Dual crosslinked silk-based materials for in SITU deposition or printing

Abstract

Disclosure herein concerns a non-crosslinked composition having capacity for double crosslinking. The non-crosslinked composition includes a population of methacrylate-modified silk fibroin fragments, each of the methacrylate-modified silk fibroin fragments comprising methacrylate side chains, a photo-crosslinker, an enzymatic crosslinker and optionally one or more enzymatic crosslinker co-reactants, and water

Description

DUAL CROSSLINKED SILK-BASED MATERIALS FOR IN SITU DEPOSITION OR PRINTINGCLAIM TO PRIORITY

[0001] This application claims priority to and the benefit of United States Provisional Application 63 / 621,921 filed in the U.S. Patent and Trademark Office on January 17, 2024. The foregoing patent application is incorporated herein by reference in its entirety for all purposes.STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

[0002] This invention was made with government support under P41EB027062 awarded by the NIH and Marie Curie Grant 101008041. The government has certain rights in the invention.BACKGROUND

[0003] The shortage of tissues and organs for transplantation is an urgent clinical concern. In situ 3D printing is aimed at printing the new tissue or organ directly in the patient. The ink for this process is central to the outcomes, and must meet specific requirements such as rapid gelation, shape integrity, stability over time, and adhesion to surrounding healthy tissues. Among natural materials, silk fibroin exhibits fascinating properties that have made it widely studied in tissue engineering and regenerative medicine. However, its low viscosity may hinder its application in in situ 3D printing unless at high concentrations or blended with other materials. Thus, a process for enabling the in situ printing of low concentration silk fibroin is needed.SUMMARY

[0004] In some aspects, the techniques described herein relate to a non-crosslinked composition having capacity for double crosslinking, the non-crosslinked composition including: a population of methacrylate-modified silk fibroin fragments, each of the methacrylate-modified silk fibroin fragments including methacrylate side chains; a photo-crosslinker; an enzymatic crosslinker and optionally one or more enzymatic crosslinker co-reactants; and water.

[0005] In some aspects, the techniques described herein relate to a non-crosslinked composition having capacity for double crosslinking, the non-crosslinked composition including: a population of methacrylate-modified silk fibroin fragments, each of the methacrylate-modified silk fibroin fragments including methacrylate side chains; a photo-crosslinker; one or more enzymatic crosslinker co-reactants and optionally an enzymatic crosslinker; and water.

[0006] In some aspects, the techniques described herein relate to a method of making a non- crosslinked composition, the method including: combining a population of methacrylate-modified silk fibroin fragments, a photo-crosslinker, an enzymatic crosslinker, optionally one or more enzymatic crosslinker co-reactants, and water to form the non-crosslinked composition.

[0007] In some aspects, the techniques described herein relate to a method of using a noncrosslinked composition, the method including: pre-crosslinking a non-crosslinked composition, thereby converting the non-crosslinked composition into a pre-crosslinked composition, wherein the non-crosslinked composition is optionally the non-crosslinked composition of any one the preceding claims.

[0008] In some aspects, the techniques described herein relate to a general method including: photo-crosslinking a composition described herein to establish a desired printing viscosity; printing the photo-crosslinked composition; and enzymatically crosslinking the printed composition to establish a final mechanical property.

[0009] These and other systems, methods, objects, features, and advantages of the present disclosure will be apparent to those skilled in the art from the following detailed description of the preferred embodiment and the drawings.

[0010] All documents mentioned herein are hereby incorporated in their entirety by reference. References to items in the singular should be understood to include items in the plural, and vice versa, unless explicitly stated otherwise or clear from the text. Grammatical conjunctions are intended to express any and all disjunctive and conjunctive combinations of conjoined clauses, sentences, words, and the like, unless otherwise stated or clear from the context.BRIEF DESCRIPTION OF THE FIGURES

[0011] The disclosure and the following detailed description of certain embodiments thereof may be understood by reference to the following figures:

[0012] Fig. 1 depicts a double-crosslinking process.

[0013] Fig. 2 depicts the design of double crosslinking process. First, Sil-Ma (methacrylate silk fibroin) was pre-photo-crosslinked with 365 nm light. Later, the pre-gel was transferred into the printing syringe for 3D printing. The printing bed temperature was 37 °C and after filament deposition the enzymatic crosslinking led to the stabilization of the structure, improving final gelation.

[0014] Fig. 3 A depicts the flow point of the pre -photo-crosslinked (PC) condition of 15 mB Sil-Ma and the filament extruded after the pre-gelation.

[0015] Fig. 3B depicts fully photo-crosslinked (FC) condition of 15 mB Sil-Ma.

[0016] Fig. 3C depicts the crossover point of the flow points in all the conditions studied for 30 mB Sil-Ma. EC represents enzymatic crosslinking with complete gelation.

[0017] Fig. 4A depicts the storage modulus G’ monitored over time for the pre PC and the double crosslinked (DC) conditions for 15 mB Sil-Ma. *p<0.05, **p<0.01, ***p<0.001.

[0018] Fig. 4B depicts G’ monitored over time for the pre PC and DC conditions for 30 mB Sil- Ma. *p<0.05, **p<0.01, ***p<0.001.

[0019] Fig. 5A depicts the printability index measured on DC Sil-Ma printed with 25 G nozzle and two different infill grids, 10% and 15%.

[0020] Fig. 5B depicts printed structures with the double crosslinking process (DC). In blue (dye) some STL 3D models of selected shapes. Scale bar 0.5 cm.

[0021] Fig. 6A depicts IR spectra of both 15 mB and 30 mB PC Sil-Ma before and after extrusion.

[0022] Fig. 6B depicts the amide I deconvolution of 15 mB and 30 mB Sil-Ma before and after extrusion.

[0023] Fig. 6C depicts the 15 mB Sil-Ma amide I deconvolution of the PC condition at day 1, 7, and 14 in medium and water.

[0024] Fig. 6D depicts amide I deconvolution of the DC condition at day 1, 7, and 14 in medium and in water.

[0025] Fig. 7A depicts swelling tests performed in medium (M) and water (W) on pre PC and DC 15 mB Sil-MA gels.

[0026] Fig. 7B depicts swelling tests performed in medium (M) and water (W) on pre PC and DC 30 mB Sil-MA gels.

[0027] Fig. 8A depicts degradation kinetics with Protease XIV performed in medium (M) and water (W) on PC and DC Sil-Ma 15 mB gels.

[0028] Fig. 8B depicts degradation kinetics with Protease XIV performed in medium (M) and water (W) on PC and DC Sil-Ma 30 mB gels.

[0029] Fig. 9A depicts the Young’s modulus of PC and DC 15 mB Sil-Ma gels after incubation in medium (M) and water (W).

[0030] Fig. 9B depicts the Young’s modulus of PC and DC 30 mB Sil-Ma gels after incubation in medium (M) and water (W).

[0031] Fig. 10A depicts an AlamarBlue Assay to monitor metabolic activity over 7 days of culture on Sil-Ma 15mB double crosslinked (DC 15mB) and pre-photo-crosslinked (PC 15mB), Sil-Ma 30mB double crosslinked (DC 30mB) and pre -photo-crosslinked (PC 30mB). ***p<0.001.

[0032] Fig. 10B depicts a confocal micrograph at day one representative of cell behavior on DC 30 mB gel.

[0033] Fig. 10C depicts a confocal micrograph at day one representative of cell behavior on DC 15 mB gel.

[0034] Fig. 10D depicts a confocal micrograph at day one representative of cell behavior on PC 30 mB gel.

[0035] Fig. 10E depicts a confocal micrograph at day seven representative of cell behavior on DC 30 mB gel.

[0036] Fig. 10F depicts a confocal micrograph at day seven representative of cell behavior on DC 15 mB gel.

[0037] Fig. 10G depicts a confocal micrograph at day seven representative of cell behavior on DC 30 mB gel.

[0038] Fig. 10H depicts a confocal micrograph at day seven representative of cell behavior on DC 15 mB gel.

[0039] Fig. 101 depicts a confocal micrograph at day seven representative of cell behavior on PC 15 mB gel.

[0040] Fig. 10L depicts a confocal micrograph at day seven representative of cell behavior on PC 30 mB.

[0041] Fig. 11 A depicts in situ printing on a chicken breast of DC Sil-Ma with 25 G nozzle.

[0042] Fig. 1 IB depicts the finished printing on a chicken breast.

[0043] Fig. 11C depicts the adhesion of DC Sil-Ma on a chicken breast.

[0044] Fig. 12 depicts the viscosity measurement (left) of Sil-Ma 15 mB and 30 mB at different shear rates and 3D printing of the ink without any pre-crosslinking (right) leading to drop formation instead of filament formation thus lacking any shape fidelity.

[0045] Fig. 13A depicts viscoelastic analysis of DC Sil-Ma 15 mB to assess ink printability. No flow points were detected.

[0046] Fig. 13B depicts viscoelastic analysis of EC Sil-Ma 15 mB to assess ink printability. No Flow points were detected.

[0047] Fig. 14A depicts PC Sil-Ma 30 mB amide I deconvolution at day 1, 7, and 14 both in medium and water.

[0048] Fig. 14B depicts DC Sil-Ma 30 mB amide I deconvolution at day 1, 7, and 14 both in medium and water.

[0049] Fig. 14C depicts the day 1 spectra of both PC and DC Sil-Ma 15 mB incubated in medium and water at 37 °C for 1 day.

[0050] Fig. 14D depicts the day 1 spectra of both PC and DC Sil-Ma 30 mB incubated in medium and water at 37 °C for 1 day.

[0051] Fig. 15 A depicts degradation kinetics without enzyme performed in medium (M) and water (W) on PC and DC Sil-Ma 15 mB gels at days 0, 1, 7, and 14.

[0052] Fig. 15B depicts degradation kinetics without enzyme performed in medium (M) and water (W) on PC and DC Sil-Ma 30 mB gels at days 0, 1, 7, and 14.

[0053] Fig. 16A depicts DC Sil-Ma 15 mB gel.

[0054] Fig. 16B depicts DC Sil-Ma 15 mB gel.

[0055] Fig. 16C depicts DC Sil-Ma 30 mB gel.

[0056] Fig. 16D depicts DC Sil-Ma 30 mB gel.DETAILED DESCRIPTION

[0057] Before the present disclosure is described in further detail, it is to be understood that the disclosure is not limited to the particular embodiments described. It is also understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. The scope of the present disclosure will be limited only by the claims. As used herein, the singular forms "a", "an", and "the" include plural embodiments unless the context clearly dictates otherwise.

[0058] In this application, unless otherwise clear from context, (i) the term “a” may be understood to mean “at least one”; (ii) the term “or” may be understood to mean “and / or”; (iii) the terms “comprising” and “including” may be understood to encompass itemized components or steps whether presented by themselves or together with one or more additional components or steps; and (iv) the terms “about” and “approximately” are used as equivalents and may be understood to permit standard variation as would be understood by those of ordinary skill in the art; and (v) where ranges are provided, endpoints are included.

[0059] Approximately: as used herein, the term “approximately” or “about,” as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 1 1%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0060] Composition: as used herein, may be used to refer to a discrete physical entity that comprises one or more specified components. In general, unless otherwise specified, a composition may be of any form - e.g., gas, gel, liquid, solid, etc. In some embodiments, “composition” may refer to a combination of two or more entities for use in a single embodiment or as part of the same article. It is not required in all embodiments that the combination of entities result in physical admixture, that is, combination as separate co-entities of each of the components of the composition is possible; however many practitioners in the field may find it advantageous to prepare a composition that is an admixture of two or more of the ingredients in a pharmaceutically acceptable carrier, diluent, orexcipient, making it possible to administer the component ingredients of the combination at the same time.

[0061] Improve, increase, or reduce: as used herein or grammatical equivalents thereof, indicate values that are relative to a baseline measurement, such as a measurement in a similar composition made according to previously known methods.

