, or final biosolidified body or the first, second, third, fourth, or final biopolymer article.

[0097] Any of the methods disclosed herein further includes a step of physically crosslinking the first, second, third, fourth, or final biosolidified body or the first, second, third, fourth, or final biopolymer article.

[0098] Any of the methods disclosed herein further includes a step of chemically etching the first, second, third, fourth, or final biosolidified body or the first, second, third, fourth, or final biopolymer article.

[0099] Any of the methods disclosed herein further comprises a step of chemically smoothing the first, second, third, fourth, or final biosolidified body or the first, second, third, fourth, or final biopolymer article.

[0100] Any of the methods disclosed herein further includes the step of extending and / or branching polymer chains in the first, second, third, fourth, or final biosolidified body or the first, second, third, fourth, or final biopolymer article.

[0101] Any of the methods disclosed herein further includes a step of chemically functionalizing the first, second, third, fourth, or final biosolidified body or the first, second, third, fourth, or final biopolymer article.

[0102] In any of the methods disclosed herein, the method further comprises a step of degassing the first, second, third, fourth and / or repeated aqueous solutions of the biopolymer and / or the first, second, third, fourth and / or repeated aqueous solutions for curing.

[0103] In any of the methods disclosed herein, the first, second, third, fourth and / or repeated aqueous solutions of the biopolymer and / or the first, second, third, fourth and / or repeated curing solutions further include additives. In one example, the first aqueous solution of the biopolymer includes additives. In one example, the second aqueous solution of the biopolymer includes additives. In one example, the third aqueous solution of the biopolymer includes additives. In one example, the fourth aqueous solution of the biopolymer includes additives. In one example, the repeated aqueous solutions of the biopolymer include additives. In one example, the first curing solution includes additives. In one example, the second curing solution includes additives. In one example, the third curing solution includes additives. In one example, the fourth curing solution includes additives. In one example, the repeated curing solution includes additives. In the examples, the additives include nanoparticles, structural modification materials, or bioactive materials. Structural modification materials may include fibers, fabrics, scaffolding, foams, plasticizers, or pologens. In one example, the additive may be a biodegradable metal mesh, such as a magnesium-based mesh (e.g., centrifugal coated). In embodiments, the mesh may be present in the mold beforehand or added during the polymerization process. Nanoparticles include silk nanoparticles, metal nanoparticles, or semiconductor nanoparticles. The bioactive material includes one or more cells or one or more growth factors.

[0104] In any of the methods disclosed herein, the mold has a structural modification material before introducing a first, second, third, fourth and / or iterative biopolymer aqueous solution and / or a first, second, third, fourth and / or iterative curing aqueous solution into the mold. The structural modification material includes fibers, fabrics, scaffolding, foams, plasticizers, or pologens.

[0105] In any of the methods disclosed herein, the mold has nanoparticles before introducing a first, second, third, fourth and / or iterative biopolymer aqueous solution and / or a first, second, third, fourth and / or iterative curing aqueous solution into the mold. The nanoparticles include silk nanoparticles, metal nanoparticles, or semiconductor nanoparticles.

[0106] In any of the methods disclosed herein, the mold has a bioactive material before introducing a first, second, third, fourth and / or iterative biopolymer aqueous solution and / or a first, second, third, fourth and / or iterative curing aqueous solution into the mold. The bioactive material comprises one or more cells or one or more growth factors.

[0107] Articles manufactured by any of the methods disclosed herein are disclosed herein. For example, an article is at least part of a medical device. For example, an article or medical device is a catheter, stent, tympanostomy tube, nasal septum button, nerve cap device, Kirschner wire, artificial lens, dental implant, bone scaffold, spinal implant, plate, screw, pin, rod, or a combination thereof.

[0108] Disclosed herein are centrifugal solidification-controlled chitosan articles, which are not films. At least a portion of the article includes at least one characteristic centrifugal property selected from the group consisting of i) uniform density over at least a portion of the article; ii) optical opacity of at least a portion of the article; iii) absence of external laminations in at least a portion of the article; iv) absence of internal laminations in at least a portion of the article; v) absence of internal voids in at least a portion of the article; vi) absence of cylindrical extrusion gaps in at least a portion of the article; and vii) absence of laminations in at least a portion of the article.

[0109] A centrifugal controlled chitosan biosolidification molding method is disclosed herein. The method comprises the following sequential steps: A) introducing a chitosan solution into a mold and, if necessary, allowing the chitosan solution to settle in the mold; B) introducing a chitosan curing solution onto the chitosan solution in the mold and, if necessary, allowing the chitosan curing solution to settle in the mold, wherein the mold has a first orientation with respect to gravity during step B); and C) producing a first biosolid by subjecting the mold and its contents to a first centrifuge and stopping after a first centrifugation period, wherein the mold is within 45° of the first orientation with respect to the first centrifuge during step C). A first biopolymer article is produced from the first biosolid by waiting for a first final biosolidification period after step C).

[0110] In one example of the method, the sedimentation step involves subjecting the mold and its contents to centrifugal sedimentation and stopping the centrifugation after the centrifugation period, thereby causing the chitosan solution to conform to the geometry of the mold, wherein the mold is within 45° of the first orientation to the first centrifuge in step B) during centrifugal sedimentation.

[0111] In one example of the method, the settling step involves making the chitosan solution conform to the geometry of the mold by tapping the mold and the contents of the mold on the surface, the mold being within 45° of the first orientation with respect to the first centrifugation in step B) during tapping.

[0112] An example of the method further includes a step of producing a first biopolymer article by waiting for a first final biosolidification period after step C).

[0113] In one example of the method, the aqueous solution for curing chitosan is sodium hydroxide, ethanol, or a combination thereof.

[0114] An example of the method further includes the following sequential steps: D) introducing a second biopolymer aqueous solution onto the first biosolid in a mold and, if necessary, allowing the second biopolymer aqueous solution to settle in the mold; E) introducing a second curing aqueous solution onto the second biopolymer aqueous solution in a mold and, if necessary, allowing the second curing aqueous solution to settle in the mold, wherein the mold has a second orientation with respect to gravity during step E); and F) producing a second biosolid containing the first biosolid by subjecting the mold and its contents to a second centrifuge and stopping after the second centrifugation period, wherein the mold is within 45° of the second orientation with respect to the second centrifuge during step F). A second biopolymer article is produced from the second biosolid by waiting for a second final biosolidification period after step F). In the example, the method further includes a step of producing a second biopolymer article by waiting for a second final biosolidification period after step F). In the example, the second orientation is the same as the first orientation. In the example, the second orientation is different from the first orientation. In this example, the method further includes the following steps: G) introducing a third biopolymer aqueous solution onto a second biosolid in a mold and, if necessary, allowing the third biopolymer aqueous solution to settle in the mold; H) introducing a third curing aqueous solution onto the third biopolymer aqueous solution in a mold and, if necessary, allowing the third curing aqueous solution to settle in the mold, wherein the mold has a third orientation with respect to gravity during step H); and I) producing a third biosolid containing the first and second biosolids by subjecting the mold and its contents to a third centrifuge and stopping after a third centrifugation period, wherein the mold is within 45° of the third orientation with respect to the third centrifuge during step I). A third biopolymer article is produced from the third biosolid by waiting for a third final biosolidification period after step I). In the example, the method further includes a step of producing a third biopolymer article by waiting for a third final solidification period after step I).In this example, the method further comprises the following sequential steps: J) introducing a fourth biopolymer aqueous solution onto a third biosolid in a mold and, if necessary, allowing the fourth biopolymer aqueous solution to settle in the mold; K) introducing a fourth curing aqueous solution onto the fourth biopolymer aqueous solution in a mold and, if necessary, allowing the fourth curing aqueous solution to settle in the mold, wherein the mold has a fourth orientation with respect to gravity during step J); and L) producing a fourth biosolid containing the first, second, and third biosolids by subjecting the mold and its contents to a fourth centrifuge and stopping after a fourth centrifugation period, wherein the mold is within 45° of the fourth orientation with respect to the fourth centrifuge during step L). A fourth biopolymer article is produced from the fourth biosolid by waiting for a fourth final biosolidification period after step L). In the example, the method further includes a step of producing a third biopolymer article by waiting for a third final solidification period after step L).

[0115] One example of the method further includes the following sequential steps: X) introducing an additional biopolymer aqueous solution onto an existing portion of the biosolid, and optionally allowing the additional biopolymer aqueous solution to settle in the mold, wherein the existing portion of the biosolid includes a first biosolid and optionally includes second, third, and fourth biosolids; Y) introducing an additional curing aqueous solution onto the additional biopolymer aqueous solution in the mold, and optionally allowing the additional curing aqueous solution to settle in the mold; and Z) centrifugating the mold and its contents for an additional period, stopping after the additional centrifugation period, thereby producing an additional biosolid containing the existing portion of the biosolid. Additional biopolymer articles are produced from the additional biosolid by waiting for an additional final biosolidification period after step Z). In this example, the method further includes producing additional biopolymer articles by waiting for an additional final solidification period after step Z).

[0116] In one example of the method, the second, third, or fourth biopolymer aqueous solution is a silk aqueous solution, an alginate aqueous solution, a fibrinogen aqueous solution, a chitosan aqueous solution, a collagen aqueous solution, or a combination thereof. If the biopolymer aqueous solution is a silk aqueous solution, the corresponding second, third, fourth, or additional curing aqueous solution is a silk crosslinking and / or hydrogel initiation aqueous solution. If the biopolymer aqueous solution is an alginate aqueous solution, the second, third, fourth, or additional curing aqueous solution is an alginate crosslinking aqueous solution. If the biopolymer aqueous solution is a fibrinogen aqueous solution, the second, third, fourth, or additional curing aqueous solution is a fibrinogen curing aqueous solution. If the biopolymer aqueous solution is a collagen aqueous solution, the second, third, fourth, or additional curing aqueous solution is a collagen curing aqueous solution. If the biopolymer aqueous solution is a chitosan aqueous solution, the second, third, fourth, or additional curing aqueous solution is a chitosan curing aqueous solution (e.g., sodium hydroxide, ethanol, or a combination thereof).

[0117] In one example of the method, a sacrificial agent is used. The sacrificial agent is acrylonitrile butadiene styrene. The method includes a step of removing the sacrificial agent using acetone or alcohol.

[0118] In one example of the method, the chitosan, a second, third, fourth and / or additional biopolymer aqueous solution and / or the chitosan, a second, third, fourth and / or additional curing aqueous solution further comprises additives (e.g., nanoparticles, structural modification materials, or bioactive materials).

[0119] Depending on the embodiment, various functionalizing agents may be used with the silk-containing embodiments described herein (e.g., silk films, silk compositions, silk articles, silk matrices, silk foams, silk microspheres, liquid compositions, whipped silk cream, silk meringue, compressed silk meringue, hot-pressed silk meringue, silk leather, silk powder, silk toner, etc.). The examples herein may describe one or a few silk-containing embodiments, but it should be understood that they may be applicable to any silk-containing embodiment where applicable. In some embodiments, the functionalizing agent may be any compound or molecule that promotes the adhesion of one or more endothelial cells to and / or development (e.g., growth) on the silk film. In some embodiments, the functionalizing agent may be any compound or molecule that promotes the adhesion of one or more megakaryocytes and / or hematopoietic progenitor cells to and / or development (e.g., growth) on the silk matrix and / or silk film. In some embodiments, the functionalizing agent may be or may include agents suitable for promoting the production of one or more leukocytes and erythrocytes.

[0120] In some embodiments, the functionalizing agent may be or may include cell adhesion mediators and / or extracellular matrix proteins, such as collagen (e.g., type I, type III, type IV, or type VI collagen), elastin, fibronectin, vitronectin, laminin, fibrinogen, von Willebrand factor, proteoglycan, decorin, perlecan, nidogen, hyaluronan, and / or peptides containing known integrin-binding domains, such as peptides containing the “RGD” integrin-binding sequence or a variant thereof, which is known to affect cell adhesion.

[0121] In some embodiments, the functionalizing agent may be any soluble molecule produced by endothelial cells. Non-limiting examples include fibroblast growth factor-1 (FGF1) and vascular endothelial growth factor (VEGF).