[0062] Substantially: as used herein, the term “substantially” refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and / or proceed to completeness or achieve or avoid an absolute result. The term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0063] It should be apparent to those skilled in the art that many additional modifications beside those already described are possible without departing from the inventive concepts. In interpreting this disclosure, all terms should be interpreted in the broadest possible manner consistent with the context. Variations of the term "comprising" should be interpreted as referring to elements, components, or steps in a non-exclusive manner, so the referenced elements, components, or steps may be combined with other elements, components, or steps that are not expressly referenced. Embodiments referenced as "comprising" certain elements are also contemplated as "consisting essentially of and "consisting of" those elements. When two or more ranges for a particular value are recited, this disclosure contemplates all combinations of the upper and lower bounds of those ranges that are not explicitly recited. For example, recitation of a value of between 1 and 10 or between 2 and 9 also contemplates a value of between 1 and 9 or between 2 and 10.

[0064] As used herein, "silk fibroin" refers to silk fibroin protein whether produced by silkworm, spider, or other insect, or otherwise generated (Lucas et al., Adv. Protein Chem., 13: 107-242 (1958)). Any type of silk fibroin can be used in different embodiments described herein. Silk fibroin produced by silkworms, such as Bombyx mori, is the most common and represents an earth-friendly, renewable resource. For instance, silk fibroin used in a silk film may be attained by extracting sericin from the cocoons of B. mori. Organic silkworm cocoons are also commercially available. There are many different silks, however, including spider silk (e.g., obtained from Nephila clavipes), transgenic silks, genetically engineered silks, such as silks from bacteria, yeast, mammalian cells, transgenic animals, or transgenic plants, and variants thereof, that can be used. See, e.g., WO 97 / 08315 and U.S. Pat. No. 5,245,012, each of which is incorporated herein by reference in their entireties.

[0065] Disclosed herein is a double crosslinking process to allow the in situ printing of low concentration silk fibroin. Silk fibroin-based inks were developed for in situ applications by exploiting a covalent crosslinking process consisting of a pre-photo-crosslinking prior to printing, or deposition otherwise, and in situ enzymatic crosslinking of the printed / deposited material, or in some embodiments, fully photo-cross linking (FC). Different silk fibroin molecular weights were characterized and the synergistic effect of the covalent bonds with shear forces enhanced the shift in silk secondary structure toward P-sheets, thus, rapid stabilization. These hydrogels exhibited good mechanical properties, stability over time, and resistance to enzymatic degradation over 14 days, with no significant changes over time in their secondary structure and swelling behavior. Additionally, adhesion to tissues in vitro was demonstrated.

[0066] This disclosure provides a printable composition (e.g., 3D printing applications) of silk fibroin of varying molecular weight at low concentration where nozzle shear stress plays a role in secondary structure. Dual crosslinked silk-based material allows direct deposition in situ. In embodiments, no additional post-extrusion step is required to induce gelation. The process is compatible with injection-based approaches (e.g., soft tissue fillers), or additive manufacturing approaches (e.g., 3D printing, handheld deposition devices).

[0067] In an aspect, a non-crosslinked composition having capacity for double crosslinking may include a population of methacrylate-modified silk fibroin fragments, a photo-crosslinker, an enzymatic crosslinker, and water. Each of the methacrylate-modified silk fibroin fragments includes methacrylate side chains. The non-crosslinked composition can optionally include one or more enzymatic crosslinker co-reactants, such as hydrogen peroxide.

[0068] Disclosed herein is a pre-crosslinked composition that is partly photo-crosslinked and has capacity for further photo-crosslinking and an additional enzymatic crosslinking to become doublecrosslinked. The pre-crosslinked composition includes the non-crosslinked composition disclosed herein having partial photo-crosslinking between methacrylate-modified silk fibroin fragments of the population of methacrylate-modified silk fibroin fragments. Partial photo-crosslinking produces an ink with numerous rheological properties and features. For example, partially photo-crosslinked gels exhibit shape fidelity after extrusion. In another example, partially photo-crosslinked gels can form a homogenous and uniform filament that can be deposited on a printing bed keeping its shape. Further, the layers avoid collapse when more than one filament is printed. Partial photo-crosslinking may also produce a storage modulus G’ that is higher than G’ ’ , where the crossover point is at most in the range of 300Pa of stress. Partially photo-crosslinking may also produce gels that must be extruded with an extrusion pressure not to exceed 400 kPa. In some embodiments, the phrase ‘partly photo-crosslinked’ may refer to a level of crosslinking where any further crosslinking can be initiated with further lightexposure. In embodiments, partial photo-crosslinking may involve between 1% and 50%, between 40% and 70%, between 1% and 99%, or between 50% and 99.9% of the methacrylate side chains, including combinations of the lower and upper bounds of these ranges (e.g., 1 -70%, 1 -99.9%, 40-99%, 40-99.9%, etc.). The pre-crosslinked composition may be adhesive to mammalian tissue (e.g., a chicken breast) or an anatomical surface, and may have an adhesive strength of at least at least 200 kPa, 500 kPa, at least 750 kPa, at least IMPa, at least 2 MPa, at least 3 MPa, or at least 5 MPa. In embodiments of a double-crosslinked composition including the pre-crosslinked composition disclosed herein, the additional enzymatic crosslinking generates enzymatic crosslinks between the methacrylate-modified silk fibroin fragments of the population of methacrylate-modified silk fibroin fragments, such as dityrosine bonds. In embodiments, a seeded, double-crosslinked composition includes the double-crosslinked composition disclosed herein and a plurality of cells seeded on the double-crosslinked composition. The plurality of cells may have a seeding density of at least IxlO5cells / mL. Without wishing to be bound by any particular theory, there is an upper limit to the seeding density, but the variation in the different cell types and tissue types makes estimating an upper limit highly challenging.

[0069] Enzymatic crosslinking may involve an oxidation reaction on the phenol groups present on tyrosine, and the activation of the enzyme may be dependent on the presence of hydrogen peroxide. Other variables that may affect the reaction kinetics are the protein concentrations, molecular weight, temperature, and the like.

[0070] In certain cases, the enzymatic crosslinker can be horseradish peroxidase (e.g., HRP). The HRP may be within the composition prior to the pre-photo-crosslinking and may remain in the composition through printing. In other embodiments, the PC gel may be used to print and then the HRP may be introduced subsequent to the printing. Similarly, co-reactants such as hydrogen peroxide may be present in the composition prior to or subsequent to printing. In embodiments, one of the HRP or the hydrogen peroxide may be present in the starting solution while the other is not and is introduced after printing.

[0071] While examples included in this disclosure may be generally directed to silk fibroin obtained after boiling for 15 minutes and 30 minutes, and corresponding to a molecular weight of 300 kDa and 20 kDa respectively, it should be understood that the double crosslinking process may be used with silk fibroins of different molecular weights or obtained after being boiled for different times, as disclosed herein. Throughout this disclosure, ‘mB’ refers to ‘minutes boiled’, For example, ’30 mB silk fibroin’ refers to silk fibroin that has been obtained after 30 minutes of boiling. Without wishing to be bound by any particular theory, the molecular weight of the silk fibroin may impact: 1) the rheological properties of the non-crosslinked solution; 2) the rheological properties of the pre -photo-crosslinked (PC) gel; and / or 3) the rheological / material properties of the double-crosslinked (DC) gel. In some aspects, decreasing the molecular weights or silk concentration may result in softer hydrogels. For example, the 30mB silk fibroin may have rheological properties that are slightly lower compared to the 15mB silk fibroin. Without wishing to be bound by any particular theory, it is postulated that the higher the molecular weight, the closer the side chains are to interact with each other.

[0072] Pre-crosslinking (e.g., the photo-driven gelation) may be modulated according to the molecular weight of the silk fibroin. For example, the lower the molecular weight of silk fibroin, the longer light exposure will be needed to allow the chains to interlock and form covalent bonds. Precrosslinking may provide the proper viscosity to achieve a filament formation and shape fidelity and it can be tuned with light exposure, light intensity, and the like. In embodiments, pre-crosslinking silk fibroins of lower molecular weights may lead to softer gels. The molecular weight of silk fibroin obtained through the degumming process is polydisperse with longer degumming times leading to higher polydispersity. In embodiments, gels created from silk fibroins obtained after a shorter degumming time (e.g., 15 mB and 30 mB silk fibroins) exhibit rheological differences in comparison to gels made from silk fibroins obtained after a longer degumming process (e.g., 60mB or 120mB silk fibroins where the distance of methacrylate sites may be farther from each other than for silk fibroins obtained after a shorter degumming time). In some embodiments, shear stress in the delivery device (e.g., nozzle, coaxial nozzle system) may further induce beta-sheet formation which may increase structural stabilization via physical crosslinking. In some embodiments, when shear stress results in a significant shift to crystalline structure, the result may be stable secondary structures over time with a reduction in differences between compositions including different molecular weight silk fibroins.

[0073] In an aspect, a method of making a non-crosslinked composition may include combining a population of methacrylate-modified silk fibroin fragments, a photo-crosslinker, an enzymatic crosslinker, optionally one or more enzymatic crosslinker co-reactants, and water to form the noncrosslinked composition.

[0074] In an aspect, a method of using a non-crosslinked composition may include pre-crosslinking a non-crosslinked composition, thereby converting the non-crosslinked composition into a precrosslinked composition. The non-crosslinked composition is optionally the non-crosslinked composition disclosed herein. The method may further include printing the pre-crosslinked composition in a predefined shape, such as internal structural features, to produce a printed, precrosslinked article. The method may further include enzymatically crosslinking the pre-crosslinked article. In embodiments, printing may be in vivo printing in a mammalian subject, such as a human subject.

[0075] In embodiments, compositions and methods disclosed herein may include silk fibroins having an average molecular weight of between 25 kDa and 500 kDa, between 50 kDa and 400 kDa, between 100 kDa and 300 Kda, or between 150 kDa and 350 kDa. In embodiments, compositions and methods disclosed herein may include silk fibroins having an average molecular weight greater than 150 kDa. In embodiments, compositions and methods disclosed herein may include silk fibroins obtained by boiling for for at least 5 minutes, at least 15 minutes, at least 20 minutes, at least 30 minutes, at least 45 minutes, or at least 60 minutes, and at least 15 minutes, at least 30 minutes, at least 60 minutes, or at least 120 minutes. In embodiments, compositions and methods disclosed herein may include silk fibroin wherein at least 50% by weight of the silk fibroin has a silk fibroin backbone with a molecular weight of at least 25 kDa. In embodiments, compositions and methods disclosed herein may include silk fibroin wherein at least 50% by weight of the silk fibroin has a silk fibroin backbone with a molecular weight of at least 50 kDa. In embodiments, compositions and methods disclosed herein may include silk fibroin wherein at least 50% by weight of the silk fibroin has a silk fibroin backbone with a molecular weight of at least 100 kDa. In embodiments, compositions and methods disclosed herein may include silk fibroin wherein at least 50% by weight of the silk fibroin has a silk fibroin backbone with a molecular weight of at least 250 kDa. In embodiments, compositions and methods disclosed herein may include silk fibroin wherein at least 50% by weight of the silk fibroin has a silk fibroin backbone with a molecular weight of at least 450 kDa. In certain aspects, an average molecular weight refers to a weight average molecular weight. In certain aspects, an average molecular weight refers to a number average molecular weight. In instances of mutual exclusivity, an average molecular weight refers to a weight average molecular weight.