[0122] According to some embodiments, multiple functionalizing agents may be used. For example, in some embodiments where platelet production is desired, the provided composition may include the use of laminin, fibronectin and / or fibrinogen, and type IV collagen to promote the adhesion and growth of endothelial cells to a silk membrane (e.g., a porous silk membrane) and / or the adhesion of megakaryocytes to the silk matrix.

[0123] In some embodiments, the functionalizing agent can be embedded in or otherwise associated with the silk film and / or silk matrix, in contrast to the functionalizing agent simply being placed along the surface of the silk film and / or silk matrix. In some embodiments, the functionalizing agent can be distributed along and / or incorporated into substantially the entire surface area of ​​the silk film / silk wall. In some embodiments, the functionalizing agent can be distributed and / or incorporated into only one or more distinct portions of the silk film / silk wall and / or silk matrix. In some embodiments, the functionalizing agent can be distributed to and / or incorporated along at least one of the luminal-side surface of the silk wall and the matrix-side surface of the silk wall.

[0124] According to various embodiments, an appropriate amount of one or more functionalizing agents may be used for any application. In some embodiments, the amount of each functionalizing agent may be between about 1 μg / ml and 1,000 μg / ml (e.g., between about 2 to 1,000, 5 to 1,000, 10 to 1,000, 10 to 500, or 10 to 100 μg / ml). In some embodiments, the amount of each functionalizing agent may be at least 1 μg / ml (e.g., at least 5, 10, 15, 20, 25, 50, 100, 200, 300, 400, 500, 600, 700, 800, or 900 μg / ml). In some embodiments, the amount of each functionalizing agent is at most 1,000 μg / ml (for example, 900, 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, or 5 μg / ml).

[0125] In some embodiments, the composition comprises one or more detection agents, e.g., detection dyes. The detection agents / detection dyes are environmentally sensitive and produce a measurable response to one or more environmental factors. In some embodiments, the environmentally sensitive agent or dye may be present in the composition in an amount effective enough to change the composition from a first physicochemical state to a second chemical-physical state in response to environmental parameters (e.g., pH, luminosity or exposure, temperature, pressure or strain, potential, physiological parameters of the subject, and / or changes in the concentration of chemical species in the surrounding environment) or externally applied stimuli (e.g., optical queries, acoustic queries, and / or applied heat). In some cases, the detection 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. Preferred concentrations of the detection agents described herein may be the concentrations of the colorants and additives described elsewhere herein. Those skilled in the art of chemical detection can determine concentrations suitable for use in detection applications of the inks described herein.

[0126] In some embodiments, the first and second chemical-physical states may be physical properties of the composition, such as mechanical properties, chemical properties, acoustic properties, electrical properties, magnetic properties, optical properties, thermal properties, radiological properties, or sensory properties. Examples of detection dyes or agents include, but are not limited to, pH-sensitive agents, thermosensitive agents, pressure-sensitive or strain-sensitive agents, photosensitive agents, or potentiometric agents.

[0127] Examples of pH-sensitive dyes or agents include cresol red, methyl violet, crystal violet, ethyl violet, malachite green, methyl green, 2-(p-dimethylaminophenylazo)pyridine, paramethyl red, methanyl yellow, 4-phenylazodiphenylamine, thymol blue, metacresol purple, orange IV, 4-o-tolylazo-o-toluindine, quinaldine red, 2,4-dinitrophenol, erythrosine disodium salt, and benzopurprine 4B. N,N-dimethyl-p-(m-tolyl azo)aniline, p-dimethylaminoazobenene, 4,4'-bis(2-amino-1-naphthylazo)-2,2'-stilbendisulfonic acid, tetrabromophenolphthalein ethyl ester, bromophenol blue, Congo red, methyl orange, ethyl orange, 4-(4-dimethylamino-1-naphthylazo)-3-methoxybenesulfonic acid (acid), bromocresol green, resazurin, 4-phenylazo-1-naphthylamine, ethyl red 2-(1-dimethylaminophenylazo)pyridine, 4-(p-ethoxyphenylazo)-m-phenylene-diamine monohydrochloride monohydrochloride), resorcinol blue, alizarin red S, methyl red, propyl red, bromocresol purple, chlorophenol red, p-nitrophenol, alizarin 2-(2,4-dinitrophenylazo)1-naphthol-3,6-disulfonic acid, bromothymol blue, 6,8-dinitro-2,4-(1H)quinazolindione, brilliant yellow, phenol red, neutral red, m-nitrophenol, cresol red, turmeric, metacresol purple, 4,Examples include, but are not limited to, 4'-bis(3-amino-1-naphthylazo)-2,2'-stilbendisulfonic acid, thymol blue, p-naphtholbenzeyne, phenolphthalein, o-cresolphthalein, ethylbis(2,4-dimethylphenyl)ethanolate, thymolphthalein, nitrazine yellow, alizarin yellow R, alizarin, p-(2,4-dihydroxyphenylazo)benzenesulfonic acid, 5,5'-indigodisulfonic acid, 2,4,6-trinitrotoluene, 1,3,5-trinitrobenzene, and Clayton yellow.

[0128] Examples of photochromic dyes or agents include, but are not limited to, triarylmethane, stilbene, azasilbene, nitrone, fulgide, spiropyran, naphtopyran, spiro-oxazine, quinone, derivatives thereof, and combinations thereof.

[0129] Examples of potentiometric dyes include, but are not limited to, substituted aminonaphthylethenylpyridinium (ANEP) dyes, such as di-4-ANEPPS, di-8-ANEPPS, and N-(4-sulfobutyl)-4-(6-(4-dibutylamino)phenyl)hexatrienyl)pyridinium (RH237).

[0130] Examples of temperature-sensitive dyes or agents include, but are not limited to, thermochromic compounds or thermochromic agents, such as thermochromic liquid crystals, leuco dyes, fluorane dyes, and octadecylphosphonic acid.

[0131] Examples of pressure-sensitive or strain-sensitive dyes or agents include, but are not limited to, spiropyran compounds and agents.

[0132] Examples of chemically sensitive dyes or agents include, but are not limited to, antibodies such as immunoglobulin G (IgG) that can change color from blue to red in response to bacterial contamination.

[0133] In some embodiments, the compound comprises one or more additives, dopants, or biological agents suitable for the desired intended purpose. In some embodiments, the additives or dopants may be present in the composition in an amount effective to impart optical or sensory properties to the composition. Examples of additives or dopants that impart optical or sensory properties include, but are not limited to, dyes / pigments, fragrances, aromatic compounds, granules, or fiber fillers.

[0134] In addition, or alternatively, additives, dopants, or bioactive agents may be present in a composition in an amount effective enough to “functionalize” the composition to impart a desired mechanical property or added functionality to the composition. Examples of additives, dopants, or bioactive agents that impart a desired mechanical property or added functionality include, but are not limited to, environmentally sensitive / detectable dyes; active biomolecules; conductive or metallic particles; micro and nanofibers (e.g., silk nanofibers, carbon nanofibers for reinforcement); nanotubes; inorganic particles (e.g., hydroxyapatite, tricalcium phosphate, bioglass); drugs (e.g., antibiotics, small molecules or low molecular weight organic compounds); proteins and their fragments or complexes (e.g., enzymes, antigens, antibodies and their antigen-binding fragments); DNA / RNA (e.g., siRNA, miRNA, mRNA); cells and their fractions (viruses and viral particles; prokaryotic cells, e.g., bacteria; eukaryotic cells, e.g., mammalian cells and plant cells; fungi).

[0135] In some embodiments, the additive or dopant includes a flavoring agent or fragrance.

[0136] Examples of flavorings include ester flavorings, amino acid flavorings, nucleic acid flavorings, organic acid flavorings, and inorganic acid flavorings, for example, but not limited to, diacetyl, acetylpropionyl, acetoin, isoamyl acetate, benzaldehyde, cinnamaldehyde, ethyl propionate, methyl anthranilate, limonene, ethyl decadienoate, allyl hexanoate, ethyl maltol, ethyl vanillin, methyl salicylate, manzanate, glutamate, glycine salt, guanylate, inosinate, acetic acid, ascorbic acid, citric acid, fumaric acid, lactic acid, malic acid, phosphoric acid, tartaric acid, derivatives and mixtures thereof.

[0137] In some embodiments, the additive or dopant includes an aromatic compound. Examples of aromatic compounds include ester aromatic compounds, terpene aromatic compounds, cyclic terpenes, and aromatic aromatic compounds, for example, but not limited to these, 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, myrecene, geraniol, nerol, citral, citronellal, citronellol, linalool, nerolidol, limonene, camphor, menthol, chalon, terpineol, alpha-ionone, thujone, eucalyptol, benzaldehyde, eugenol, cinnamaldehyde, ethyl maltol, vanillin, anisole, anethole, estragol, and thymol.

[0138] In some embodiments, the additive or dopant includes a colorant, such as a dye or pigment. In some embodiments, the dye or pigment imparts color or grayscale to the composition. The colorant may differ from the detection agents and / or detection dyes described below. Any organic and / or inorganic pigments and dyes may be included in the ink. Examples of pigments suitable for use in this disclosure include those in the International Color Index, namely CI Pigment Black 1, 7, 11 and 31; CI Pigment Black 15, 15:1, 15:2, 15:3, 15:4, 15:6, 16, 27, 29, 61 and 62; CI Pigment Green 7, 17, 18 and 36; CI Pigment Orange 5, 13, 16, 34 and 36; CI Pigment Violet 3, 19, 23 and 27; CI Pigment Red 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, CI Pigments Yellow 1, 2, 3, 12, 13, 14, 17, 42, 65, 73, 74, 75, 83, 30, 93, 109, 110, 128, 138, 139, 147, 142, 151, 154 and 180, D&C Red 7, D&C Red 6 and D&C Red 34, Carbon Black Pigment (e.g., Regal Examples include 330 (Cabot Corporation), quinacridone pigment (quinacridone magenta (228-0122), available from Sun Chemical Corporation, Fort Lee, NJ), diallylide yellow pigment (e.g., AAOT Yellow (274-1788), available from Sun Chemical Corporation); and phthalocyanine blue pigment (e.g., Blue 15:3 (294-1298), available from Sun Chemical Corporation). Classes of dyes suitable for use in the present invention may be selected from acid dyes, natural dyes, direct dyes (either cationic or anionic), basic dyes, and reactive dyes.Acid dyes, also considered anionic dyes, are soluble in water and primarily insoluble in organic solvents, and are selected from yellow acid dyes, orange acid dyes, red acid dyes, purple acid dyes, blue acid dyes, green acid dyes, and black acid dyes. European Patent 0745651, incorporated herein by reference, describes several acid dyes suitable for use in this disclosure. Examples of yellow acid dyes include Acid Yellow 1 (International Color Index, i.e., CI 10316), Acid Yellow 7 (CI 56295), Acid Yellow 17 (CI 18965), Acid Yellow 23 (CI 19140), Acid Yellow 29 (CI 18900), Acid Yellow 36 (CI 13065), Acid Yellow 42 (CI 22910), Acid Yellow 73 (CI 45350), Acid Yellow 99 (CI 13908), Acid Yellow 194, and Food Yellow 3 (CI 15985). Examples of orange acid dyes include Acid Orange 1 (CI 13090 / 1), Acid Orange 10 (CI 16230), Acid Orange 20 (CI 14603), Acid Orange 76 (CI 18870), Acid Orange 142, Food Orange 2 (CI 15980), and Orange B.

[0139] Examples of red acid dyes include Acid Red 1 (CI 18050); Acid Red 4 (CI 14710); Acid Red 18 (CI 16255); Acid Red 26 (CI 16150); Acid Red 2.7 (CI 16255); Acid Red 51 (CI 45430, BASF) Examples of acid red dyes (available from Corporation, Mt. Olive, NJ) include Acid Red 52 (CI 45100); Acid Red 73 (CI 27290); Acid Red 87 (CI 45380); Acid Red 94 (CI 45440); Acid Red 194; and Food Red 1 (CI 14700). Exemplary purple acid dyes include Acid Violet 7 (CI 18055); and Acid Violet 49 (CI 42640). Exemplary blue acid dyes include Acid Blue 1 (CI 42045); Acid Blue 9 (CI 42090); Acid Blue Examples of acid dyes include -22 (CI 42755), Acid Blue 74 (CI 73015), Acid Blue 93 (CI 42780), and Acid Blue 158A (CI 15050). Exemplary green acid dyes include Acid Green 1 (CI 10028), Acid Green 3 (CI 42085), Acid Green 5 (CI 42095), Acid Green 26 (CI 44025), and Food Green 3 (CI 42053). Exemplary black acid dyes include Acid Black 1 (CI 20470) and Acid Black 194 (Basantol® X80, available from BASF Corporation, an azo / 1:2 CR complex).

[0140] Examples of direct dyes for use in this disclosure include Direct Blue 86 (CI 74180); Direct Blue 199; Direct Black 168; Direct Red 253; and Direct Yellow 107 / 132 (CI unassigned).

[0141] Examples of natural dyes for use in this disclosure include alkanet (CI 75520, 75530); annatto (CI 75120); carotene (CI 75130); chestnut; cochineal (CI 75470); kutch (CI 75250, 75260); zibizibi; fastic (CI 75240); hypernic (CI 75280); logwood (CI 75200); osage orange (CI 75660); paprika; quer citron (CI 75720); sanrou (CI 75100); sandalwood (CI 75510, 75540, 75550, 75560); smack; and turmeric (CI 75300). Examples of reactive dyes for use in this disclosure include Reactive Yellow 37 (monoazo dye); Reactive Black 31 (disazo dye); Reactive Blue 77 (phthalocyanine dye); and Reactive Red 180 and Reactive Red 108 dyes. Colorants described in The Printing Ink Manual (5th ed., Leach et al. eds. (2007), pages 289-299) are also suitable. Other organic and inorganic pigments and dyes, as well as combinations thereof, can be used to obtain desired colors.

[0142] In addition to, or instead of, visible colorants, the compositions provided herein may contain ETV fluorophores that are excited in the ETV range and emit light at higher wavelengths (typically 400 nm and above). Examples of ETV fluorophores include, but are not limited to, coumarin, benzoxazole, rhodamine, napthalimide, perylene, benzoanthrone, benzoxanthone, or materials derived from the benzothia-xanthone family. The addition of UV fluorophores (e.g., optical glossants) may help maintain maximum visible light transmittance. The amount of colorant, if present, is generally between 0.05% and 5% or between 0.1% and 1% based on the weight of the composition.

[0143] For non-white compositions, the amount of pigment / dye is generally present in an amount of 0.1% by weight or about 0.1% to 20% by weight or about 20% by weight, based on the weight of the composition. In some applications, non-white inks may contain 15% by weight or less of pigment / dye, or 10% by weight or less of pigment / dye, or 5% by weight of pigment / dye, or 1% by weight of pigment / dye, based on the weight of the composition. In some applications, non-white inks may contain 1% to 10% by weight, or 5% to 15% by weight, or 10% to 20% by weight of pigment / dye, based on the weight of the composition. In some applications, non-white inks may contain amounts of dye / pigment that are 1% by weight, 2% by weight, 3% by weight, 4% by weight, 5% by weight, 6% by weight, 7% by weight, 8% by weight, 9% by weight, 10% by weight, 11% by weight, 12% by weight, 13% by weight, 14% by weight, 15% by weight, 16% by weight, 17% by weight, 18% by weight, 19% by weight, or 20% by weight, based on the weight of the composition.

[0144] For white compositions, the amount of white pigment is generally 1% by weight or about 1% by weight to 60% by weight or about 60% by weight, based on the weight of the composition. In some applications, more than 60% by weight of white pigment may be present. Preferred white pigments include titanium dioxide (antalite and rutile), zinc oxide, lithopone (calcined coprecipitate of barium sulfate and zinc sulfide), zinc sulfide, blankfix and hydrated alumina, and combinations thereof, any of which may be combined with calcium carbonate. In some applications, the white ink may contain 60% by weight or less of white pigment, or 55% by weight or less of white pigment, or 50% by weight of white pigment, or 45% by weight of white pigment, or 40% by weight of white pigment, or 35% by weight of white pigment, or 30% by weight of white pigment, or 25% by weight of white pigment, or 20% by weight of white pigment, or 15% by weight of white pigment, or 10% by weight of white pigment, based on the weight of the composition. In some applications, white inks may contain 5% to 60% by weight of white pigment, or 5% to 55% by weight, or 10% to 50% by weight, or 10% to 25% by weight, or 25% to 50% by weight, or 5% to 15% by weight, or 40% to 60% by weight, based on the weight of the composition. In some applications, non-white inks may contain 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32% by weight, based on the weight of the composition. The amount of dye / pigment may be 1% by weight, 33% by weight, 34% by weight, 35%, 36% by weight, 37% by weight, 38% by weight, 39% by weight, 40% by weight, 41% by weight, 42% by weight, 43% by weight, 44% by weight, 45%, 46% by weight, 47% by weight, 48% by weight, 49% by weight, 50% by weight, 51% by weight, 52% by weight, 53% by weight, 54% by weight, 55%, 56% by weight, 57% by weight, 58% by weight, 59% by weight, or 60% by weight.

[0145] In some embodiments, the additive or dopant includes a conductive additive. Examples of conductive additives include, but are not limited to, graphite, graphite powder, carbon nanotubes, and metal particles or nanoparticles, such as gold nanoparticles. In some embodiments, the conductive additive is biocompatible and nontoxic.

[0146] In some embodiments, the additive is a biologically active agent. The term “biologically active agent,” as used herein, refers to any molecule that exerts at least one biological effect in vivo. For example, a biologically active agent may be a therapeutic agent for treating or preventing a disease or condition in a subject. Biologically active agents include, but are not limited to, organic molecules, inorganic molecules, 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 compositions provided herein include, but are not limited to, anticancer agents, antibiotics, analgesics, anti-inflammatory agents, immunosuppressants, enzyme inhibitors, antihistamines, anticonvulsants, hormones, muscle relaxants, antispasmodics, ophthalmic agents, prostaglandins, antidepressants, antipsychotics, nutrients, bone-inducing proteins, growth factors, and vaccines.

[0147] The term “active agent” may also be used herein to refer to a biological sample (e.g., a tissue or bodily fluid sample, such as blood) or its components, and / or to a biologically active entity or compound, and / or to a structurally or functionally unstable entity.

[0148] Examples of active agents include, but are not limited to, therapeutic agents, diagnostic agents (e.g., contrast agents), and any combination thereof. In some embodiments, active agents present in a silk matrix (e.g., silk microspheres) or composition may include unstable active agents, such as agents that may undergo chemical, physical, or biological changes, degradation, and / or inactivation after exposure to certain conditions, such as high temperature, high humidity, exposure, and any combination thereof. In some embodiments, active agents present in a silk matrix (e.g., silk microspheres) or composition may include temperature-sensitive active agents, such as agents that lose at least about 30% or more of their original activity or biological activity upon exposure to temperatures of at least about 10°C or higher, including temperatures of at least about 15°C or higher, at least near room temperature or higher, or at least near body temperature (e.g., about 37°C) or higher.

[0149] The active agent may generally be present in a silk matrix (e.g., silk microspheres) or composition in amounts of about 0.01% (w / w) to about 70% (w / w), or about 0.1% (w / w) to about 50% (w / w), or about 1% (w / w) to about 30% (w / w). The active agent may be present on the surface of the silk matrix (e.g., silk microspheres) or composition, and / or may be encapsulated and dispersed uniformly, non-uniformly, or in a gradient manner within the silk matrix (e.g., silk microspheres) or composition. In some embodiments, the active agent may be added to a silk solution and then subjected to the methods described herein for preparing a silk matrix (e.g., silk microspheres) or composition. In some embodiments, the active agent may be coated onto the surface of the silk matrix (e.g., silk microspheres) or composition. In some embodiments, the active agent can be loaded onto a silk matrix (e.g., silk microspheres) or a composition by incubating the active agent in a solution for a period of time during which a certain amount of the active agent can diffuse into the silk matrix (e.g., silk microspheres) or a composition, and thus be distributed into the silk matrix (e.g., silk microspheres) or a composition.

[0150] In some embodiments, additives are therapeutic agents. As used herein, the term “therapeutic agent” means a molecule, group of molecules, complex or substance administered to an organism for diagnostic, therapeutic, preventive medicine or veterinary purposes. As used herein, the term “therapeutic agent” includes “drug” or “vaccine.” This term includes topical, localized and systemic human and animal medicinal products, treatment agents, therapeutic means, nutritional supplements, medicinal cosmetics, biological products, devices, diagnostic agents and contraceptives, including preparations useful in clinical and veterinary screening, prevention, disease prevention, cure, wellness, detection, imaging, diagnosis, treatment, surgery, monitoring, cosmetics, prosthetics and orthotics, and forensic science. This term may also be used to refer to therapeutic agents or therapeutic means for agricultural, workplace, military, industrial and environmental use, including or achievable contact with plants, animals and / or humans, including cell receptors, membrane receptors, hormone receptors, therapeutic receptors, microorganisms, viruses or selected molecules or selected nucleic acid sequences capable of recognizing selected targets. This term may particularly include nucleic acids and nucleic acid-containing compounds, including, for example, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), nucleic acid analogs (e.g., locked nucleic acid (LNA), peptide nucleic acid (PNA), xeno nucleic acid (XNA)), or mixtures or combinations thereof, which produce therapeutic effects, such as DNA nanoplexes, siRNA, microRNA, shRNA, aptamers, ribozymes, decoy nucleic acids, antisense nucleic acids, and RNA activators. In general, any therapeutic agent can be included in the compositions provided herein.

[0151] The term “therapeutic agent” also includes drugs that can produce local or systemic biological, physiological, or therapeutic effects in the biological system to which they are applied. For example, therapeutic agents can act, among many functions, to control infection or inflammation, to promote cell growth and tissue regeneration, to control tumor growth, to act as an analgesic, to promote anti-cell adhesion, and to promote bone growth. Other suitable therapeutic agents include antiviral agents, hormones, antibodies, or therapeutic proteins. Other therapeutic agents include prodrugs, which are drugs that are not biologically active when administered but are converted into biologically active drugs by metabolism or some other mechanism once administered to a target. In addition, silk-based drug delivery compositions may contain one therapeutic agent or a combination of two or more therapeutic agents.

[0152] Therapeutic agents may include a wide variety of different compounds, including compounds and mixtures of compounds, e.g., small organic or inorganic molecules; sugars; oligosaccharides; polysaccharides; biomacromolecules, e.g., peptides, proteins, and peptide analogs and derivatives; peptide mimetic drugs; antibodies and their antigen-binding fragments; nucleic acids; nucleic acid analogs and derivatives; extracts produced from biological materials such as bacteria, plants, fungi, or animal cells; animal tissues; naturally occurring or synthetic compositions; and any combination thereof. In some embodiments, the therapeutic agent is a small molecule.

[0153] When used herein in relation to active agents, the term “bioactivity” generally refers to the ability of an active agent to interact with and / or produce an effect on a biological target. For example, bioactivity may include, but is not limited to, eliciting an irritant, inhibitory, modulating, toxic, or lethal response in a biological target. A biological target may be a molecule or a cell. For example, bioactivity may refer to the ability of an active agent to modulate the effect / activity of an enzyme, to block a receptor, to stimulate a receptor, to modulate the expression level of one or more genes, to modulate cell proliferation, to modulate cell division, to modulate cell morphology, or any combination thereof. In some cases, bioactivity may 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 target proteins or other molecules by cells; activation of membrane surface molecules, including receptor activation; transmembrane ion transport; transcriptional regulation; changes in cell viability; changes in cell morphology; changes in the presence or expression of intracellular components; changes in gene expression or transcripts; changes in the activity of enzymes produced within cells; and changes in the presence or expression of ligands and / or receptors (e.g., protein expression and / or binding activity). Methods for assaying different cellular responses, such as Western blotting to determine the presence or changes in the expression of endogenous proteins in cells, or microscopy to monitor cell morphology in response to active agents, or FISH and / or qPCR for the detection and quantification of nucleic acid changes, are well known to those skilled in the art. In some embodiments, biological activity may be determined, for example, by assaying cellular responses.