[0076] In embodiments, methacrylate side chains may be present on the methacrylate-modified silk fibroin disclosed herein in an amount by weight that may be defined by the number of lysine residues (e.g., 0.3-4 % mol along silk fibroin sequence). Methacrylate side chains may be have a degree of substitution (DS) on the methacrylate-modified silk fibroin between 0. 1 % to 99.9%, between 30% and 80%, between 50% and 75%, or between 60% and 70%. Methacrylate coverage may be tuned depending on the concentration of glycidyl methacrylate and how long the silk is in contact with it. In embodiments, the methacrylate-modified silk fibroin has a silk fibroin backbone with a molecular weight of between 25 kDa and 500 kDa, between 50 kDa and 400 kDa, between 100 kDa and 300 kDa, or between 150 kDa and 350 kDa. In embodiments, the methacrylate-modified silk fibroin has a silk fibroin backbone having an average molecular weight greater than 150 kDa. In embodiments, the methacrylate-modified silk fibroin includes a silk fibroin obtained by boiling for at most 15 minutes, at most 20 minutes, at most 30 minutes, at most 45 minutes, or at most 60 minutes, at least 15 minutes, at least 30 minutes, at least 60 minutes, or at least 120 minutes. In embodiments, at least 50% by weightof the methacrylate-modified silk fibroin has a silk fibroin backbone with a molecular weight of at least 25 kDa. In embodiments, at least 50% by weight of the methacrylate-modified silk fibroin has a silk fibroin backbone with a molecular weight of at least 50 kDa. In embodiments, at least 50% by weight of the methacrylate-modified silk fibroin has a silk fibroin backbone with a molecular weight of at least 100 kDa. In embodiments, at least 50% by weight of the methacrylate-modified silk fibroin has a silk fibroin backbone with a molecular weight of at least 250 kDa. In embodiments, at least 50% by weight of the methacrylate-modified silk fibroin has a silk fibroin backbone with a molecular weight of at least 450 kDa.

[0077] In embodiments, the methacrylate-modified silk fibroin may be present in the noncrosslinked composition in an amount by weight of between 2% and 10%, at least 2%, at least 3%, at least 4%, or at least 5% and at most 10%, at most 9%, at most 8%, at most 7%, at most 6%, or at most 5%.

[0078] In embodiments, the photo-crosslinker may be present in the non-crosslinked composition in an amount by weight of between 0.001% and 0.1%, at least 0.001%, at least 0.005%, or at least 0.01% and at most 0.5%, at most 0.1%, at most 0.08%, or at most 0.05%. The photo-crosslinker may be lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP)

[0079] In embodiments, the enzymatic crosslinker is present in the non-crosslinked composition in an amount providing enzymatic activity of between 5 U / mL and 20 U / mL, at least 2 U / mL, at least 5 U / mL or at least 10 U / mL and at most 20 U / mL, at most 15 U / mL, or at most 10 U / mL.

[0080] In embodiments, water may be present in the non-crosslinked composition in an amount by weight of between 85% and 95%, including but not limited to, between 90% and 95%.

[0081] In embodiments, a general method may include photo-crosslinking a composition described herein to establish a desired printing viscosity, printing the photo-crosslinked composition, and enzymatically crosslinking the printed composition to establish a final mechanical property. In some embodiments, the process of printing the composition may alter a structural property of the photocrosslinked composition.

[0082] In some embodiments, the methacrylate content of regenerated methacrylate silk fibroin (Sil- Ma) can be tuned (e.g., the number of methacrylate side chains may be altered) to enable full photocrosslinking. However, since the methacrylation is performed on lysine residues, which typically make up 0.3-4 % mol along the silk fibroin sequence, the decrease of the methacrylation may result in the need for very long exposure time under light, photo-oxidation of the protein, and weak crosslinking.

[0083] Throughout this disclosure, examples include printing with, or otherwise extruding or depositing, a PC gel, however, it may be possible to extrude a gel after complete photo-crosslinking(e.g., full photo-crosslinking, or FC) with lower molecular weight silk fibroins, although the nozzle / delivery device shear stress may cause damage to a fully crosslinked composition.

[0084] Given the massive crystalline secondary structure of the gels, in combination with covalent crosslinking, the gels experience in vivo stability with slow degradation (e.g., on the order of months and years). Shear stress in the nozzle induces beta-sheet formation via physical entanglement, and pre- photo-crosslinking the enzymatic crosslinking induce the formation of covalent bonds, which are difficult to digest when compared to physical bonds. In the PC gel, the crosslinking is not completed and the methacrylate sites are not widespread along the sequence, thus, the covalent bonds are insufficient to resist degradation over a testing time period (e.g., two weeks). Enzymatic crosslinking (e.g., full enzymatic crosslinking, EC) creates further covalent bonds via tyrosine along the silk sequence, and the resulting gel is more resistant to degradation than the PC gel. In some embodiments, the PC gel in combination with the shear stress in the nozzle may be used for applications requiring faster degradation kinetics compared to the double-crosslinked (DC) ones.

[0085] Silk fibroin exhibits extraordinary properties for biomedical applications. However, 3D printing and deposition techniques require biomaterials to possess specific characteristics, particularly appropriate viscosity and shape fidelity. Silk fibroin presents challenges in meeting these requirements, as its gelation demands precise control and stability over the process. However, its low viscosity and risk of unwanted gelation, especially at high concentrations, have historically limited its applications in bioprinting. This disclosure overcomes these limitations by developing low -concentration silk fibroin-based inks that provide both shape fidelity and stability after printing through two key steps.

[0086] A photo-driven partial pre-crosslinking process of methacrylated silk fibroin achieves optimal viscosity and gelation properties for printing. This pre-crosslinking step, combined with the pressure (shear stresses) applied during extrusion through the printing nozzle, enhances silk molecule rearrangement and subsequent gelation, resulting in stable printed structures.

[0087] Following the pre-crosslinking step, an in situ enzymatic crosslinking mechanism can be activated. This secondary process forms additional covalent bonds, producing hydrogels with enhanced resistance to enzymatic degradation.

[0088] The versatility of this methodology has been demonstrated using two different silk fibroin molecular weights. This development enables printing pristine silk structures that form stable hydrogels with tunable degradation kinetics that are adaptable to various applications and holds significant potential for bioprinting companies developing bioinks or three-dimensional protein-based matrices for biomaterials deposition with final hydrogels fabrication.

[0089] The shortage of tissues and organs for transplantation is an urgent clinical concern. In situ 3D printing is an advanced 3D printing technique aimed at printing the new tissue or organ directlyin the patient. The ink for this process is central to the outcomes, and must meet specific requirements such as rapid gelation, shape integrity, stability over time, and adhesion to surrounding healthy tissues. Among natural materials, silk fibroin exhibits fascinating properties that have made it widely studied in tissue engineering and regenerative medicine. However, further improvements in silk fibroin inks are needed to match the requirements for in situ 3D printing. In the present disclosure, silk fibroin-based inks were developed for in situ applications by exploiting covalent crosslinking process consisting of a pre-photo-crosslinking prior to printing and in situ enzymatic crosslinking. Two different silk fibroin molecular weights were characterized and the synergistic effect of the covalent bonds with shear forces enhanced the shift in silk secondary structure toward 0- sheets, thus, rapid stabilization. These hydrogels exhibited good mechanical properties, stability over time, and resistance to enzymatic degradation over 14 days, with no significant changes over time in their secondary structure and swelling behavior. Additionally, adhesion to tissues in vitro was demonstrated.

[0090] The shortage of tissues / organs transplantation remains a global challenge, with only 10% of needs being met. To potentially fill some of this gap, 3D (bio)printing is a technology that can offer a solution. 3D printing enables reproducible, standardized, and personalized manufacturing of 3D architectures to mimic native tissue and organ structures. Numerous 3D printing techniques have been developed including extrusion-based, and light-based techniques such as digital light processing, stereolithography, volumetric 3D printing. However, there are intrinsic limitations in the application of in vitro 3D printing to optimize clinical translation, including mismatches between the fabricated implant and the expected / unexpected defect size in vivo, limitations with the biological evaluation performed in vitro, infection risks due to manipulation to transfer the implant in the surgical setting and during implantation, as well as integration of the printed materials with the native healthy tissue at the implant site in vivo. Thus, more advanced options are needed, and in situ 3D printing as a personalized medicine approach is an evolving strategy. Here, 3D models can be elaborated starting from the patients’ specific defect size and shape via images, to guide direct printing of the new tissue in the patient in the operating room, with anatomical accuracy and fidelity using minimally invasive routes. By printing directly in the patient, there is the control in real time of the defect size, by inducing crosslinking of the ink in situ, and the body can then work as natural bioreactor. In this scenario, two main approaches have been developed to achieve success. The first one is based on a portable device, such as the Biopen, and the second, robotic arms to print with high precision along three axes via a surgeon-controlled console. Both approaches exhibit some limitations, such as shape fidelity dependent on surgeon skills and only superficial tissues such as skin can be reached by the robotic approach, along with the overall complexity of the procedure.

[0091] Ex situ and in situ 3D printing approaches applied to cartilage regeneration were compared. Compared to canonical 3D in vitro printing techniques, there remain many issues with in situ applications. The in situ approach not only provides shape fidelity but offers more direct and rapid integration to the surrounding tissues, thereby avoiding complications with in vitro printed materials that require time to integrate and can lose interfaces with the surrounding tissues, resulting in complications in regeneration. A key point in the development of in situ printing is the ink. The ink must exhibit specific requirements including suitable rheological properties, adhesion to tissues, shape fidelity after deposition in the defect, and rapid gelation. Moreover, compared to in vitro 3D printing, inks designed for in situ applications require a fixed printing bed temperature at 37°C to simulate body temperature, whereas photo-crosslinking in situ may be problematic since irradiation in the body can be harmful and light penetration into the depth of tissues is limited in order to reach internal organs.

[0092] Silk fibroin at relatively low concentrations exhibits low viscosity which can limit extrusion 3D printing. Higher concentrations are prone to aggregation that can block printer heads. To exploit the potential of pristine silk fibroin for in situ 3D printing, it is necessary to avoid nozzle clogging when using high concentrations of the protein, overcome the low viscosity of silk solutions, and induce rapid crosslinking, among other challenges.

[0093] The aim was to design silk-based inks for in situ applications through a reliable, repeatable, and versatile process, without any postprocessing manipulation and to overcome the various challenges highlighted above. A double crosslinking process was developed to address this goal, combining pre-gelation via incomplete photo-crosslinking followed by in situ HRP-driven gelation. The versatility of the process was demonstrated with two different silk molecular weights to show feasibility for in situ 3D printing. Additionally, the fundamental role of physical crosslinking induced by extrusion stress in the print nozzle was a focus towards rapid transition of random coils and 0-turn structures to -sheet formation, further stabilizing the gels over time. The printed hydrogels resulted in mechanically stable systems that supported bone marrow-derived human mesenchymal stem cell adhesion and viability, and good adhesion on mock tissues (e.g., raw chicken breast). An overview of the process is shown in Fig. 1.

[0094] Overall, pristine silk fibroin has good printability at low concentrations. Nozzle shear stress plays a key role in secondary structure. Two molecular weight silk fibroins were demonstrated, proving this is a versatile system. Silk fibroin is a suitable candidate for in situ 3D printing applications.

[0095] In some aspects, a non-crosslinked composition has a capacity for double crosslinking. The non-crosslinked composition may include a population of methacrylate-modified silk fibroinfragments, with each of the methacrylate-modified silk fibroin fragments including methacrylate side chains. The composition may also include a photo-crosslinker, an enzymatic crosslinker and optionally one or more enzymatic crosslinker co-reactants, and water. The enzymatic crosslinker coreactant may be hydrogen peroxide.

[0096] In some aspects, a non-crosslinked composition has a capacity for double crosslinking. The non-crosslinked composition may include a population of methacrylate-modified silk fibroin fragments, with each of the methacrylate-modified silk fibroin fragments including methacrylate side chains, a photo-crosslinker, one or more enzymatic crosslinker co-reactants, optionally an enzymatic crosslinker, and water. The enzymatic crosslinker co-reactant may be hydrogen peroxide.

[0097] In some aspects, a pre-crosslinked composition that is partly photo-crosslinked may have capacity for further photo-crosslinking and additional enzymatic crosslinking to become doublecrosslinked. The pre-crosslinked composition may comprise the non-crosslinked composition mentioned herein having partial photo-crosslinking between methacrylate-modified silk fibroin fragments. The partial photo-crosslinking may produce an ink that one or more of: exhibits shape fidelity after extrusion, forms a homogenous and uniform filament that can be deposited on a printing bed keeping its shape and avoids the layers collapsing when more than one is printed, has a storage modulus G’ higher than G”, has a crossover point at most in the range of 300 GPa of stress, or the ink can be extruded with an extrusion pressure that does not exceed 400 kPa. The partial photocrosslinking may involve between 1% and 50%, between 40% and 70%, between 1% and 99%, or between 50% and 99.9% of the methacrylate side chains. The pre-crosslinked composition may be adhesive to mammalian tissue or an anatomical surface. The additional enzymatic crosslinking may generate enzymatic crosslinks between the methacrylate-modified silk fibroin fragments of the population of methacrylate-modified silk fibroin fragments to produce a double-crosslinked composition. The enzymatic crosslinks may be dityrosine bonds. The double-crosslinked composition may have a plurality of cells seeded to form a seeded, double-crosslinked composition. The cells may have a seeding density of at least IxlO5cells / mL.

[0098] A non-crosslinked composition is made by combining a population of methacrylate- modified silk fibroin fragments, a photo-crosslinker, an enzymatic crosslinker, optionally one or more enzymatic crosslinker co-reactants, and water.

[0099] A non-crosslinked composition is used by pre-crosslinking the non-crosslinked composition, thereby converting the non-crosslinked composition into a pre-crosslinked composition. The pre-crosslinked article may be enzymatically crosslinked. The non-crosslinked composition may be the non-crosslinked composition described herein. The pre-crosslinked composition may be printed into a predefined shape to produce a printed, pre-crosslinked article. Theprinting may be in vivo printing in a mammalian subject, which may optionally be a human subject. The predefined shape may include internal structural features.

[0100] In some aspects, the methacrylate side chains may be present on the methacry late-modified silk fibroin in an amount by weight of between 0.3% mol and 4% mol of the silk fibroin. The methacrylate-modified silk fibroin may have a silk fibroin backbone with a molecular weight between 25 and 500 kDa, between 50 and 400 kDa, between 100 kDa and 300 kDa, or between 150 kDa and 350 kDa. The methacrylate-modified silk fibroin may have a silk fibroin backbone having an average molecular weight greater than 150 kDa. The methacrylate-modified silk fibroin may include a silk fibroin obtained by boiling for at most 15 minutes, at most 20 minutes, at most 30 minutes, at most 45 minutes, or at most 60 minutes, at least 15 minutes, at least 30 minutes, at least 60 minutes, or at least 120 minutes. At least 50% by weight of the methacrylate-modified silk fibroin may have a silk fibroin backbone with a molecular weight of at least 25 kDa, at least 50 kDa, at least 100 kDa, at least 250 kDa, or at least 450 kDa.

[0101] The methacrylate-modified silk fibroin may be present in the non-crosslinked composition in an amount by weight of between 2% and 10%. More specifically, the methacrylate-modified silk fibroin may be present in the non-crosslinked composition in an amount by weight of at least 2%, at least 3%, at least 4%, or at least 5% and at most 10%, at most 9%, at most 8%, at most 7%, at most 6%, or at most 5%. The photo-crosslinker may be present in the non-crosslinked composition in an amount by weight of between 0.001% and 0.1%. More specifically, the photo-crosslinker is present in the non-crosslinked composition in an amount by weight of at least 0.001%, at least 0.005%, or at least 0.01 % and at most 0.5%, at most 0.1 %, at most 0.08%, or at most 0.05%. The enzymatic crosslinker may be present in the non-crosslinked composition in an amount providing enzymatic activity of between 5 U / mL and 20 U / mL. More specifically, the enzymatic crosslinker may be present in the non-crosslinked composition in an amount providing enzymatic activity of at least 2 U / mL, at least 5 U / mL, or at least 10 U / mL, and at most 20 U / mL, at most 15 U / mL, or at most 10 U / mL.

[0102] In some aspects, the photo-crosslinker is lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP). The water may be present in the non-crosslinked composition in an amount by weight of between 85% and 95%, including but not limited to between 90% and 95%.

[0103] A general method includes photo-crosslinking a composition described herein to establish a desired printing viscosity, printing the photo-crosslinked composition, and enzymatically crosslinking the printed composition to establish a final mechanical property.

[0104] According to various embodiments, a variety of functionalizing agents may be used with the silk-containing embodiments described herein (e.g., silk membrane, silk composition, silk articles, silkmatrix, silk foam, silk microsphere, liquid composition, whipped silk cream, silk meringue, compressed silk meringue, hot-pressed silk meringue, silk leather, silk powder, silk toner, edible silkbased films, etc.). It should be understood that the examples herein may recite one or a few silkcontaining embodiments but are applicable to any silk-containing embodiment, as applicable. In some embodiments, a functionalizing agent may be any compound or molecule that facilitates the attachment to and / or development (e.g., growth) of one or more endothelial cells on a silk membrane. In some embodiments, a functionalizing agent may be any compound or molecule that facilitates the attachment and / or development (e.g., growth) of one or more megakaryocytes and / or hematopoietic progenitor cells on a silk matrix and / or silk membrane. In some embodiments, a functionalizing agent may be or comprise an agent suitable for facilitating the production of one or more of white blood cells and red blood cells.

[0105] In some embodiments, a functionalizing agent may be or comprise a cell attachment mediator and / or an extracellular matrix protein, for example: collagen (e.g., collagen type I, collagen type III, collagen type IV or collagen type VI), elastin, fibronectin, vitronectin, laminin, fibrinogen, von Willebrand factor, proteoglycans, decorin, perlecan, nidogen, hyaluronan, and / or peptides containing known integrin binding domains e.g. “RGD” integrin binding sequence, or variations thereof, that are known to affect cellular attachment.

[0106] In some embodiments, a functionalizing agent may be any soluble molecule produced by endothelial cells. Non-limiting examples include fibroblast growth factor- 1 (FGF1) and vascular endothelial growth factors (VEGF).

[0107] According to some embodiments, a plurality of functionalizing agents may be used. For example, in some embodiments wherein production of platelets is desired, provided compositions may comprise the use of laminin, fibronectin and / or fibrinogen, and type IV collagen in order to facilitate the attachment and growth of endothelial cells on a silk membrane (e.g., a porous silk membrane) and / or attachment of megakaryocytes to a silk matrix.

[0108] In some embodiments, a functionalizing agent may be embedded or otherwise associated with a silk membrane and / or silk matrix such that at least a portion of the functionalizing agent is surrounded by a silk membrane and / or silk matrix as contrasted to a functionalizing agent simply being positioned along the surface of a silk membrane and / or silk matrix. In some embodiments, a functionalizing agent is distributed along and / or incorporated in substantially the entire surface area of a silk membrane / silk wall. In some embodiments, a functionalizing agent is distributed and / or incorporated only at one or more discrete portions of a silk membrane / wall and / or silk matrix. In some embodiments, a functionalizing agent is distributed in and / or along at least one of the lumenfacing side of a silk wall and the matrix-facing side of a silk wall.

[0109] According to various embodiments, any application-appropriate amount of one or more functionalizing agents may be used. In some embodiments, the amount of an individual functionalizing agent may be between about 1 pg / ml and 1,000 pg / ml (e.g., between about 2 and 1,000, 5 and 1,000, 10 and 1,000, 10 and 500, 10 and 100 pg / ml). In some embodiments, the amount of an individual functionalizing agent may be at least 1 pg / ml (e.g., at least 5, 10, 15, 20 25, 50, 100, 200, 300 400, 500, 600, 700, 800, or 900 pg / ml ). In some embodiments, the amount of an individual functionalizing agent is at most 1,000 pg / ml (e.g., 900, 800, 700, 600, 500, 400, 300 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, or 5 pg / ml ).

[0110] In some aspects, the composition comprises one or more sensing agents, such as a sensing dye. The sensing agents / sensing dyes are environmentally sensitive and produce a measurable response to one or more environmental factors. In some aspects, the environmentally- sensitive agent or dye may be present in the composition in an effective amount to alter the composition from a first chemical -physical state to a second chemical -physical state in response to an environmental parameter (e.g., a change in pH, light intensity or exposure, temperature, pressure or strain, voltage, physiological parameter of a subject, and / or concentration of chemical species in the surrounding environment) or an externally applied stimulus (e.g., optical interrogation, acoustic interrogation, and / or applied heat). In some cases, the sensing dye is present to provide one optical appearance under one given set of environmental conditions and a second, different optical appearance under a different given set of environmental conditions. Suitable concentrations for the sensing agents described herein can be the concentrations for the colorants and additives described elsewhere herein. A person having ordinary skill in the chemical sensing arts can determine a concentration that is appropriate for use in a sensing application of the inks described herein.

[0111] In some aspects, the first and second chemical-physical state may be a physical property of the composition, such as mechanical property, a chemical property, an acoustical property, an electrical property, a magnetic property, an optical property, a thermal property, a radiological property, or an organoleptic property. Exemplary sensing dyes or agents include, but are not limited to, a pH sensitive agent, a thermal sensitive agent, a pressure or strain sensitive agent, a light sensitive agent, or a potentiometric agent.

[0112] Exemplary pH sensitive dyes or agents include, but are not limited to, cresol red, methyl violet, crystal violet, ethyl violet, malachite green, methyl green, 2-(p- dimethylaminophenylazo) pyridine, paramethyl red, metanil yellow, 4-phenylazodiphenylamine, thymol blue, metacresol purple, orange IV, 4-o-Tolylazo-o-toluindine, quinaldine red, 2,4- dinitrophenol, erythrosine disodium salt, benzopurpurine 4B, N,N-dimethyl-p-(m-tolylazo) aniline, p- dimethylaminoazobenene, 4,4'-bis(2-amino-l-naphthylazo)-2,2'-stilbenedisulfonic acid,tetrabromophenolphthalein ethyl ester, bromophenol blue, Congo red, methyl orange, ethyl orange, 4-(4-dimethylamino-l-naphylazo)-3-methoxybenesulfonic acid, bromocresol green, resazurin, 4- phenylazo-l-napthylamine, ethyl red 2-(l-dimethylaminophenyazo) pyridine, 4-(p- ethoxypehnylazo)-m-phenylene-diamine monohydrochloride, resorcin blue, alizarin red S, methyl red, propyl red, bromocresol purple, chlorophenol red, p-nitrophenol, alizarin 2-(2,4- dinitrophenylazo) l-napthol-3,6-disulfonic acid, bromothymol blue, 6,8-dinitro-2,4-(lH) quinazolinedione, brilliant yellow, phenol red, neutral red, m-nitrophenol, cresol red, turmeric, metacresol purple, 4,4'-bis(3-amino-l-naphthylazo)-2,2'-stilbenedisulfonic acid, thymol blue, p- naphtholbenzein, phenolphthalein, o-cresolphthalein, ethyl bis(2,4-dimethylphenyl) ethanoate, thymolphthalein, nitrazine yellow, alizarin yellow R, alizarin, p-(2,4-dihydroxyphenylazo) benzenesulfonic acid, 5,5'-indigodisulfonic acid, 2,4,6-trinitrotoluene, 1,3,5-trinitrobenezne, and clayton yellow.

[0113] Exemplary light responsive dyes or agents include, but are not limited to, photochromic compounds or agents, such as triarylmethanes, stilbenes, azasilbenes, nitrones, fulgides, spiropyrans, napthopyrans, spiro-oxzines, quinones, derivatives and combinations thereof.

[0114] Exemplary potentiometric dyes include, but are not limited to, substituted amiononaphthylehenylpridinium (ANEP) dyes, such as di-4-ANEPPS, di-8-ANEPPS, and N-(4- Sulfobutyl)-4-(6-(4-(Dibutylamino)phenyl)hexatrienyl)Pyridinium (RH237).

[0115] Exemplary temperature sensitive dyes or agents include, but are not limited to, thermochromic compounds or agents, such as thermochromic liquid crystals, leuco dyes, fluoran dyes, octadecylphosphonic acid.

[0116] Exemplary pressure or strain sensitive dyes or agents include, but are not limited to, spiropyran compounds and agents.

[0117] Exemplary chemi- sensitive dyes or agents include, but are not limited to, antibodies such as immunoglobulin G (IgG) which may change color from blue to red in response to bacterial contamination.