[0154] With respect to antibodies, the term “bioactivity” includes, but is not limited to, epitope or antigen-binding affinity, in vivo and / or in vitro stability of the antibody, immunogenicity of the antibody when administered to, for example, 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 techniques known in the art, such as scintillation proximity assays; ELISA; ORIGEN immunoassay (IGEN); fluorescence quenching; fluorescence ELISA; competitive ELISA; SPR analysis including, but not limited to, SPR analysis using BIAcore biosensors; in vitro and in vivo neutralization assays (see, e.g., International Publication No. WO2006 / 062685); receptor binding; and immunohistochemical tests using tissue sections from different sources, including humans, primates, or any other source as appropriate. With respect to immunogen, “bioactivity” includes immunogenicity, the definition of immunogenicity will be discussed in detail later. With respect to viruses, "bioactivity" includes infectivity, the definition of infectivity will be discussed in detail later. With respect to contrast agents, such as dyes, "bioactivity" refers to the ability of the contrast agent to enhance the contrast of structures or fluids within a subject when administered to that subject. The bioactivity of a contrast agent also includes, but is not limited to, its ability to interact with the biological environment and / or influence the response of other molecules under certain conditions.

[0155] As used herein, the term “small molecule” may refer to a compound that is “natural product-like,” but is not limited to such compounds. More precisely, small molecules are typically characterized by containing several carbon-carbon bonds and having a molecular weight of less than 5000 daltons (5 kDa), preferably less than 3 kDa, more preferably less than 2 kDa, and most preferably less than 1 kDa. In some cases, small molecules preferably have a molecular weight of 700 daltons or less.

[0156] Examples of therapeutic agents include, but are not limited to, those found in Harrison's Principles of Internal Medicine, 13th Edition, Eds. TR Harrison et al. McGraw-Hill NY, NY; Physicians' Desk Reference, 50th Edition, 1997, Oradell New Jersey, Medical Economics Co.; Pharmacological Basis of Therapeutics, 8th Edition, Goodman and Gilman, 1990; and United States Pharmacopeia, The National Formulary, ETSP XII NF XVII, 1990, all of which are incorporated herein by reference in their entirety.

[0157] Therapeutic agents include the categories and specific examples disclosed herein. The categories are not intended to be limited by specific examples. Those skilled in the art will also recognize a great many other compounds that fall into the categories and are useful in accordance with this disclosure. Examples include radiosensitizers, steroids, xanthines, beta-2-agonist bronchodilators, anti-inflammatory agents, analgesics, calcium channel blockers, angiotensin-converting enzyme inhibitors, beta-blockers, centrally acting alpha-agonists, alpha-1-antagonists, anticholinergic / antispasmodics, vasopressin analogs, antiarrhythmics, antiparkinsonian agents, antianginic / antihypertensive agents, anticoagulants, antiplatelet agents, sedatives, anxiolytic agents, peptide agents, biopolymers, antineoplastic agents, laxatives, antidiarrheal agents, antimicrobial agents, antifungal agents, vaccines, proteins, or nucleic acids. In a further embodiment, the pharmaceutically active agent is an anti-inflammatory agent, including coumarins, albumins, steroids such as betamethasone, dexamethasone, methylprednisolone, prednisolone, prednisone, triamcinolone, budesonide, hydrocortisone, and pharmaceutically acceptable hydrocortisone derivatives; xanthines such as theophylline and doxophiline; beta-2-bronchodilators such as salbutamol, fenterol, clenbuterol, bambuterol, salmeterol, fenoterol; and anti-asthmatic anti-inflammatory agents, anti-arthritis anti-inflammatory agents and nonsteroidal anti-inflammatory agents, the examples of which include Anti-inflammatory agents, including but not limited to sulfides, mesalamine, butezonide, sulfasalazine, diclofenac, pharmaceutically acceptable diclofenac salts, nimeslide, naproxen, acetaminophen, ibuprofen, ketoprofen, and piroxicam; analgesics, e.g., salicylates; calcium channel blockers, e.g., nifedipine, amlodipine, and nicardipine; angiotensin-converting enzyme inhibitors, e.g., captopril, benazepril hydrochloride, hosinopril sodium, trandolapril, ramipril, lisinopril, enalapril, quinapril hydrochloride, and moexipril hydrochloride;Beta-blockers (i.e., beta-adrenergic blockers), e.g., sotalol hydrochloride, timolol maleate, esmolol hydrochloride, carteolol, propanolol hydrochloride, betaxolol hydrochloride, penbutrol sulfate, metoprolol tartrate, metoprolol succinate, acebutrol hydrochloride, atenolol, pindolol, and bisoprolol fumarate; centrally acting alpha-2-agonists, e.g., clonidine; alpha-1-antagonists, e.g., doxazosin and prazosin; anticholinergic / antispasmodics, e.g., dicyclomine hydrochloride, scopolamine hydrobromide, glycol Pyroleates, clidinium bromide, flavoxates, and oxybutynin; vasopressin analogs, e.g., vasopressin and desmopressin; antiarrhythmic agents, e.g., quinidine, lidocaine, tocainide hydrochloride, mexiletine hydrochloride, digoxin, verapamil hydrochloride, propafenone hydrochloride, flecainide acetate, procainamide hydrochloride, morricidine hydrochloride, and disopyramide phosphate; antiparkinson's disease agents, e.g., dopamine, L-dopa / carbido Pa, selegiline, dihydroergocriptine, pergolide, lislide, apomorphine, and bromocriptine; antianginal and antihypertensive agents, e.g., isosorbide mononitrate, isosorbide dinitrate, propranolol, atenolol, and verapamil; anticoagulants and antiplatelet agents, e.g., coumadin, warfarin, acetylsalicylic acid, and ticlopidine; sedatives, e.g., benzodiazepines and barbiturates; anxiolytics Agents, e.g., lorazepam, bromazepam, and diazepam; peptide and biopolymer agents, e.g., calcitonin, leuprolide, and other LHRH agonists, hirudin, cyclosporine, insulin, somatostatin, protirelin, interferon, desmopressin, somatotropin, thymopentin, pyrotimod, erythropoietin, interleukin, melatonin, granulocyte / macrophage-CSF, and heparin;Antineoplastic agents, e.g., etoposide, etoposide phosphate, cyclophosphamide, methotrexate, 5-fluorouracil, vincristine, doxorubicin, cisplatin, hydroxyurea, leucovorin calcium, tamoxifen, flutamide, asparaginase, altoretamine, mitotane, and procarbazine hydrochloride; laxatives, e.g., senna concentrate, casanthranol, bisacodyl, and sodium picosulfate; antidiarrheal agents, e.g., diphenoxine hydrochloride, loperamide hydrochloride, furazolidone, diphenoxylate hydrochloride These may include hydrochloride, microorganisms; vaccines, e.g., bacterial and viral vaccines; antimicrobial agents, e.g., penicillin-based antimicrobial agents, cephalosporin-based antimicrobial agents, and macrolide-based antimicrobial agents; antifungal agents, e.g., imidazole and triazole derivatives; and nucleic acids, e.g., DNA sequences encoding biological proteins, and antisense oligonucleotides.

[0158] Anticancer agents include alkylating agents, platinum compounds, antimetabolites, topoisomerase inhibitors, antitumor antibiotics, mitotic inhibitors, 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, hormones and antihormone agents, photodynamic agents, and tyrosine kinase inhibitors.

[0159] Antibiotics include aminoglycoside antibiotics (e.g., gentamicin, tobramycin, netylmycin, streptomycin, amikacin, neomycin), bacitracin, carbapenem antibiotics (e.g., imipenem / cislastatin), cephalosporin antibiotics (e.g., colistin, methenamine), monobactam antibiotics (e.g., aztreonam), penicillin (e.g., penicillin G, penicillin V, methicillin, nafcillin, oxacillin, cloxacillin, dicloxacillin, ampicillin, ammonium), This includes xylin, carbenicillin, ticarcillin, piperacillin, mezlocillin, azlocillin), polymyxin B, quinolones, and vancomycin; as well as bacteriostatic agents, such as chloramphenicol, clindamycin, macrolide antibiotics (e.g., erythromycin, azithromycin, clarithromycin), lincomycin, nitrofurantoin, sulfonamide antibiotics, tetracycline antibiotics (e.g., tetracycline, doxycycline, minocycline, demeclocycline), and trimethoprim. Metronidazole, fluoroquinolones, and ritampin are also included.

[0160] Enzyme inhibitors are substances that inhibit enzyme reactions. Examples of enzyme inhibitors include edrophonium chloride, N-methylphysostigmine, neostigmine bromide, physostigmine sulfate, tacrine, 1-hydroxymaleate, iodotubercidin, p-bromotetramisole, 10-(alpha-diethylaminopropionyl)-phenothiazine hydrochloride, carmidazolium chloride, hemicolinium-3,3,5-dinitrocatechol, diacylglycerol kinase inhibitor I, diacylglycerol kinase inhibitor II, 3-phenylpropargylamine, and N°-monomethyl-L-arginine acetate. acetate), carbidopa, 3-hydroxybenzylhydrazine, hydralazine, chlorgiline, deprenyl, hydroxylamine, iproniazide phosphate, 6-MeO-tetrahydro-9H-pyridoindole, niaramid, pargiline, quinacrine, semicarbazide, tranylcypromine, N,N-diethylaminoethyl-2,2-diphenylvalerate, 3-isobutyl-1-methylxanthine (3-isobutyl- Examples include l-methylxanthne, papaverine, indomethacin, 2-cyclooctyl-2-hydroxyethylamine hydrochloride, 2,3-dichloro-α-methylbenzylamine (DCMB), 8,9-dichloro-2,3,4,5-tetrahydro-1H-2-benzazepine hydrochloride, p-aminoglutethimide, p-aminoglutethimide tartrate, 3-iodotyrosine, alpha-methyltyrosine, azetazolamide, dichlorphenamide, 6-hydroxy-2-benzothiazole sulfonamide, and allopurinol.

[0161] Among the many antihistamines, pyriramine, chlorpheniramine, and tetrahydrazoline are particularly noteworthy.

[0162] Anti-inflammatory agents include corticosteroids, non-steroidal anti-inflammatory drugs (e.g., aspirin, phenylbutazone, indomethacin, sulindac, tolmetin, ibuprofen, piroxicam, and fenamate), acetaminophen, phenacetin, gold salts, chloroquine, D-penicillamine, methotrexate, colchicine, allopurinol, probenecid, and sulfinpyrazone.

[0163] Muscle relaxants include mephenesin, methocarbamol, cyclobenzaprine hydrochloride, trihexylphenidyl hydrochloride, levodopa / carbidopa, and biperiden.

[0164] Antispasmodics include atropine, scopolamine, oxyphenonium, and papaverine.

[0165] Analgesics include aspirin, phenylbutazone, indomethacin, sulindac, tolmetic, ibuprofen, piroxicam, fenamate, acetaminophen, phenacetin, morphine sulfate, codeine sulfate, meperidine, nalorphine, opioids (e.g., codeine sulfate, fentanyl citrate, hydrocodone bitartrate, loperamide, morphine sulfate, noscapine, norcodeine, normorphine, thebaine, nornaltolfimine, buprenorphine, chlornaltrexamine, funaltrexamione, nalbufine, nalorphine, naloxone, naloxonazine, naltrexone, and naltrindol), procaine, lidocaine, tetracaine, and dibucaine. Ophthalmic preparations include sodium fluorescein, rose bengal, metacholine, adrenaline, cocaine, atropine, alpha-chymotrypsin, hyaluronidase, betaxalol, pilocarpine, timolol, timolol salts, and combinations thereof.

[0166] Prostaglandins are a class of naturally occurring, chemically related, long-chain hydroxy fatty acids that are recognized in the art and have various biological effects.

[0167] Antidepressants are substances that can prevent or alleviate depression.

[0168] Examples of antidepressants include imipramine, amitriptyline, nortriptyline, protriptyline, desipramine, amoxapine, doxepin, maprotiline, tranylcypromine, phenelzine, and isocarboxazide.

[0169] Nutrients are factors whose continuous presence improves the viability or lifespan of cells. Nutrients include, but are not limited to, platelet-derived growth factor (PDGP), neutrophil-activating protein, monocyte chemotactic 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 growth factor, insulin-like growth factor, glial-derived growth neurotrophic factor, ciliary body-derived neurotrophic factor, nerve growth factor, bone growth / cartilage-inducing factors (alpha and beta), osteomorphic proteins, interleukins (e.g., interleukin inhibitors, or interleukin receptors including interleukin 1 to 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 factor; and transforming growth factors (beta), including beta-1, beta-2, beta-3, inhibin, and activin.

[0170] Hormone preparations include estrogens (e.g., estradiol, estrone, estriol, diethylstibestrol, kinestrol, chlorotrianicene, ethinylestradiol, mestranol), antiestrogens (e.g., clomiphene, tamoxifen), progestins (e.g., medroxyprogesterone, norethindrone, hydroxyprogesterone, norgestrel), antiprogestins (mifepristone), and These include hormones (e.g., testosterone cypionic acid, fluoxymesterone, danazol, testolactone), antiandrogens (e.g., cyproterone acetate, flutamide), thyroid hormones (e.g., triiodothyronne, thyroxine, propylthiouracil, methimazole, and iodyxode), and pituitary hormones (e.g., corticotropin, somatotropin, oxytocin, and vasopressin). Hormone preparations are commonly used in hormone replacement therapy and / or for fertility control. Steroid hormones, such as prednisone, are also used as immunosuppressants and anti-inflammatory agents. In some embodiments, the additives are agents that stimulate tissue formation, as well as / or the healing and regrowth of natural tissues, and any combination thereof. Agents that increase the formation of new tissue and / or stimulate the healing or regrowth of native tissue at the injection site include, but are not limited to, fibroblast growth factor (FGF), transforming growth factor-beta (TGF-beta), platelet-derived growth factor (PDGF), epidermal growth factor (EGF), connective tissue activating peptide (CTAP), bone morphogenetic proteins and other bone morphogenetic factors, heparin, angiotensin II (A-II) and its fragments, insulin-like growth factor, tumor necrosis factor, interleukin, colony-stimulating factor, erythropoietin, nerve growth factor, interferon, bioactive analogs, fragments and derivatives of such growth factors, and any combination thereof.

[0171] In some embodiments, the silk composition may further include at least one additional material for soft tissue enhancement, such as dermal fillers, hyaluronic acid, commercially available dermal fillers, such as BOTOX® (from Allergan), DYSPORT®, COSMODERM®, EVOLENCE®, RADIESSE®, RESTYLANE®, JUVEDER® (from Allergan), SCULPTRA®, PERLANE®, and CAPTIQEIE®, as well as any combination thereof.

[0172] In some embodiments, the additive is a wound healing agent. As used herein, “wound healing agent” is a compound or composition that actively promotes the wound healing process.

[0173] Examples of wound-healing agents include dexpanthenol; growth factors; enzymes, hormones; povidone-iodide; fatty acids; anti-inflammatory agents; antibiotics; antimicrobial agents; disinfectants; cytokines; thrombin; analgesics; opioids; aminoxyl; floxane; nitrosothiols; nitrates and anthocyanins; nucleosides, e.g., adenosine; and nucleotides, e.g., adenosine diphosphate (ADP) and adenosine triphosphate (ATP); neurotransmitters. Examples of mitters / neuronal modulators, e.g., acetylcholine and 5-hydroxytryptamine (serotonin / 5-HT); histamine and catecholamines, e.g., adrenaline and noradrenaline; lipid molecules, e.g., 5-sphingosine-1-phosphate and lysophosphatidic acid; amino acids, e.g., arginine and lysine; peptides, e.g., bradykinin, substance P and calcium gene-related peptide (CGRP); nitrogen oxides; and any combination thereof, but not limited to these.

[0174] In certain embodiments, the active agent provided herein is an immunogen. In one embodiment, the immunogen is a vaccine. Most vaccines are sensitive to the environmental conditions in which they are stored and / or transported. For example, freezing may increase the reactivity (e.g., the ability to elicit an immunological response) and / or loss of efficacy for some vaccines (e.g., HepB, and DTaP / IPV / FQB) or may cause cracking of the container, leading to contamination. Furthermore, some vaccines (e.g., BCG, varicella, and MMR) are sensitive to heat. Many vaccines (e.g., BCG, MMR, varicella, group C meningococcal conjugate, and most DTaP-containing vaccines) are photosensitive. For example, see Galazka et al., Thermostability of vaccines, in Global Programme for Vaccines & Immunization (World Health Organization, Geneva, 1998); and Peettermans et al., Stability of freeze-dried rubella virus vaccine (Cendehill strain) at various temperatures, 1 J. Biological Standardization 179 (1973). Therefore, the compositions and methods provided herein also provide vaccine stability regardless of cold chain and / or other environmental conditions.

[0175] In some embodiments, the additive is a cell, for example, a living cell. Cells useful for incorporation into the composition may originate from any source, such as mammals, insects, plants, etc. In some embodiments, the cell may be a human, rat, or mouse cell. In general, the cell to be used with the compositions provided herein may be any type of cell. In general, the cell should be viable when encapsulated in the composition. In some embodiments, 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 embodiments, exemplary cells that can be used with the composition include platelets, activated platelets, stem cells, totipotent cells, pluripotent cells, and / or embryonic stem cells. In some embodiments, exemplary cells that can be encapsulated in the composition include, but are not limited to, primary cells and / or cell lines from any tissue. For example, cardiomyocytes, muscle cells, 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, spermatids, oocytes, liver cells, epithelial cells from the lungs, epithelial cells from the gut, epithelial cells from the intestine, epithelial cells from the liver, epithelial cells from the skin, and / or hybrids thereof may be included in the silk / platelet compositions disclosed herein. It will be apparent to those skilled in the art that the cells listed herein are illustrative, non-exclusive, lists of cells. Cells may be obtained from a donor (allogeneic) or from a recipient (autologous). Cells may be obtained, in non-limiting examples, by biopsy or other surgical means known to those skilled in the art.

[0176] In some embodiments, the cells may be genetically modified cells. Cells can be genetically modified to express and secrete desired compounds, such as bioactive drugs, growth factors, differentiation factors, and cytokines. Methods for genetically modifying cells to express and secrete compounds of interest are known in the art and can be readily adapted by those skilled in the art.

[0177] Differentiated cells that have been reprogrammed into stem cells can also be used.

[0178] For example, human skin cells reprogrammed into embryonic stem cells by transduction of Oct3 / 4, Sox2, c-Myc, and Klf4 (Junying Yu, et ah., Science, 2007, 318, 1917-1920 and Takahashi K. et ah., Cell, 2007, 131, 1-12).

[0179] All references cited herein, including published documents, patent applications, and patents, are incorporated herein by reference to the same extent as if they were individually and specifically indicated as being incorporated herein by reference in their entirety.

[0180] Preferred embodiments of the present invention, including the best methods known to the inventors for carrying out the invention, are described herein. By reading the above description, variations of these preferred embodiments may become apparent to those skilled in the art. The inventors anticipate that those skilled in the art will appropriately utilize such variations, and they intend that the invention may be carried out in ways other than those specifically described herein. Accordingly, the invention includes all modifications and equivalents of the subject matter described in the claims appended herein as permitted by applicable law. Furthermore, any combination of the above elements, in all possible variations thereof, is encompassed by the invention unless otherwise indicated herein or unless there is a clear and distinct inconsistency in the context. Although the present invention has been illustrated and described in detail in the preceding drawings and description, this should be considered to aid understanding and not to be inherently limiting, and it should be understood that it is desirable to protect the embodiments shown and described, as well as all modifications and alterations that fall within the spirit of the invention, as merely embodiments that aid understanding the present invention. For example, any feature or function of any embodiment disclosed herein can be incorporated into any of the other embodiments disclosed herein. [Examples]

[0181] (Example 1)

[0182] This describes an exemplary procedure for producing an article using a single biopolymer to produce a highly homogeneous article. The following steps were taken: 1) A 25 wt% silk solution was prepared according to a previously reported method (Rockwood, D., Preda, R., Yucel, T. et al. Materials fabrication from Bombyx mori silk fibroin. Nat Protoc 6, 1612-1631 (2011)) and stored in a syringe with care taken to reduce or eliminate the presence of air bubbles; 2) 4M 1) Prepare a solution of NaCl and 0.5M dipotassium hydrogen phosphate; 2) Deposit 1.5 mL of the silk solution into the mold; 3) Centrifuge the mold and its contents at 2000 × g for 3 minutes to force the silk solution to conform to the mold and fill all empty spaces; 4) Carefully deposit 1.5 mL of the ionic solution on top of the silk solution, taking care not to disturb the settled layer, and wait for polymerization to begin; 5) Centrifuge the mold and its contents at 2000 × g for 3 minutes to drive polymerization / bath osmosis, forcing the solution to conform to the mold and thus during solidification. 7) Counteract any shrinkage and geometric changes that occur; gently aspirate the supernatant and polymerization solution, taking care not to interfere with the polymerization of the silk; 8) Verify that the silk solution has not completely polymerized (this can be visually observed by the transition from yellow to white, but other methods are also known. If the previously deposited layer is overpolymerized, an undesirable, clearly distinguishable layer will be formed; 9) Repeat steps 3-8) until the mold is filled; and 10) Deposit the ionic solution on top of the mold and leave for 24 hours to polymerize completely.

[0183] The amount of material to be deposited and the number of cycles can be customized for specific material and volume requirements.

[0184] The article possessed desirable material properties. The material was very strong, bulky, and uniform. The article could be used for bone applications because it is stronger than other silk techniques, yet more elastic than heat-treated silk, thus offering several advantages over previous methods.

[0185] (Example 2)

[0186] This describes one exemplary procedure for producing an article, specifically a procedure for producing a highly uniform article using two different biopolymers. The following steps were taken: 1) 1.5 mL of chitosan dissolved in acetic acid was introduced into a mold; 2) The mold and its contents were centrifuged at 2000 × g for 3 minutes to allow the chitosan solution to conform to the mold; 3) 1.5 mL of a 5% NaOH and 70% ethanol solution was deposited into the mold to initiate the polymerization process; 4) The mold and its contents were centrifuged at 2000 × g for 3 minutes to drive the polymerization process, forcing the solution to conform to the mold and thereby counteracting any shrinkage and geometric changes that occur during solidification; 5) The mold was left to stand until the polymerization of the chitosan was complete (24 hours); 6) The polymerized chitosan and the inside of the mold were rinsed with water (preferably DI water). Wash again to remove any remaining NaOH; 7) Deposit 1.5 mL of the above silk solution directly on top of the chitosan in the mold; 8) Centrifuge the mold and its contents at 2000 × g for 3 minutes to force the silk solution to conform to the mold and fill any empty spaces; 9) Initiate the crystallization of the silk by depositing 1.5 mL of the above ionic solution on top of the silk solution, taking care not to disturb the settled layer; Centrifuge the mold and its contents at 2000 × g for 3 minutes to drive polymerization / bath immersion, force the solution to conform to the mold, and thereby counteract any shrinkage and geometric changes that occur during solidification; 11) Leave the mold to stand until the polymerization of the silk is complete (24 hours).

[0187] Similar to Example 1, the amount of material to be deposited and the number of cycles can be customized for specific material and volume requirements.

[0188] The article possessed desirable material properties. The article exhibited a duality of material properties, most notably the strength and robustness of silk, but also solubility enhanced by another layer of chitosan.

[0189] (Example 3)

[0190] Referring to Figure 4, first, the biopolymer solution is deposited into a pre-designed mold. Centrifugal force is used to conform the viscous bioink to the mold (adapting it to any desired geometry). Next, the polymerization bath / polymerizer is deposited locally onto the centrifuged biopolymer layer, and the mold is centrifuged again. By centrifuging the polymerizer and polymer together, polymerization is driven by compressive force and by the centrifugal force that permeates the polymerization bath agent into the biopolymer layer. The centrifugal force and time applied for each step of conforming the biopolymer and inducing polymerization in the salt bath are optimized, in particular for each material and polymerization method. After centrifuging the polymerization bath, the supernatant and polymerization bath are aspirated. Excess solution may be removed from the mold because liquid or gas pockets tend to form when the next layer of biopolymer is deposited locally. This can impair the structural integrity of the molded structure. These steps are repeated until the mold is completely filled. In some embodiments, the mold is overfilled, and then all excess material is scraped off.