[0118] In some aspects, the compositions comprise one or more additive, dopant, or biologically active agent suitable for a desired intended purpose. In some aspects, the additive or dopant may be present in the composition in an amount effective to impart an optical or organoleptic property to the composition. Exemplary additives or dopants that impart optical or organoleptic properties include, but are not limited to, dyes / pigments, flavorants, aroma compounds, granular or fibrous fillers.

[0119] Additionally or alternatively, the additive, dopant, or biologically active agent may be present in the composition in an amount effective to "functionalize" the composition to impart a desired mechanical property or added functionality to the composition. Exemplary additive, dopants,or biologically active agent that impart the desired mechanical property or added functionality include, but are not limited to: environmentally sensitive / sensing dyes; active biomolecules; conductive or metallic particles; micro and nanofibers (e.g., silk nanofibers for reinforcement, carbon nanofibers); nanotubes; inorganic particles (e.g., hydroxyapatite, tricalcium phosphate, bioglasses); drugs (e.g., antibiotics, small molecules or low molecular weight organic compounds); proteins and fragments or complexes thereof (e.g., enzymes, antigens, antibodies and antigen-binding fragments thereof); DNA / RNA (e.g., siRNA, miRNA, mRNA); cells and fractions thereof (viruses and viral particles; prokaryotic cells such as bacteria; eukaryotic cells such as mammalian cells and plant cells; fungi).

[0120] In some aspects, the additive or dopant comprises a flavoring agent or flavorant.

[0121] Exemplary flavorants include ester flavorants, amino acid flavorants, nucleic acid flavorants, organic acid flavorants, and inorganic acid flavorants, such as, but not limited to, diacetyl, acetylpropionyl, acetoin, isoamyl acetate, benzaldehyde, cinnamaldehyde, ethyl propionate, methyl anthranilate, limonene, ethyl decadienoate, allyl hexanoate, ethyl maltol, ethylvanillin, methyl salicylate, manzanate, glutamic acid salts, glycine salts, guanylic acids salts, inosinic acid salts, acetic acid, ascorbic acid, citric acid, fumaric acid, lactic acid, malic acid, phosphoric acid, tartaric acid, derivatives, and mixtures thereof.

[0122] In some aspects, the additive or dopant comprises an aroma compound. Exemplary aroma compounds include ester aroma compounds, terpene aroma compounds, cyclic terpenes, and aromatic aroma compounds, such as, but not limited to, geranyl acetate, methyl formate, metyl acetate, methyl propionate, methyl butyrate, ethyl acetate, ethyl butyrate, isoamyl acetate, pentyl butrate, pentyl pentanoate, octyl acetate, benzyl acetate, methyl anthranilate, myrecene, geraniol, nerol, citral, cironellal, cironellol, linalool, nerolidol, limonene, camphor, menthol, carone, terpineol, alpha-lonone, thujone, eucalyptol, benzaldehyde, eugenol, cinnamaldehyde, ethyl maltol, vanillin, anisole, anethole, estragole, thymol.

[0123] In some aspects, the additive or dopant comprises a colorant, such as a dye or pigment. In some aspects, the dye or pigment imparts a color or grayscale to the composition. The colorant can be different than the sensing agents and / or sensing dyes below. Any organic and / or inorganic pigments and dyes can be included in the inks. Exemplary pigments suitable for use in the present disclosure include International Color Index or C.I. Pigment Black Numbers 1 , 7, 1 1 and 31 , C.I. Pigment Blue Numbers 15, 15 : 1 , 15 :2, 15 :3, 15 :4, 15 :6, 16, 27, 29, 61 and 62, C.I. Pigment Green Numbers 7, 17, 18 and 36, C.I. Pigment Orange Numbers 5, 13, 16, 34 and 36, C.I. Pigment Violet Numbers 3, 19, 23 and 27, C.I. Pigment Red Numbers 3, 17, 22, 23, 48: 1 , 48:2, 57: 1 , 81 : 1 , 81 :2, 81 :3, 81 :5, 101 , 1 14, 122, 144, 146, 170, 176, 179, 181 , 185, 188, 202, 206, 207, 210 and249, C.I. Pigment Yellow Numbers 1 , 2, 3, 12, 13, 14, 17, 42, 65, 73, 74, 75, 83, 30, 93, 109, 1 10, 128, 138, 139, 147, 142, 151 , 154 and 180, D&C Red No. 7, D&C Red No. 6 and D&C Red No. 34, carbon black pigment (such as Regal 330, Cabot Corporation), quinacridone pigments (Quinacridone Magenta (228-0122), available from Sun Chemical Corporation, Fort Lee, N.J.), diarylide yellow pigment (such as AAOT Yellow (274- 1788) available from Sun Chemical Corporation); and phthalocyanine blue pigment (such as Blue 15 :3 (294-1298) available from Sun Chemical Corporation). The classes of dyes suitable for use in present invention can be selected from acid dyes, natural dyes, direct dyes (either cationic or anionic), basic dyes, and reactive dyes. The acid dyes, also regarded as anionic dyes, are soluble in water and mainly insoluble in organic solvents and are selected, from yellow acid dyes, orange acid dyes, red acid dyes, violet acid dyes, blue acid dyes, green acid dyes, and black acid dyes. European Patent 0745651, incorporated herein by reference, describes a number of acid dyes that are suitable for use in the present disclosure. Exemplary yellow acid dyes include Acid Yellow 1 International Color Index or C.I. 10316); Acid Yellow 7 (C.I. 56295); Acid Yellow 17 (C.I. 18965); Acid Yellow 23 (C.I. 19140); Acid Yellow 29 (C.I. 18900); Acid Yellow 36 (C.I. 13065); Acid Yellow 42 (C.I. 22910); Acid Yellow 73 (C.I. 45350); Acid Yellow 99 (C.I. 13908); Acid Yellow 194; and Food Yellow 3 (C.I. 15985). Exemplary orange acid dyes include Acid Orange 1 (C.I. 13090 / 1); Acid Orange 10 (C.I. 16230); Acid Orange 20 (C.I. 14603); Acid Orange 76 (C.I. 18870); Acid Orange 142; Food Orange 2 (C.I. 15980); and Orange B.

[0124] Exemplary red acid dyes include Acid Red 1. (C.I. 18050); Acid Red 4 (C.I. 14710); Acid Red 18 (C.I. 16255), Acid Red 26 (C.I. 16150); Acid Red 2.7 (C.I. as Acid Red 51 (C.I. 45430, available from BASF Corporation, Mt. Olive, N.J.) Acid Red 52 (C.I. 45100); Acid Red 73 (C.I. 27290); Acid Red 87 (C. I. 45380); Acid Red 94 (C.I. 45440) Acid Red 194; and Food Red 1 (C.I. 14700). Exemplary violet acid dyes include Acid Violet 7 (C.I. 18055); and Acid Violet 49 (C.I. 42640). Exemplary blue acid dyes include Acid Blue 1 (C.I. 42045); Acid Blue 9 (C.I. 42090); Acid Blue 22 (C.I. 42755); Acid Blue 74 (C.I. 73015); Acid Blue 93 (C.I. 42780); and Acid Blue 158A (C.I. 15050). Exemplary green acid dyes include Acid Green 1 (C.I. 10028); Acid Green 3 (C.I. 42085); Acid Green 5 (C.I. 42095); Acid Green 26 (C.I. 44025); and Food Green 3 (C.I. 42053). Exemplary black acid dyes include Acid Black 1 (C.I. 20470); Acid Black 194 (Basantol® X80, available from BASF Corporation, an azo / 1 :2 CR-complex.

[0125] Exemplary direct dyes for use in the present disclosure include Direct Blue 86 (C.I. 74180); Direct Blue 199; Direct Black 168; Direct Red 253; and Direct Yellow 107 / 132 (C.I. Not Assigned).

[0126] Exemplary natural dyes for use in the present disclosure include Alkanet (C.I.75520,75530); Annafto (C.I. 75120); Carotene (C.I. 75130); Chestnut; Cochineal (C.I.75470); Cutch (C.I. 75250, 75260); Divi-Divi; Fustic (C.I. 75240); Hypemic (C.I. 75280); Logwood (C.I. 75200);Osage Orange (C.I. 75660); Paprika; Quercitron (C.I. 75720); Sanrou (C.I. 75100) ; Sandal Wood (C.I. 75510, 75540, 75550, 75560); Sumac; and Tumeric (C.I. 75300). Exemplary reactive dyes for use in the present disclosure include Reactive Yellow 37 (monoazo dye); Reactive Black 31 (disazo dye); Reactive Blue 77 (phthalo cyanine dye) and Reactive Red 180 and Reactive Red 108 dyes. Suitable also are the colorants described in The Printing Ink Manual (5th ed., Leach et al. eds.(2007), pages 289-299. Other organic and inorganic pigments and dyes and combinations thereof can be used to achieve the colors desired.

[0127] In addition to or in place of visible colorants, compositions provided herein can contain ETV fluorophores that are excited in the ETV range and emit light at a higher wavelength (typically 400 nm and above). Examples of ETV fluorophores include but are not limited to materials from the coumarin, benzoxazole, rhodamine, napthalimide, perylene, benzanthrones, benzoxanthones or benzothia- xanthones families. The addition of a UV fluorophore (such as an optical brightener for instance) can help maintain maximum visible light transmission. The amount of colorant, when present, generally is between 0.05% to 5% or between 0.1% and 1% based on the weight of the composition.

[0128] For non- white compositions, the amount of pigment / dye generally is present in an amount of from at or about 0.1 wt% to at or about 20 wt% based on the weight of the composition. In some applications, a non-white ink can include 15 wt% or less pigment / dye, or 10 wt% or less pigment / dye or 5 wt% pigment / dye, or 1 wt% pigment / dye based on the weight of the composition. In some applications, a non-white ink can include 1 wt% to 10 wt%, or 5 wt% to 15 wt%, or 10 wt% to 20 wt% pigment / dye based on the weight of the composition. In some applications, a non-white ink can contain an amount of dye / pigment that is 1 wt%, 2 wt%, 3 wt%, 4 wt%, 5%, 6 wt%, 7 wt%, 8 wt%, 9 wt%, 10 wt%, 11 wt%, 12 wt%, 13 wt%, 14 wt%, 15%, 16 wt%, 17 wt%, 18 wt%, 19 wt% or 20 wt% based on the weight of the composition.

[0129] For white compositions, the amount of white pigment generally is present in an amount of from at or about 1 wt% to at or about 60 wt% based on the weight of the composition. In some applications, greater than 60 wt% white pigment can be present. Preferred white pigments include titanium dioxide (anatase and rutile), zinc oxide, lithopone (calcined coprecipitate of barium sulfate and zinc sulfide), zinc sulfide, blanc fixe and alumina hydrate and combinations thereof, although any of these can be combined with calcium carbonate. In some applications, a white ink can include 60 wt% or less white pigment, or 55 wt% or less white pigment, or 50 wt% white pigment, or 45 wt% white pigment, or 40 wt% white pigment, or 35 wt% white pigment, or 30 wt% white pigment, or 25 wt% white pigment, or 20 wt% white pigment, or 15 wt% white pigment, or 10 wt% white pigment, based on the weight of the composition. In some applications, a white ink can include 5wt% to 60 wt%, or 5 wt% to 55 wt%, or 10 wt% to 50 wt%, or 10 wt% to 25 wt%, or 25 wt% to 50 wt%, or 5 wt% to 15 wt%, or 40 wt% to 60 wt% white pigment based on the weight of the composition. In some applications, a non- white ink can an amount of dye / pigment that is 5%, 6 wt%, 7 wt%, 8 wt%, 9 wt%, 10 wt%, 11 wt%, 12 wt%, 13 wt%, 14 wt%, 15%, 16 wt%, 17 wt%, 18 wt%, 19 wt%, 20 wt%, 21 wt%, 22 wt%, 23 wt%, 24 wt%, 25%, 26 wt%, 27 wt%, 28 wt%, 29 wt%, 30 wt%, 31 wt%, 32 wt%, 33 wt%, 34 wt%, 35%, 36 wt%, 37 wt%, 38 wt%, 39 wt%, 40 wt%, 41 wt%, 42 wt%, 43 wt%, 44 wt%, 45%, 46 wt%, 47 wt%, 48 wt%, 49 wt%, 50 wt%, 51 wt%, 52 wt%, 53 wt%, 54 wt%, 55%, 56 wt%, 57 wt%, 58 wt%, 59 wt% or 60 wt% based on the weight of the composition.