[0191] Multi-material structures: Systems and methods for producing structures having clearly distinguishable composite layers are disclosed herein. This can be controlled by the degree of polymerization of subsequent layers. There are combinations of materials that can be incorporated into a single mold. Silk and chitosan, because silk has a negative charge and chitosan has a positive charge, can adhere to each other and form a composite structure having clearly distinguishable layers. After the deposition of the previous material, the supernatant and the previous polymerization bath can be aspirated by normal use. The molding process with the next desired material is then repeated by the same process.

[0192] Solvent bath additives: Since centrifugal force promotes the penetration of any material into the previous layer, additives can be incorporated into the material layer using this method. Often, this will be controlled by the density of the additive in the solution, the force applied, and the degree of polymerization of the previous layer.

[0193] (Example 4)

[0194] This describes one exemplary procedure for producing an article using chitosan to produce a highly uniform article. The following steps were taken: 1) Chitosan dissolved in acetic acid is introduced into a mold; 2) The mold and its contents are centrifuged, for example, at 2000 × g for 3 minutes, so that the chitosan solution conforms to the female mold; 3) NaOH and ethanol solution are deposited into the mold to initiate the polymerization process; 4) The mold and its contents are centrifuged to drive the polymerization process, forcing the solution to conform to the female mold and thereby counteracting any shrinkage and geometric changes that occur during solidification; 5) The mold is left to stand until the polymerization of the chitosan is complete (e.g., 24 hours); 6) The polymerized chitosan and the inside of the mold are repeatedly washed with water (preferably DI water) to remove any remaining NaOH. The amount of material to be deposited and the number of cycles of steps 1-6 can be repeated and / or customized for specific material and volume requirements.

[0195] While this disclosure is made in connection with preferred embodiments described in detail, various modified and improved forms of this disclosure will be readily apparent to those skilled in the art. Therefore, the essence and scope of this disclosure should not be limited by the examples given above, but should be interpreted in the broadest sense permitted by law.

[0196] In addition to the features described above and elsewhere in this specification, this disclosure also includes the following provisions: 1. A centrifugal solidification controlled biopolymer article, wherein the article is not a film, and at least a portion of the article is i) Uniform density over at least a portion of the article; ii) At least a portion of the article is optically opaque; iii) At least a portion of the article is free from external lamination; iv) At least a portion of the article is free from internal lamination; v) At least a portion of the article is free from internal voids; vi) No cylindrical extrusion gaps in at least a portion of the article; and vii) At least a portion of the article is free from lamination. A centrifugal solidification-controlled biopolymer article comprising at least one characteristic centrifugal property selected from the group consisting of the following. 2. The centrifugal solidification controlled biopolymer article according to claim 1, wherein at least one characteristic centrifugal property is i) uniform density over at least a portion of the article. 3. A centrifugal solidification-controlled biopolymer article according to claim 1 or 2, wherein at least one characteristic centrifugal property is ii) at least a portion of the article is optically opaque. 4. A centrifugal solidification-controlled biopolymer article according to any one of the preceding paragraphs, wherein at least one characteristic centrifugal property is iii) that at least a portion of the article is free of external lamination. 5. A centrifugal solidification-controlled biopolymer article according to any one of the preceding paragraphs, wherein at least one characteristic centrifugal property is iv) that at least a portion of the article is free of internal lamination. 6. A centrifugal solidification-controlled biopolymer article according to any one of the preceding paragraphs, wherein at least one characteristic centrifugal property is v) that at least a portion of the article is free of internal voids. 7. A centrifugal solidification-controlled biopolymer article according to any one of the preceding paragraphs, wherein at least one characteristic centrifugal property is vi) that at least a portion of the article is free of cylindrical extrusion gaps. 8. A centrifugal solidification-controlled biopolymer article according to any one of the preceding paragraphs, wherein at least one characteristic centrifugal property is vii) that at least a portion of the article is not laminated. 9. The item is i) Uniform density over at least a portion of the article; ii) At least a portion of the article is optically opaque; iii) At least a portion of the article is free from external lamination; iv) At least a portion of the article is free from internal lamination; v) At least a portion of the article is free from internal voids; vi) No cylindrical extrusion gaps in at least a portion of the article; and vii) At least a portion of the article is free from lamination. A centrifugal solidification-controlled biopolymer article according to any one of the above claims, comprising at least two, at least three, at least four, at least five, at least six, or at least seven of the at least one characteristic centrifugal properties selected from the group consisting of the above. 10. A centrifugal controlled biosolidification molding method comprising the following sequential steps: A) A step of introducing a first biopolymer aqueous solution into the mold and, if necessary, allowing the first biopolymer aqueous solution to settle in the mold; B) A step of introducing a first curing aqueous solution onto a first biopolymer aqueous solution in a mold, and, if necessary, allowing the first curing aqueous solution to settle in the mold, wherein the mold has a first orientation relative to gravity during step B); and C) A step of producing a first biosolidified body by subjecting the mold and the contents of the mold to a first centrifuge and stopping after the first centrifugation period, wherein the mold is within 45° of a first orientation to the first centrifuge during step C). A method comprising step C) in which a first biopolymer article is produced from a first biosolidified body by waiting for a first final biosolidification period after step C). 11. The method according to item 10, wherein step A) includes the step of settling a first biopolymer aqueous solution into a mold. 12. The method according to claim 11, wherein the settling step includes subjecting the mold and the contents of the mold to centrifugal sedimentation and stopping the centrifugal sedimentation after a period of time, thereby causing the first biopolymer solution to conform to the geometry of the mold, and the mold is within 45° of the first orientation with respect to the first centrifuge in step B) during centrifugal sedimentation. 13. The method according to claim 11, wherein the settling step includes causing the first biopolymer solution to conform to the geometry of the mold by tapping the mold and the contents of the mold on the surface, and the mold is within 45° of the first orientation with respect to the first centrifugal force in step B) during tapping. 14. The method according to paragraph 10, further comprising the step of producing a first biopolymer article by waiting for a first final biosolidification period after step C). 15. The method according to paragraph 10, wherein the first biopolymer aqueous solution is a silk aqueous solution, an alginate aqueous solution, a fibrinogen aqueous solution, a chitosan aqueous solution, a collagen aqueous solution, or a combination thereof. 16. The method according to claim 15, wherein the first biopolymer aqueous solution is a silk aqueous solution, and the first curing aqueous solution is a silk crosslinking and / or hydrogel initiation aqueous solution. 17. The method according to paragraph 15, wherein the first aqueous biopolymer solution is an aqueous alginate solution, and the first curing aqueous solution is an aqueous alginate crosslinking solution. 18. The method according to paragraph 15, wherein the first biopolymer aqueous solution is a fibrinogen aqueous solution and the first curing aqueous solution is a fibrinogen curing aqueous solution. 19. The method according to paragraph 15, wherein the first biopolymer aqueous solution is a collagen aqueous solution, and the first curing aqueous solution is a collagen curing aqueous solution. 20. The method according to claim 15, wherein the first biopolymer aqueous solution is a chitosan aqueous solution and the first curing aqueous solution is a chitosan curing aqueous solution. 21. The method according to the preceding paragraph, wherein the aqueous solution for curing chitosan is sodium hydroxide, ethanol, or a combination thereof. 22. The following sequential steps: D) A step of introducing a second biopolymer aqueous solution onto the first bio-solidified body in the mold, and, if necessary, allowing the second biopolymer aqueous solution to settle in the mold; E) A step of introducing a second curing aqueous solution onto a second biopolymer aqueous solution in a mold, and, if necessary, allowing the second curing aqueous solution to settle in the mold, wherein the mold has a second orientation relative to gravity during step E); and F) A step of producing a second biosolid containing a first biosolid, by subjecting the mold and the contents of the mold to a second centrifugation and stopping after the second centrifugation period, wherein the mold is within 45° of the second orientation to the second centrifugation during step F), It further includes, After step F), a second final bio-solidification period is observed, after which a second biopolymer article is produced from the second bio-solidified body. The method described in paragraph 10. 23. The method according to claim 22, further comprising the step of producing a second biopolymer article by waiting for a second final biosolidification period after step F). 24. The method according to paragraph 22, wherein the second orientation is the same as the first orientation. 25. The method according to paragraph 22, wherein the second orientation is different from the first orientation. 26. The following steps: G) A step of introducing a third biopolymer aqueous solution onto the second biosolidified body in the mold, and, if necessary, allowing the third biopolymer aqueous solution to settle in the mold; H) A step of introducing a third curing aqueous solution onto a third biopolymer aqueous solution in a mold, and, if necessary, allowing the third curing aqueous solution to settle in the mold, wherein the mold has a third orientation relative to gravity during step H); and I) A step of producing a third biosolid containing the first and second biosolids by subjecting the mold and the contents of the mold to a third centrifuge and stopping after the third centrifuge period, wherein the mold is within 45° of the third orientation to the third centrifuge during step I), It further includes, After step I), a third final bio-solidification period is observed, after which a third biopolymer article is produced from the third bio-solidified body. The method described in paragraph 22. 27. The method according to claim 26, further comprising the step of producing a third biopolymer article by waiting for a third final solidification period after step I). 28. The following sequential steps: J) A step of introducing a fourth biopolymer aqueous solution onto the third bio-solidified body in the mold, and, if necessary, allowing the fourth biopolymer aqueous solution to settle in the mold; K) A step of introducing a fourth curing aqueous solution onto the fourth biopolymer aqueous solution in the mold, and, if necessary, allowing the fourth curing aqueous solution to settle in the mold, wherein the mold has a fourth orientation relative to gravity during step J); and L) A step of producing a fourth biosolid containing the first, second and third biosolids by subjecting the mold and the contents of the mold to a fourth centrifuge and stopping after the fourth centrifuge period, wherein the mold is within 45° of the fourth orientation with respect to the fourth centrifuge during step L), It further includes, After step L), a fourth final bio-solidification period is awaited, thereby producing a fourth biopolymer article from the fourth bio-solidified body. The method described in paragraph 26. 29. The method according to claim 28, further comprising the step of producing a third biopolymer article by waiting for a third final solidification period after step L). 30. The following sequential steps: X) A step of introducing an additional biopolymer aqueous solution onto an existing portion of the biosolid, and, if necessary, allowing the additional biopolymer aqueous solution to settle in the mold, wherein the existing portion of the biosolid includes a first solid and, if necessary, a second, third, and fourth biosolid; Y) The step of introducing an additional curing aqueous solution onto the additional biopolymer aqueous solution in the mold, and, if necessary, allowing the additional curing aqueous solution to settle in the mold; and Z) A process to produce additional biosolids, including the existing portion of the biosolids, by subjecting the mold and its contents to additional centrifugation and stopping after the additional centrifugation period. It further includes, After step Z), an additional final biosolidification period is allowed to occur, resulting in the production of additional biopolymer articles from the additional biosolidified material. The method described in any one of the paragraphs from paragraph 10 to the immediately preceding paragraph. 31. The method according to paragraph 30, further comprising a step of producing additional biopolymer articles by waiting for an additional final solidification period after step Z). 32. A method for producing a biopolymer article, comprising the following steps: X) A step of introducing a repeating biopolymer aqueous solution into a mold and, if necessary, allowing the repeating biopolymer aqueous solution to settle in the mold; Y) A step of introducing a repeating curing aqueous solution onto the repeating biopolymer aqueous solution in the mold, and, if necessary, allowing the repeating curing aqueous solution to settle in the mold; and Z) Repeatedly adding the mold and its contents to the existing portion of the solidified body by subjecting them to repeated centrifugation and stopping the centrifugation after the repeated centrifugation period. This includes at least two iterative cycles, A method for producing a biopolymer article, comprising repeatedly introducing step X), introducing step Y), and introducing step Z) until a final biosolid is formed, and waiting for a final biosolidification period after the formation of the final biosolid. 33. The method according to claim 32, further comprising a step of producing a biopolymer article by waiting for a final biosolidification period after step Z). 34. A method comprising the step of forming a first bio-solidified body by centrifuging a biopolymer aqueous solution in a mold to a first late solidification stage. 35. The method according to claim 34, further comprising the step of forming a second biosolid containing a first biosolid by centrifuging a second initial solidification aqueous solution of a biopolymer over a first biosolid in a mold to a second late solidification stage. 36. The method described in any one of the paragraphs from paragraph 10 to the immediately preceding paragraph, wherein the mold is three-dimensionally printed. 37. The method according to any one of the items from item 10 to the immediately preceding item, wherein the method includes the step of three-dimensionally printing a mold before step A). 38. A method of any one of the paragraphs from paragraph 10 to the immediately preceding paragraph, wherein the sacrificial type is used in the method. 39. The method described in the preceding paragraph, wherein the sacrificial type is acrylonitrile butadiene styrene. 40. The method of the preceding paragraph, wherein the method includes a step of removing the sacrificial form using acetone or alcohol. 41. The method according to any one of the claims from claim 10 to the preceding claim, wherein the upper mold is present during one or more centrifugal steps to introduce additional molding into one or more of the biosolidified articles. 42. The method according to any one of the claims from 10 to the immediately preceding claim, wherein the first biopolymer in the first biopolymer aqueous solution, the second biopolymer in the second biopolymer solution, the third biopolymer in the third biopolymer solution, the fourth biopolymer in the fourth biopolymer solution, and / or the repeating biopolymer in the repeating biopolymer solution are selected from the group consisting of silk fibroin, alginate, fibrinogen, chitosan, collagen, and combinations thereof. 43. The method according to paragraph 42, wherein the first, second, third, fourth and / or repeating biopolymer is silk fibroin. 44. The method according to the preceding section, wherein the first, second, third, fourth and / or repeated curing solution is a beta-sheet initiation or hydrogel initiation composition. 45. The method according to paragraph 42, wherein the first, second, third, fourth and / or repeating biopolymer is an alginate. 46. ​​The method according to the preceding paragraph, wherein the first, second, third, fourth, and / or repeated curing solution is a calcium ion solution. 47. The method according to paragraph 42, wherein the first, second, third, fourth and / or repeating biopolymer is a fibrinogen. 48. The method according to the preceding section, wherein the first, second, third, fourth and / or repeated curing solution is a thrombin solution that cleaves and polymerizes fibrinogen to form fibrin. 49. The method according to paragraph 42, wherein the first, second, third, fourth and / or repeating biopolymer is chitosan. 50. The method according to the preceding paragraph, wherein the chitosan is acidic, and the first, second, third, fourth, and / or repeated curing solution is a neutralizing agent that neutralizes the acid and solidifies the chitosan. 51. The method according to the preceding paragraph, wherein the first, second, third, fourth and / or repeated curing solution is at least one of sodium hydroxide, a mixture of sodium hydroxide and ethanol, ethanol, urea (carbamide), carbonic acid, or sodium tripolyphosphate. 52. The method according to paragraph 42, wherein the first, second, third, fourth and / or repeating biopolymer is collagen. 53. The method according to the preceding paragraph, wherein the collagen is acidic, and the first, second, third, fourth, and / or repeated curing solutions are pH adjusters that direct the self-assembly of the collagen. 54. The method according to any one of the claims from 10 to the immediately preceding claim, further comprising the step of chemically crosslinking the first, second, third, fourth or final biosolidified body or the first, second, third, fourth or final biopolymer article. 55. The method according to any one of the claims from 10 to the preceding claim, further comprising the step of physically crosslinking the first, second, third, fourth or final biosolidified body or the first, second, third, fourth or final biopolymer article. 56. The method according to any one of the claims from 10 to the immediately preceding claim, further comprising the step of chemically etching the first, second, third, fourth or final biosolidified body or the first, second, third, fourth or final biopolymer article. 57. The method according to any one of the claims from 10 to the immediately preceding claim, further comprising the step of chemically smoothing the first, second, third, fourth or final biosolidified body or the first, second, third, fourth or final biopolymer article. 58. The method according to any one of the claims from 10 to the preceding claim, further comprising the step of extending and / or branching polymer chains in the first, second, third, fourth or final biosolidified body or the first, second, third, fourth or final biopolymer article. 59. The method according to any one of the claims from 10 to the immediately preceding claim, further comprising a step of chemically functionalizing the first, second, third, fourth or final biosolidified body or the first, second, third, fourth or final biopolymer article. 60. The method according to any one of the claims from 10 to the immediately preceding claim, further comprising a step of degassing the first, second, third, fourth and / or repeated aqueous solutions of the biopolymer and / or the first, second, third, fourth and / or repeated aqueous solutions for curing. 61. The method according to any one of the claims from 10 to the immediately preceding claim, wherein the first, second, third, fourth and / or repeated aqueous solutions of a biopolymer and / or the first, second, third, fourth and / or repeated aqueous solutions for curing further include additives. 62. The method according to item 61, wherein the first aqueous solution of the biopolymer contains an additive. 63. The method according to item 61, wherein the second aqueous solution of the biopolymer contains an additive. 64. The method according to item 61, wherein the aqueous solution of the third biopolymer includes an additive. 65. The method according to item 61, wherein the aqueous solution of the fourth biopolymer contains an additive. 66. The method according to claim 61, wherein a repeated aqueous solution of a biopolymer contains an additive. 67. The method according to item 61, wherein the first curing aqueous solution contains an additive. 68. The method according to item 61, wherein the second curing aqueous solution contains an additive. 69. The method according to item 61, wherein the third curing aqueous solution contains an additive. 70. The method according to item 61, wherein the fourth curing aqueous solution contains an additive. 71. The method according to item 61, wherein the repeated curing aqueous solution includes an additive. 72. The method according to any one of claims 61 to 71, wherein the additive comprises nanoparticles. 73. The method according to any one of the claims 61 to 71, wherein the additive includes a structural modification material. 74. The method according to any one of the claims 61 to 71, wherein the additive comprises a biologically active material. 75. The method according to any one of the above paragraphs, wherein the mold has a structural modification material before introducing the first, second, third, fourth and / or repeated biopolymer aqueous solution and / or the first, second, third, fourth and / or repeated curing aqueous solution into the mold. 76. The method described in paragraph 73 or 75, wherein the modified material includes fibers. 77. The method described in heading 73 or 75, wherein the structural modification material includes fabric. 78. Structural modification materials, including scaffolding, as described in paragraph 73 or 75. 79. The method described in paragraph 73 or 75, wherein the structural modification material includes a foam. 80. The method described in paragraph 73 or 75, wherein the structural modification material includes a plasticizer. 81. The method described in paragraph 73 or 75, wherein the modified material includes a pologen. 82. The method according to any one of the above, wherein the mold has nanoparticles before introducing the first, second, third, fourth and / or repeated biopolymer aqueous solutions and / or the first, second, third, fourth and / or repeated curing aqueous solutions into the mold. 83. The method according to item 72 or 82, wherein the nanoparticles include silk nanoparticles. 84. The method according to item 72 or 82, wherein the nanoparticles include metal nanoparticles. 85. The method according to item 72 or 82, wherein the nanoparticles include semiconductor nanoparticles. 86. The method according to any one of the above paragraphs, wherein the mold has a biologically active material before introducing the first, second, third, fourth and / or repeated biopolymer aqueous solutions and / or the first, second, third, fourth and / or repeated curing aqueous solutions into the mold. 87. The method according to item 74 or 86, wherein the biologically active material comprises one or more cells. 88. The method according to item 74 or 86, wherein the biologically active material comprises one or more growth factors. 89. Articles produced by any one of the methods described in any of the paragraphs from paragraph 10 to the preceding paragraph. 90. The article described in the preceding paragraph, which is at least a part of a medical device. 91. Articles or medical devices described in the preceding paragraph, which include catheters, stents, tympanostomy tubes, nasal septum buttons, nerve cap devices, Kirschner wires, artificial lenses, dental implants, bone scaffolds, spinal implants, plates, screws, pins, rods, or combinations thereof. 92. A centrifugal solidification controlled type chitosan article, wherein the article is not a film, and at least a portion of the article is i) Uniform density over at least a portion of the article; ii) At least a portion of the article is optically opaque; iii) At least a portion of the article is free from external lamination; iv) At least a portion of the article is free from internal lamination; v) At least a portion of the article is free from internal voids; vi) No cylindrical extrusion gaps in at least a portion of the article; and vii) At least a portion of the article is free from lamination. A centrifugal solidification-controlled chitosan article comprising at least one characteristic centrifugal property selected from the group consisting of the following. 93. A centrifugal controlled method for solidifying chitosan bio, comprising the following sequential steps: A) A step of introducing a chitosan solution into the mold and, if necessary, allowing the chitosan solution to settle within the mold; B) A step of introducing a chitosan curing solution onto the chitosan solution in the mold, and, if necessary, allowing the chitosan curing solution to settle in the mold, wherein the mold has a first orientation relative to gravity during step B); and C) A step of producing a first biosolidified body by subjecting the mold and the contents of the mold to a first centrifuge and stopping after the first centrifugation period, wherein the mold is within 45° of a first orientation to the first centrifuge during step C). Includes, A method for producing a first biopolymer article from a first biosolidified body by waiting for a first final biosolidification period after step C). 94. The method according to claim 93, wherein the settling step includes subjecting the mold and the contents of the mold to centrifugal sedimentation and stopping the centrifugal sedimentation after a period of time, thereby causing the chitosan solution to conform to the geometry of the mold, and the mold is within 45° of a first orientation with respect to the first centrifuge in step B) during centrifugal sedimentation. 95. The method according to claim 93, wherein the settling step includes causing the chitosan solution to conform to the geometry of the mold by tapping the mold and the contents of the mold on the surface, and the mold is within 45° of the first orientation with respect to the first centrifugation in step B) during tapping. The method according to claim 93, further comprising the step of producing a first biopolymer article by waiting for a first final biosolidification period after step C). 97. The method according to paragraph 93, wherein the aqueous solution for curing chitosan is sodium hydroxide, ethanol, or a combination thereof. 98. The following sequential steps: D) A step of introducing a second biopolymer aqueous solution onto the first bio-solidified body in the mold, and, if necessary, allowing the second biopolymer aqueous solution to settle in the mold; E) A step of introducing a second curing aqueous solution onto a second biopolymer aqueous solution in a mold, and, if necessary, allowing the second curing aqueous solution to settle in the mold, wherein the mold has a second orientation relative to gravity during step E); and F) A step of producing a second biosolid containing a first biosolid, by subjecting the mold and the contents of the mold to a second centrifugation and stopping after the second centrifugation period, wherein the mold is within 45° of the second orientation to the second centrifugation during step F), It further includes, After step F), a second final bio-solidification period is observed, after which a second biopolymer article is produced from the second bio-solidified body. The method described in paragraph 93. The method according to claim 98, further comprising the step of producing a second biopolymer article by waiting for a second final biosolidification period after step F). 100. The method according to paragraph 98, wherein the second orientation is the same as the first orientation. 101. The method according to paragraph 98, wherein the second orientation is different from the first orientation. 102. The following steps: G) A step of introducing a third biopolymer aqueous solution onto the second biosolidified body in the mold, and, if necessary, allowing the third biopolymer aqueous solution to settle in the mold; H) A step of introducing a third curing aqueous solution onto a third biopolymer aqueous solution in a mold, and, if necessary, allowing the third curing aqueous solution to settle in the mold, wherein the mold has a third orientation relative to gravity during step H); and I) A step of producing a third biosolid containing the first and second biosolids by subjecting the mold and the contents of the mold to a third centrifuge and stopping after the third centrifuge period, wherein the mold is within 45° of the third orientation to the third centrifuge during step I), It further includes, After step I), a third final bio-solidification period is observed, after which a third biopolymer article is produced from the third bio-solidified body. The method described in paragraph 98. 103. The method according to claim 102, further comprising the step of producing a third biopolymer article by waiting for a third final solidification period after step I). 104. The following sequential steps: J) A step of introducing a fourth biopolymer aqueous solution onto the third bio-solidified body in the mold, and, if necessary, allowing the fourth biopolymer aqueous solution to settle in the mold; K) A step of introducing a fourth curing aqueous solution onto the fourth biopolymer aqueous solution in the mold, and, if necessary, allowing the fourth curing aqueous solution to settle in the mold, wherein the mold has a fourth orientation relative to gravity during step J); and L) A step of producing a fourth biosolid containing the first, second and third biosolids by subjecting the mold and the contents of the mold to a fourth centrifuge and stopping after the fourth centrifuge period, wherein the mold is within 45° of the fourth orientation with respect to the fourth centrifuge during step L), It further includes, After step L), a fourth final bio-solidification period is awaited, thereby producing a fourth biopolymer article from the fourth bio-solidified body. The method described in paragraph 102. 105. The method according to claim 104, further comprising the step of producing a third biopolymer article by waiting for a third final solidification period after step L). 106. The following sequential steps: X) A step of introducing an additional biopolymer aqueous solution onto an existing portion of the biosolid, and, if necessary, allowing the additional biopolymer aqueous solution to settle in the mold, wherein the existing portion of the biosolid includes a first biosolid, and, if necessary, includes second, third, and fourth biosolids; Y) The step of introducing an additional curing aqueous solution onto the additional biopolymer aqueous solution in the mold, and, if necessary, allowing the additional curing aqueous solution to settle in the mold; and Z) A process to produce additional biosolids, including the existing portion of the biosolids, by subjecting the mold and its contents to additional centrifugation and stopping after the additional centrifugation period. Furthermore, by waiting for an additional final biosolidification period after step Z), additional biopolymer articles are produced from the additional biosolidified material. The method described in any one of the paragraphs from paragraph 93 to the immediately preceding paragraph. 107. The method according to claim 106, further comprising the step of producing an additional biopolymer article by waiting for an additional final solidification period after step Z). 108. The method according to any of the claims from 93 to the preceding claim, wherein the second, third, or fourth aqueous biopolymer solution is an aqueous silk solution, an aqueous alginate solution, an aqueous fibrinogen solution, an aqueous chitosan solution, an aqueous collagen solution, or a combination thereof. 109. The method according to claim 108, wherein the second, third, fourth or additional biopolymer aqueous solution is a silk aqueous solution, and the corresponding second, third, fourth or additional curing aqueous solution is a silk crosslinking and / or hydrogel initiation aqueous solution. 110. The method according to claim 108, wherein the second, third, fourth or additional biopolymer aqueous solution is an alginate aqueous solution, and the second, third, fourth or additional curing aqueous solution is an alginate crosslinking aqueous solution. 111. The method according to claim 108, wherein the second, third, fourth or additional biopolymer aqueous solution is a fibrinogen aqueous solution, and the second, third, fourth or additional curing aqueous solution is a fibrinogen curing aqueous solution. 112. The method according to claim 108, wherein the second, third, fourth or additional biopolymer aqueous solution is a collagen aqueous solution, and the second, third, fourth or additional curing aqueous solution is a collagen curing aqueous solution. 113. The method according to claim 108, wherein the second, third, fourth or additional biopolymer aqueous solution is a chitosan aqueous solution, and the second, third, fourth or additional curing aqueous solution is a chitosan curing aqueous solution. 114. The method according to the preceding paragraph, wherein the aqueous solution for curing chitosan is sodium hydroxide, ethanol, or a combination thereof. 115. A method of any one of the paragraphs from paragraph 93 to the immediately preceding paragraph, wherein the sacrificial type is used in the method. 116. The method described in the preceding paragraph, wherein the sacrificial type is acrylonitrile butadiene styrene. 117. The method of the preceding paragraph, wherein the method includes a step of removing the sacrificial form using acetone or alcohol. 118. The method according to any one of the claims from 93 to the immediately preceding claim, further comprising chitosan, a second, third, fourth and / or additional aqueous biopolymer solution and / or chitosan, a second, third, fourth and / or additional curing aqueous solution as an additive. 119. The method described in the preceding paragraph, wherein the additive comprises nanoparticles, structurally modified materials, or biologically active materials.