[0130] In some aspects, the additive or dopant comprises a conductive additive. Exemplary conductive additives include, but are not limited to graphite, graphite powder, carbon nanotubes, and metallic particles or nanoparticles, such as gold nanoparticles. In some aspects, the conductive additive is biocompatible and non-toxic.

[0131] In some aspects, the additive is a biologically active agent. The term “biologically active agent” as used herein refers to any molecule which exerts at least one biological effect in vivo. For example, the biologically active agent can be a therapeutic agent to treat or prevent a disease state or condition in a subject. Biologically active agents include, without limitation, organic molecules, inorganic materials, proteins, peptides, nucleic acids (e.g., genes, gene fragments, gene regulatory sequences, and antisense molecules), nucleoproteins, polysaccharides, glycoproteins, and lipoproteins. Classes of biologically active compounds that can be incorporated into the composition provided herein include, without limitation, anticancer agents, antibiotics, analgesics, antiinflammatory agents, immunosuppressants, enzyme inhibitors, antihistamines, anti-convulsants, hormones, muscle relaxants, antispasmodics, ophthalmic agents, prostaglandins, anti-depressants, anti-psychotic substances, trophic factors, osteoinductive proteins, growth factors, and vaccines.

[0132] The term “active agent” may also be used herein to refer to a biological sample (e.g., a sample of tissue or fluid, such as for instance blood) or a component thereof, and / or to a biologically active entity or compound, and / or to a structurally or functionally labile entity.

[0133] Exemplary active agents include, but are not limited to, therapeutic agents, diagnostic agents (e.g., contrast agents), and any combinations thereof. In some embodiments, the active agent present in a silk matrix (e.g., a silk microsphere), composition, or the like can include a labile active agent, e.g., an agent that can undergo chemical, physical, or biological change, degradation and / or deactivation after exposure to a specified condition, e.g., high temperatures, high humidity, light exposure, and any combinations thereof. In some embodiments, the active agent present in the silk matrix (e.g., a silk microsphere), composition, or the like can include a temperature-sensitive activeagent, e.g., an active agent that will lose at least about 30% or more, of its original activity or bioactivity, upon exposure to a temperature of at least about 10° C. or above, including at least about 15° C. or above, at least about room temperature or above, or at least about body temperature (e.g., about 37° C.) or above.

[0134] The active agent can be generally present in the silk matrix (e.g., a silk microsphere), composition, or the like in an amount of about 0.01% (w / w) to about 70% (w / w), or about 0.1% (w / w) to about 50% (w / w), or about 1% (w / w) to about 30% (w / w). The active agent can be present on a surface of the silk matrix (e.g., a silk microsphere), composition, or the like and / or encapsulated and dispersed in the silk matrix (e.g., a silk microsphere), composition, or the like homogeneously or heterogeneously or in a gradient. In some embodiments, the active agent can be added into the silk solution, which is then subjected to the methods described herein for preparing a silk matrix (e.g., a silk microsphere), composition, or the like. In some embodiments, the active agent can be coated on a surface of the silk matrix (e.g., a silk microsphere), composition, or the like. In some embodiments, the active agent can be loaded in a silk matrix (e.g., a silk microsphere), composition, or the like by incubating the silk microsphere in a solution of the active agent for a period of time, during which an amount of the active agent can diffuse into the silk matrix (e.g., a silk microsphere), composition, or the like, and thus distribute within the silk matrix (e.g., a silk microsphere), composition, or the like.

[0135] In some aspects, the additive is a therapeutic agent. As used herein, the term “therapeutic agent” means a molecule, group of molecules, complex or substance administered to an organism for diagnostic, therapeutic, preventative medical, or veterinary purposes. As used herein, the term “therapeutic agent” includes a “drug” or a “vaccine.” This term include externally and internally administered topical, localized and systemic human and animal pharmaceuticals, treatments, remedies, nutraceuticals, cosmeceuticals, biologicals, devices, diagnostics and contraceptives, including preparations useful in clinical and veterinary screening, prevention, prophylaxis, healing, wellness, detection, imaging, diagnosis, therapy, surgery, monitoring, cosmetics, prosthetics, forensics and the like. This term can also be used in reference to agriceutical, workplace, military, industrial and environmental therapeutics or remedies comprising selected molecules or selected nucleic acid sequences capable of recognizing cellular receptors, membrane receptors, hormone receptors, therapeutic receptors, microbes, viruses or selected targets comprising or capable of contacting plants, animals and / or humans. This term can also specifically include nucleic acids and compounds comprising nucleic acids that produce a therapeutic effect, for example deoxyribonucleic acid (DNA), ribonucleic acid (RNA), nucleic acid analogues (e.g., locked nucleic acid (LNA), peptide nucleic acid (PNA), xeno nucleic acid (XNA)), or mixtures or combinations thereof, including, for example, DNA nanoplexes, siRNA, microRNA, shRNA, aptamers, ribozymes, decoynucleic acids, antisense nucleic acids, RNA activators, and the like. Generally, any therapeutic agent can be included in the composition provided herein.

[0136] The term “therapeutic agent” also includes an agent that is capable of providing a local or systemic biological, physiological, or therapeutic effect in the biological system to which it is applied. For example, the therapeutic agent can act to control infection or inflammation, enhance cell growth and tissue regeneration, control tumor growth, act as an analgesic, promote anti-cell attachment, and enhance bone growth, among other functions. Other suitable therapeutic agents can include anti-viral agents, hormones, antibodies, or therapeutic proteins. Other therapeutic agents include prodrugs, which are agents that are not biologically active when administered but, upon administration to a subject are converted to biologically active agents through metabolism or some other mechanism. Additionally, a silk-based drug delivery composition can contain one therapeutic agent or combinations of two or more therapeutic agents.

[0137] A therapeutic agent can include a wide variety of different compounds, including chemical compounds and mixtures of chemical compounds, e.g., small organic or inorganic molecules; saccharines; oligosaccharides; polysaccharides; biological macromolecules, e.g., peptides, proteins, and peptide analogs and derivatives; peptidomimetics; antibodies and antigen binding fragments thereof; nucleic acids; nucleic acid analogs and derivatives; an extract made from biological materials such as bacteria, plants, fungi, or animal cells; animal tissues; naturally occurring or synthetic compositions; and any combinations thereof. In some aspects, the therapeutic agent is a small molecule.

[0138] The term “bioactivity,” as used herein in reference to an active agent, generally refers to the ability of an active agent to interact with a biological target and / or to produce an effect on a biological target. For example, bioactivity can include, without limitation, elicitation of a stimulatory, inhibitory, regulatory, toxic or lethal response in a biological target. The biological target can be a molecule or a cell. For example, a bioactivity can refer to the ability of an active agent to modulate the effect / activity of an enzyme, block a receptor, stimulate a receptor, modulate the expression level of one or more genes, modulate cell proliferation, modulate cell division, modulate cell morphology, or any combination thereof. In some instances, a bioactivity can refer to the ability of a compound to produce a toxic effect in a cell. Exemplary cellular responses include, but are not limited to, lysis, apoptosis, growth inhibition, and growth promotion; production, secretion, and surface expression of a protein or other molecule of interest by the cell; membrane surface molecule activation including receptor activation; transmembrane ion transports; transcriptional regulations; changes in viability of the cell; changes in cell morphology; changes in presence or expression of an intracellular component of the cell; changes in gene expression or transcripts; changes in the activityof an enzyme produced within the cell; and changes in the presence or expression of a ligand and / or receptor (e.g., protein expression and / or binding activity). Methods for assaying different cellular responses are well known to one of skill in the art, e.g., western blot for determining changes in presence or expression of an endogenous protein of the cell, or microscopy for monitoring the cell morphology in response to the active agent, or FISH and / or qPCR for the detection and quantification of changes in nucleic acids. Bioactivity can be determined in some embodiments, for example, by assaying a cellular response.

[0139] In reference to an antibody, the term “bioactivity” includes, but is not limited to, epitope or antigen binding affinity, the in vivo and / or in vitro stability of the antibody, the immunogenic properties of the antibody, e.g., when administered to a human subject, and / or the ability to neutralize or antagonize the bioactivity of a target molecule in vivo or in vitro. The aforementioned properties or characteristics can be observed or measured using art-recognized techniques including, but not limited to, scintillation proximity assays, ELISA, ORIGEN immunoassay (IGEN), fluorescence quenching, fluorescence ELISA, competitive ELISA, SPR analysis including, but not limited to, SPR analysis using a BIAcore biosensor, in vitro and in vivo neutralization assays (see, for example, International Publication No. WO 2006 / 062685), receptor binding, and immunohistochemistry with tissue sections from different sources including human, primate, or any other source as needed. In reference to an immunogen, the “bioactivity” includes immunogenicity, the definition of which is discussed in detail later. In reference to a virus, the “bioactivity” includes infectivity, the definition of which is discussed in detail later. In reference to a contrast agent, e.g., a dye, the “bioactivity” refers to the ability of a contrast agent when administered to a subject to enhance the contrast of structures or fluids within the subject's body. The bioactivity of a contrast agent also includes, but is not limited to, its ability to interact with a biological environment and / or influence the response of another molecule under certain conditions.

[0140] As used herein, the term “small molecule” can refer to compounds that are “natural product-like,” however, the term “small molecule” is not limited to “natural product-like” compounds. Rather, a small molecule is typically characterized in that it contains several carbon — carbon bonds, and has a molecular weight of less than 5000 Daltons (5 kDa), preferably less than 3 kDa, still more preferably less than 2 kDa, and most preferably less than 1 kDa. In some cases it is preferred that a small molecule have a molecular weight equal to or less than 700 Daltons.

[0141] Exemplary therapeutic agents include, but are not limited to, those found in Harrison’s Principles of Internal Medicine, 13th Edition, Eds. T.R. Harrison et al. McGraw-Hill N.Y., NY; Physicians’ Desk Reference, 50th Edition, 1997, Oradell New Jersey, Medical Economics Co.; Pharmacological Basis of Therapeutics, 8th Edition, Goodman and Gilman, 1990; United StatesPharmacopeia, The National Formulary, ETSP XII NF XVII, 1990, the complete contents of all of which are incorporated herein by reference.

[0142] Therapeutic agents include the herein disclosed categories and specific examples. It is not intended that the category be limited by the specific examples. Those of ordinary skill in the art will recognize also numerous other compounds that fall within the categories and that are useful according to the present disclosure. Examples include a radiosensitizer, a steroid, a xanthine, a beta- 2-agonist bronchodilator, an anti-inflammatory agent, an analgesic agent, a calcium antagonist, an angiotensin-converting enzyme inhibitors, a beta-blocker, a centrally active alpha- agonist, an alpha - 1 -antagonist, an anticholinergic / antispasmodic agent, a vasopressin analogue, an anti arrhythmic agent, an antiparkinsonian agent, an antiangina / antihypertensive agent, an anticoagulant agent, an antiplatelet agent, a sedative, an ansiolytic agent, a peptidic agent, a biopolymeric agent, an antineoplastic agent, a laxative, an antidiarrheal agent, an antimicrobial agent, an antifungal agent, a vaccine, a protein, or a nucleic acid. In a further aspect, the pharmaceutically active agent can be coumarin, albumin, steroids such as betamethasone, dexamethasone, methylprednisolone, prednisolone, prednisone, triamcinolone, budesonide, hydrocortisone, and pharmaceutically acceptable hydrocortisone derivatives; xanthines such as theophylline and doxophylline; beta-2- agonist bronchodilators such as salbutamol, fenterol, clenbuterol, bambuterol, salmeterol, fenoterol; antiinflammatory agents, including antiasthmatic anti-inflammatory agents, antiarthritis antiinflammatory agents, and non-steroidal antiinflammatory agents, examples of which include but are not limited to sulfides, mesalamine, budesonide, salazopyrin, diclofenac, pharmaceutically acceptable diclofenac salts, nimesulide, naproxene, acetaminophen, ibuprofen, ketoprofen and piroxicam; analgesic agents such as salicylates; calcium channel blockers such as nifedipine, amlodipine, and nicardipine; angiotensin converting enzyme inhibitors such as captopril, benazepril hydrochloride, fosinopril sodium, trandolapril, ramipril, lisinopril, enalapril, quinapril hydrochloride, and moexipril hydrochloride; beta-blockers (i.e., beta adrenergic blocking agents) such as sotalol hydrochloride, timolol maleate, esmolol hydrochloride, carteolol, propanolol hydrochloride, betaxolol hydrochloride, penbutolol sulfate, metoprolol tartrate, metoprolol succinate, acebutolol hydrochloride, atenolol, pindolol, and bisoprolol fumarate; centrally active alpha-2-agonists such as clonidine; alpha- 1 -antagonists such as doxazosin and prazosin; anticholinergic / antispasmodic agents such as dicyclomine hydrochloride, scopolamine hydrobromide, glycopyrrolate, clidinium bromide, flavoxate, and oxybutynin; vasopressin analogues such as vasopressin and desmopressin; antiarrhythmic agents such as quinidine, lidocaine, tocainide hydrochloride, mexiletine hydrochloride, digoxin, verapamil hydrochloride, propafenone hydrochloride, flecainide acetate, procainamide hydrochloride, moricizine hydrochloride, and disopyramide phosphate;antiparkinsonian agents, such as dopamine, L-Dopa / Carbidopa, selegiline, dihydroergocryptine, pergolide, lisuride, apomorphine, and bromocry ptine; antiangina agents and antihypertensive agents such as isosorbide mononitrate, isosorbide dinitrate, propranolol, atenolol and verapamil; anticoagulant and antiplatelet agents such as Coumadin, warfarin, acetylsalicylic acid, and ticlopidine; sedatives such as benzodiazapines and barbiturates; ansiolytic agents such as lorazepam, bromazepam, and diazepam; peptidic and biopolymeric agents such as calcitonin, leuprolide and other LHRH agonists, hirudin, cyclosporin, insulin, somatostatin, protirelin, interferon, desmopressin, somatotropin, thymopentin, pidotimod, erythropoietin, interleukins, melatonin, granulocyte / macrophage-CSF, and heparin; antineoplastic agents such as etoposide, etoposide phosphate, cyclophosphamide, methotrexate, 5 -fluorouracil, vincristine, doxorubicin, cisplatin, hydroxyurea, leucovorin calcium, tamoxifen, flutamide, asparaginase, altretamine, mitotane, and procarbazine hydrochloride; laxatives such as senna concentrate, casanthranol, bisacodyl, and sodium picosulphate; antidiarrheal agents such as difenoxine hydrochloride, loperamide hydrochloride, furazolidone, diphenoxylate hdyrochloride, and microorganisms; vaccines such as bacterial and viral vaccines; antimicrobial agents such as penicillins, cephalosporins, and macrolides, antifungal agents such as imidazolic and triazolic derivatives; and nucleic acids such as DNA sequences encoding for biological proteins, and antisense oligonucleotides.

[0143] Anti-cancer agents include alkylating agents, platinum agents, antimetabolites, topoisomerase inhibitors, antitumor antibiotics, antimitotic agents, aromatase inhibitors, thymidylate synthase inhibitors, DNA antagonists, farnesyltransferase inhibitors, pump inhibitors, histone acetyltransferase inhibitors, metalloproteinase inhibitors, ribonucleoside reductase inhibitors, TNF alpha agonists / antagonists, endothelinA receptor antagonists, retinoic acid receptor agonists, immuno-modulators, hormonal and antihormonal agents, photodynamic agents, and tyrosine kinase inhibitors.

[0144] Antibiotics include aminoglycosides (e.g., gentamicin, tobramycin, netilmicin, streptomycin, amikacin, neomycin), bacitracin, corbapenems (e.g., imipenem / cislastatin), cephalosporins, colistin, methenamine, monobactams (e.g., aztreonam), penicillins (e.g., penicillin G, penicillinV, methicillin, natcillin, oxacillin, cioxacillin, dicloxacillin, ampicillin, amoxicillin, carbenicillin, ticarcillin, piperacillin, mezlocillin, azlocillin), polymyxin B, quinolones, and vancomycin; and bacteriostatic agents such as chloramphenicol, clindanyan, macrolides (e.g., erythromycin, azithromycin, clarithromycin), lincomyan, nitrofurantoin, sulfonamides, tetracyclines (e.g., tetracycline, doxycycline, minocycline, demeclocyline), and trimethoprim. Also included are metronidazole, fluoroquinolones, and ritampin.

[0145] Enzyme inhibitors are substances which inhibit an enzymatic reaction. Examples of enzyme inhibitors include edrophonium chloride, N-methylphysostigmine, neostigmine bromide, physostigmine sulfate, tacrine, tacrine, 1 -hydroxy maleate, iodo tubercidin, p- bromotetramiisole, 10- (alpha-diethylaminopropionyl)-phenothiazine hydrochloride, calmidazolium chloride, hemicholinium-3,3,5-dinitrocatechol, diacylglycerol kinase inhibitor I, diacylglycerol kinase inhibitor II, 3-phenylpropargylamine, N°-monomethyl-Larginine acetate, carbidopa, 3- hydroxybenzylhydrazine, hydralazine, clorgyline, deprenyl, hydroxylamine, iproniazid phosphate, 6- MeO-tetrahydro-9H-pyrido-indole, nialamide, pargyline, quinacrine, semi carb azide, tranylcypromine, N,N-diethylaminoethyl-2,2-diphenylvalerate hydrochloride, 3 - isobutyl- 1- methylxanthne, papaverine, indomethacind, 2-cyclooctyl-2 -hydroxy ethylamine hydrochloride, 2,3- dichloro-a-methylbenzylamine (DCMB), 8,9-dichloro-2,3,4, 5 -tetrahydro- lH-2-benzazepine hydrochloride, p-amino glutethimide, p-aminoglutethimide tartrate, 3- iodotyrosine, alphamethyltyrosine, acetazolamide, dichlorphenamide, 6-hydroxy-2- benzothiazolesulfonamide, and allopurinol.

[0146] Antihistamines include pyrilamine, chlorpheniramine, and tetrahydrazoline, among others.

[0147] Anti-inflammatory agents include corticosteroids, nonsteroidal anti-inflammatory drugs (e.g., aspirin, phenylbutazone, indomethacin, sulindac, tolmetin, ibuprofen, piroxicam, and fenamates), acetaminophen, phenacetin, gold salts, chloroquine, D-Penicillamine, methotrexate colchicine, allopurinol, probenecid, and sulfinpyrazone.

[0148] Muscle relaxants include mephenesin, methocarbomal, cyclobenzaprine hydrochloride, trihexylphenidyl hydrochloride, levodopa / carbidopa, and biperiden.

[0149] Anti-spasmodics include atropine, scopolamine, oxyphenonium, and papaverine.

[0150] Analgesics include aspirin, phenybutazone, idomethacin, sulindac, tolmetic, ibuprofen, piroxicam, fenamates, acetaminophen, phenacetin, morphine sulfate, codeine sulfate, meperidine, nalorphine, opioids (e.g., codeine sulfate, fentanyl citrate, hydrocodone bitartrate, loperamide, morphine sulfate, noscapine, norcodeine, normorphine, thebaine, nor- binaltorphimine, buprenorphine, chlomaltrexamine, funaltrexamione, nalbuphine, nalorphine, naloxone, naloxonazine, naltrexone, and naltrindole), procaine, lidocain, tetracaine and dibucaine. Ophthalmic agents include sodium fluorescein, rose bengal, methacholine, adrenaline, cocaine, atropine, alpha-chymotrypsin, hyaluronidase, betaxalol, pilocarpine, timolol, timolol salts, and combinations thereof.

[0151] Prostaglandins are art recognized and are a class of naturally occurring chemically related long-chain hydroxy fatty acids that have a variety of biological effects.

[0152] Anti-depressants are substances capable of preventing or relieving depression.

[0153] Examples of anti-depressants include imipramine, amitriptyline, nortriptyline, protriptyline, desipramine, amoxapine, doxepin, maprotiline, tranylcypromine, phenelzine, and isocarboxazide.

[0154] Trophic factors are factors whose continued presence improves the viability or longevity of a cell trophic factors include, without limitation, platelet-derived growth factor (PDGP), neutrophilactivating protein, monocyte chemoattractant protein, macrophage- inflammatory protein, platelet factor, platelet basic protein, and melanoma growth stimulating activity; epidermal growth factor, transforming growth factor (alpha), fibroblast growth factor, platelet- derived endothelial cell growth factor, insulin-like growth factor, glial derived growth neurotrophic factor, ciliary neurotrophic factor, nerve growth factor, bone growth / cartilage- inducing factor (alpha and beta), bone morphogenetic proteins, interleukins (e.g., interleukin inhibitors or interleukin receptors, including interleukin 1 through interleukin 10), interferons (e.g., interferon alpha, beta and gamma), hematopoietic factors, including erythropoietin, granulocyte colony stimulating factor, macrophage colony stimulating factor and granulocyte- macrophage colony stimulating factor; tumor necrosis factors, and transforming growth factors (beta), including beta-1, beta-2, beta-3, inhibin, and activin.

[0155] Hormones include estrogens (e.g., estradiol, estrone, estriol, diethylstibestrol, quinestrol, chlorotrianisene, ethinyl estradiol, mestranol), anti-estrogens (e.g., clomiphene, tamoxifen), progestins (e.g., medroxyprogesterone, norethindrone, hydroxyprogesterone, norgestrel), antiprogestin (mifepristone), androgens (e.g, testosterone cypionate, fluoxymesterone, danazol, testolactone), anti- androgens (e.g., cyproterone acetate, flutamide), thyroid hormones (e.g., triiodothyronne, thyroxine, propylthiouracil, methimazole, and iodixode), and pituitary hormones (e.g., corticotropin, sumutotropin, oxytocin, and vasopressin). Hormones are commonly employed in hormone replacement therapy and / or for purposes of birth control. Steroid hormones, such as prednisone, are also used as immunosuppressants and anti-inflammatories. In some aspects, the additive is an agent that stimulates tissue formation, and / or healing and regrowth of natural tissues, and any combinations thereof. Agents that increase formation of new tissues and / or stimulates healing or regrowth of native tissue at the site of injection can include, but are not limited to, fibroblast growth factor (FGF), transforming growth factor-beta (TGF-beta, platelet-derived growth factor (PDGF), epidermal growth factors (EGFs), connective tissue activated peptides (CTAPs), osteogenic factors including bone morphogenic proteins, heparin, angiotensin II (A-II) and fragments thereof, insulin-like growth factors, tumor necrosis factors, interleukins, colony stimulating factors, erythropoietin, nerve growth factors, interferons, biologically active analogs, fragments, and derivatives of such growth factors, and any combinations thereof.

[0156] In some aspects, the silk composition can further comprise at least one additional material for soft tissue augmentation, e.g., dermal filler materials, including, but not limited to, poly(methylmethacrylate) microspheres, hydroxylapatite, poly(L-lactic acid), collagen, elastin, and glycosaminoglycans, hyaluronic acid, commercial dermal filler products such as BOTOX® (from Allergan), DYSPORT®, COSMODERM®, EVOLENCE®, RADIESSE®,RESTYLANE®, JUVEDERM® (from Allergan), SCULPTRA®, PERLANE®, and CAPTIQEIE®, and any combinations thereof.

[0157] In some aspects, the additive is a wound healing agent. As used herein, a “wound healing agent" is a compound or composition that actively promotes wound healing process.