Claims

1. A centrifugal solidification controlled biopolymer article, wherein the article is not a film, and at least a portion of the article is i) Uniform density over at least a portion of the article; ii) At least a portion of the article is optically opaque; iii) At least a portion of the article is free from external lamination; iv) At least a portion of the article is free from internal lamination; v) The article has no internal voids in at least a portion of it; vi) At least a portion of the article is free from cylindrical extrusion gaps; and vii) At least a portion of the article is free from lamination. Includes at least one characteristic centrifugal characteristic selected from the group consisting of, Centrifugal solidification controlled biopolymer articles.

2. A centrifugal controlled biosolidification molding method comprising the following sequential steps: A) A step of introducing a first biopolymer aqueous solution into a mold, and, if necessary, allowing the first biopolymer aqueous solution to settle in the mold; B) A step of introducing a first curing aqueous solution onto the first biopolymer aqueous solution in the mold, and, if necessary, allowing the first curing aqueous solution to settle in the mold, wherein the mold has a first orientation relative to gravity during step B); and C) A step of producing a first biosolidified body by subjecting the mold and the contents of the mold to a first centrifuge and stopping after the first centrifuge period, wherein the mold is within 45° of the first orientation with respect to the first centrifuge during step C). Includes, A method for producing a first biopolymer article from the first biosolidified body by waiting for a first final biosolidification period after step C).

3. The method according to claim 2, wherein step A) includes the step of settling the first biopolymer aqueous solution into the mold.

4. The method according to claim 3, wherein the settling step includes subjecting the mold and the contents of the mold to centrifugal sedimentation and stopping the centrifugal sedimentation after a period of time, thereby causing the first biopolymer solution to conform to the geometry of the mold, and the mold is within 45° of the first orientation with respect to the first centrifuge in step B) during the centrifugal sedimentation.

5. The method according to claim 3, wherein the settling step includes making the first biopolymer solution conform to the geometry of the mold by tapping the mold and the contents of the mold on the surface, and the mold is within 45° of the first orientation with respect to the first centrifugal force in step B) during the tapping.

6. The method according to claim 2, further comprising the step of producing the first biopolymer article by waiting for the first final biosolidification period after step C).

7. The method according to claim 2, wherein the first biopolymer aqueous solution is a silk aqueous solution, an alginate aqueous solution, a fibrinogen aqueous solution, a chitosan aqueous solution, a collagen aqueous solution, or a combination thereof.

8. The above method comprises the following sequential steps: D) A step of introducing a second biopolymer aqueous solution onto the first biosolidified body in the mold, and, if necessary, allowing the second biopolymer aqueous solution to settle in the mold; E) A step of introducing a second curing aqueous solution onto the second biopolymer aqueous solution in the mold, and, if necessary, allowing the second curing aqueous solution to settle in the mold, wherein the mold has a second orientation relative to gravity during step E); and F) A step of producing a second biosolidified body including the first biosolidified body by subjecting the mold and the contents of the mold to a second centrifuge and stopping after the second centrifuge period, wherein the mold is within 45° of the second orientation with respect to the second centrifuge during step F), It further includes, After step F), a second final bio-solidification period is observed, thereby producing a second biopolymer article from the second bio-solidified body. The method according to claim 2.

9. The method according to claim 8, further comprising the step of producing the second biopolymer article by waiting for the second final biosolidification period after step F).

10. The method according to claim 8, wherein the second orientation is the same as the first orientation.

11. The method according to claim 8, wherein the second orientation is different from the first orientation.

12. The above method comprises the following steps: G) A step of introducing a third biopolymer aqueous solution onto the second biosolidified body in the mold, and, if necessary, allowing the third biopolymer aqueous solution to settle in the mold; H) A step of introducing a third curing aqueous solution onto the third biopolymer aqueous solution in the mold, and, if necessary, allowing the third curing aqueous solution to settle in the mold, wherein the mold has a third orientation relative to gravity during step H); and I) A step of producing a third biosolidified body including the first and second biosolidified bodies by subjecting the mold and the contents of the mold to a third centrifuge and stopping after the third centrifuge period, wherein the mold is within 45° of the third orientation with respect to the third centrifuge during step I), It further includes, After step I), a third final bio-solidification period is observed, thereby producing a third biopolymer article from the third bio-solidified body. The method according to claim 8.

13. The method according to claim 12, further comprising the step of producing the third biopolymer article by waiting for the third final solidification period after step I).

14. The above method comprises the following sequential steps: J) A step of introducing a fourth biopolymer aqueous solution onto the third biosolidified body in the mold, and, if necessary, allowing the fourth biopolymer aqueous solution to settle in the mold; K) A step of introducing a fourth curing aqueous solution onto the fourth biopolymer aqueous solution in the mold, and, if necessary, allowing the fourth curing aqueous solution to settle in the mold, wherein the mold has a fourth orientation relative to gravity during step J); and L) A step of producing a fourth biosolid containing the first, second and third biosolids by subjecting the mold and the contents of the mold to a fourth centrifuge and stopping after the fourth centrifuge period, wherein the mold is within 45° of the fourth orientation with respect to the fourth centrifuge during step L). It further includes, After step L), a fourth final bio-solidification period is awaited, thereby producing a fourth biopolymer article from the fourth bio-solidified body. The method according to claim 12.

15. The method according to claim 14, further comprising the step of producing the third biopolymer article by waiting for the third final solidification period after step L).

16. The above method comprises the following sequential steps: X) A step of introducing an additional biopolymer aqueous solution onto an existing portion of the biosolid, and, if necessary, allowing the additional biopolymer aqueous solution to settle in the mold, wherein the existing portion of the biosolid includes the first biosolid and, if necessary, includes the second, third, and fourth biosolids; Y) A step of introducing an additional curing aqueous solution onto the additional biopolymer aqueous solution in the mold, and, if necessary, allowing the additional curing aqueous solution to settle in the mold; and Z) A step of producing an additional biosolid including the existing portion of the biosolid by subjecting the mold and the contents of the mold to additional centrifugation and stopping after the additional centrifugation period. It further includes, After step Z), an additional final biosolidification period is allowed to occur, from which additional biopolymer articles are produced. The method according to any one of the claims from claim 2 to the immediately preceding claim.

17. The method according to claim 16, further comprising the step of producing the additional biopolymer article by waiting for the additional final solidification period after step Z).

18. A method for producing a biopolymer article, comprising the following steps: X) A step of introducing a repeating biopolymer aqueous solution into a mold and, if necessary, allowing the repeating biopolymer aqueous solution to settle in the mold; Y) A step of introducing a repeating curing aqueous solution onto the repeating biopolymer aqueous solution in the mold, and, if necessary, allowing the repeating curing aqueous solution to settle in the mold; and Z) Repeatedly adding to the existing portion of the solidified body by subjecting the mold and the contents of the mold to repeated centrifugation and stopping after the repeated centrifugation period. This includes at least two iterative cycles, A method for producing the biopolymer article, wherein the iterative process of introducing step X), introducing step Y), and providing step Z) is continued until a final biosolidified body is formed, and a final biosolidification period is observed after the formation of the final biosolidified body.

19. The method according to claim 18, further comprising the step of producing the biopolymer article by waiting for the final biosolidification period after step Z).

20. A method comprising the step of forming a first bio-solidified body by centrifuging a biopolymer aqueous solution in a mold to a first late solidification stage.

21. An article manufactured by the method described in any one of the claims from claim 2 to the immediately preceding claim.

22. A centrifugal solidification controlled type chitosan article, wherein the article is not a film, and at least a portion of the article is i) Uniform density over at least a portion of the article; ii) At least a portion of the article is optically opaque; iii) At least a portion of the article is free from external lamination; iv) At least a portion of the article is free from internal lamination; v) The article has no internal voids in at least a portion of it; vi) At least a portion of the article is free from cylindrical extrusion gaps; and vii) At least a portion of the article is free from lamination. Includes at least one characteristic centrifugal characteristic selected from the group consisting of, Centrifugal solidification controlled type chitosan product.

23. A centrifugal controlled chitosan biosolidification molding method comprising the following sequential steps: A) A step of introducing a chitosan solution into a mold and, if necessary, allowing the chitosan solution to settle within the mold; B) A step of introducing a chitosan curing solution onto the chitosan solution in the mold, and, if necessary, allowing the chitosan curing solution to settle in the mold, wherein the mold has a first orientation relative to gravity during step B); and C) A step of producing a first biosolidified body by subjecting the mold and the contents of the mold to a first centrifuge and stopping after the first centrifuge period, wherein the mold is within 45° of the first orientation with respect to the first centrifuge during step C). Includes, A method for producing a first biopolymer article from the first biosolidified body by waiting for a first final biosolidification period after step C).

24. The method according to claim 23, wherein the settling step includes subjecting the mold and the contents of the mold to centrifugal sedimentation and stopping the centrifugal sedimentation after a period of time, thereby causing the chitosan solution to conform to the geometric shape of the mold, and the mold is within 45° of the first orientation with respect to the first centrifuge in step B) during the centrifugal sedimentation.

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