[0158] Exemplary wound healing agents include, but are not limited to dexpanthenol; growth factors; enzymes, hormones; povidon- iodide; fatty acids; anti-inflammatory agents; antibiotics; antimicrobials; antiseptics; cytokines; thrombin; angalgesics; opioids; aminoxyls; furoxans; nitrosothiols; nitrates and anthocyanins; nucleosides, such as adenosine; and nucleotides, such as adenosine diphosphate (ADP) and adenosine triphosphate (ATP); neutotransmitter / neuromodulators, such as acetylcholine and 5 -hydroxy tryptamine (serotonin / 5- HT); histamine and catecholamines, such as adrenalin and noradrenalin; lipid molecules, such as 5 sphingosine- 1 -phosphate and lysophosphatidic acid; amino acids, such as arginine and lysine; peptides such as the bradykinins, substance P and calcium gene-related peptide (CGRP); nitric oxide; and any combinations thereof.

[0159] In certain aspects, the active agents provided herein are immunogens. In one aspect, the immunogen is a vaccine. Most vaccines are sensitive to environmental conditions under which they are stored and / or transported. For example, freezing may increase reactogenicity (e.g., capability of causing an immunological reaction) and / or loss of potency for some vaccines (e.g., HepB, and DTaP / IPV / FQB), or cause hairline cracks in the container, leading to contamination. Further, some vaccines (e.g., BCG, Varicella, and MMR) are sensitive to heat. Many vaccines (e.g., BCG, MMR, Varicella, Meningococcal C Conjugate, and most DTaP-containing vaccines) are light sensitive. See, e.g., Galazka et al., Thermostability of vaccines, in Global Programme for Vaccines & Immunization (World Health Organization, Geneva, 1998); Peetermans et al., Stability of freeze-dried rubella virus vaccine (Cendehill strain) at various temperatures, 1 J. Biological Standardization 179 (1973). Thus, the compositions and methods provided herein also provide for stabilization of vaccines regardless of the cold chain and / or other environmental conditions.

[0160] In some aspects, the additive is a cell, e.g., a biological cell. Cells useful for incorporation into the composition can come from any source, e.g., mammalian, insect, plant, etc. In some aspects, the cell can be a human, rat or mouse cell. In general, cells to be used with the compositions provided herein can be any types of cells. In general, the cells should be viable when encapsulated within compositions. In some aspects, cells that can be used with the composition include, but are not limited to, mammalian cells (e.g. human cells, primate cells, mammalian cells, rodent cells, etc.),avian cells, fish cells, insect cells, plant cells, fungal cells, spore cells, bacterial cells, and hybrid cells. In some aspects, exemplary cells that can be can be used with the compositions include platelets, activated platelets, stem cells, totipotent cells, pluripotent cells, and / or embryonic stem cells. In some aspects, exemplary cells that can be encapsulated within compositions include, but are not limited to, primary cells and / or cell lines from any tissue. For example, cardiomyocytes, myocytes, hepatocytes, keratinocytes, melanocytes, neurons, astrocytes, embryonic stem cells, adult stem cells, hematopoietic stem cells, hematopoietic cells (e.g. monocytes, neutrophils, macrophages, etc.), ameloblasts, fibroblasts, chondrocytes, osteoblasts, osteoclasts, neurons, sperm cells, egg cells, liver cells, epithelial cells from lung, epithelial cells from gut, epithelial cells from intestine, liver, epithelial cells from skin, etc., and / or hybrids thereof, can be included in the silk / platelet compositions disclosed herein. Those skilled in the art will recognize that the cells listed herein represent an exemplary, not comprehensive, list of cells. Cells can be obtained from donors (allogenic) or from recipients (autologous). Cells can be obtained, as a non-limiting example, by biopsy or other surgical means known to those skilled in the art.

[0161] In some aspects, the cell can be a genetically modified cell. A cell can be genetically modified to express and secrete a desired compound, e.g. a bioactive agent, a growth factor, differentiation factor, cytokines, and the like. Methods of genetically modifying cells for expressing and secreting compounds of interest are known in the art and easily adaptable by one of skill in the art.

[0162] Differentiated cells that have been reprogrammed into stem cells can also be used.

[0163] For example, human skin cells reprogrammed into embryonic stem cells by the transduction of Oct3 / 4, Sox2, c-Myc and Klf4 (Junying Yu, et. ah, Science, 2007, 318 , 1917-1920 and Takahashi K. et. ah, Cell, 2007, 131 , 1-12).

[0164] Unless otherwise specified or indicated by context, the terms “a”, “an”, and “the” mean “one or more.” For example, “a molecule” should be interpreted to mean “one or more molecules.”

[0165] As used herein, “about”, “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” and “approximately” will mean plus or minus <10% of the particular term and “substantially” and “significantly” will mean plus or minus >10% of the particular term.

[0166] As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.” The terms “comprise” and “comprising” should be interpreted as being “open” transitional terms that permit the inclusion of additional components further to thosecomponents recited in the claims. The terms “consist” and “consisting of’ should be interpreted as being “closed” transitional terms that do not permit the inclusion of additional components other than the components recited in the claims. The term “consisting essentially of’ should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter.

[0167] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0168] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0169] Preferred aspects of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred aspects may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect a person having ordinary skill in the art to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the abovedescribed elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0170] While the invention has been illustrated and described in detail in the foregoing drawings and description, the same is to be considered as illustrative and not restrictive in character, it being understood that only illustrative embodiments thereof have been shown and described and that all changes and modifications that come within the spirit of the invention are desired to be protected. For example, any of the features or functions of any of the embodiments disclosed herein may be incorporated into any of the other embodiments disclosed herein.

[0171] The following examples illustrate some embodiments and aspects of the invention. It will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like can be performed without altering the spirit or scope of the invention, and such modifications and variations are encompassed within the scope of the invention as defined in the claims which follow. The following examples do not in any way limit the invention.

[0172] EXAMPLES

[0173] Example 1

[0174] For extrusion 3D printing techniques, the ink must exhibit shear thinning behavior, filament formation during extrusion, and a suitable viscosity (30 mPa*s to 6xl07mPa*s). Silk fibroin does not meet these parameters. Indeed, in Figure 12, viscosity values of the Sil-Ma solution at the two molecular weights are reported. Therefore, the first step in our design was to overcome the viscosity limitations. Two different silk molecular weights were selected, 15mB and 30mB, equal to ca. 300 kDa and 200 kDa, respectively. To develop the ink, crosslinking processes leading to covalent bond formation were chosen to achieve more control over the structure, degradation kinetics, and mechanical properties. A double crosslinking process was designed, composed of a pre-gelation phase to make the silk fibroin extrudable, and an in situ second crosslinking to stabilize the printed structure as shown in Fig. 2.

[0175] Since pre-gelation was achieved via photo-crosslinking, TNBS assay was performed to investigate if the methacrylation reaction with the GM A was successfully performed, thus, to quantify the degree of substitution (DS) of the methacrylate group on primary amines. The DS resulted to be equal to 67% ± 3%. After this, for pre-crosslinking, methacrylate silk fibroin (Sil-Ma) was photo-crosslinked leading to incomplete, covalently crosslinked gel formation (PC). The gelation was intentionally not completed to avoid the formation of fully-crosslinked gels which could otherwise clog during printing. This pre-crosslinking process was controllable, and the reaction activation was dependent on the time of light exposure time. In contrast, after printing, HRP-driven enzymatic crosslinking has been selected to stabilize and induce complete gelation at 37°C by forming stable and covalent dityrosine bonds in situ. In details, phenols on tyrosine are oxidized when the HRP is in presence of the H2O2. This reaction forms an intermediate compound with an oxyferryl center and a porphyrin-based cation radical. The now activated HRP undergoes two single electron oxidation reactions in the presence of tyrosine’s phenol groups, generating phenol free radicals, thus forming dityrosine bonds. Enzymatic crosslinking (EC) depends on many variables such as temperature, free radicals, silk concentration, peroxide concentration, as well as silk molecular weight, leading to heterogeneity when used as a pre-crosslinking process. Considering the silk fibroin amino acid composition, tyrosine represents ca. 5% of the total composition, and the formation of inter and intra-dityrosine bonds allows the formation of highly stable, elastic hydrogels.

[0176] To investigate if the double crosslinking (DC) approach was printable, and to verify if enzymatic crosslinking (EC) was leading to the complete gelation, rheological analyses were performed. For the assessment of printability, viscoelasticity studies were carried out with amplitude sweeps on the different compositions to investigate flow behavior. In particular, the flow point is thecrossover point between the curves of loss and storage moduli (G" and G', respectively), corresponding to the conditions at which the material starts to flow. This is an important parameter for printability, specifically if shear can unfold and align the protein chains to allow flow through the nozzle. However, high shear stresses might impact cell encapsulation in bioinks, or lead to irreversible ink deformation (e.g., in the case of silk crystallization). The double crosslinking (DC) condition was compared to the pre-photo-crosslinking condition (PC), adding as controls the fully photo-crosslinked gels (FC), and fully enzymatically crosslinked gels (EC). All the conditions exhibited viscoelastic behavior and had G’ higher than G”. In Fig. 3A, the 15mB PC exhibited a flow point at a stress value of 82.3 Pa ± 35.3 Pa. All other conditions, DC, FC, and EC did not show a flow point in the oscillation stress range analyzed. The behavior of the fully photo-crosslinked condition (FC) is shown as an example in Fig. 3B. Although the flow point was not detected, the presence of a yield point identified the limit of the linear viscoelastic region. This also suggests that at higher stresses there may be a flow point. The same behavior was observed for fully enzymatic and double crosslinking conditions (Fig. 13A, Fig. 13B). These results confirmed the initial hypothesis (Fig. 3 A) that the 15mB pre-crosslinking led to an extrudable, homogenous filament, whereas the other conditions resulted in clogging of the nozzle, heterogenous gelation (as for the EC), and higher extrusion pressures.

[0177] The 30mB behavior was different compared to the 15mB (Fig. 3C). All the conditions exhibited a flow point, however, only the PC flow point (Fig. 3D) occurred at relatively low stress (52.6 Pa + 10.5 Pa), with values significantly different compared to the other conditions. These results confirmed the expectation regarding the 30mB Sil-Ma. First, this was due to the lower molecular weight compared to the 15mB Sil-Ma and the low concentration used in this work. Indeed, the photo-crosslinking of the methacrylation polymer takes place on primary amine groups, present mainly on lysine residues, which represent a low content in the silk (0.3 %mol) and the lower the MW, the less is the entanglement and length of the chains, thus affecting the kinetics. For this reason, photo-crosslinking efficiency is dependent on silk molecular weight and concentration, resulting in weaker gels compared to the Sil-Ma prepared at higher molecular weight. However, although in the 30 mB molecular weight, all the conditions exhibited a flow point, the high pressures required to extrude the EC and FC and their variability among the replicates, made the choice of precrosslinked conditions clear, repeatable, and resulted in more homogenous filament formation.

[0178] After the study of the viscoelasticity properties, rheological tests were performed over time in the LVE region to demonstrate that the pre-crosslinking led to the formation of a pre-gel with low storage modulus. In contrast, the combination of this pre-gel with the HRP crosslinking (double crosslinked condition - DC) led to the formation of a homogenous hydrogel, with significantlyhigher G’ compared to the former condition (Fig. 4). The 15 mB storage modulus increased over time, reaching values around 1594.0 Pa ± 44.9 Pa (Fig. 4A), whereas the 30mB modulus was 879.0 Pa + 37.2 Pa (Fig. 4B). It is worth noting that HRP crosslinking has been widely applied to silk fibroin gelation. Indeed, among all the parameters, gelation is mainly affected by the molecular weight and concentration of silk. The enzyme has an initial delay phase where gelation is not detectable, and the protein is still liquid. Then, dityrosine bond formation takes place, followed by interchain protein crosslinking. These kinetics proceed over time, leading to further polymerization until the full covalent formation of dityrosine bonds. This is reflected in the increased storage modulus in the double crosslinked condition (DC) up to 72 h. Importantly, the PC condition did not exhibit significant variations over time, and the excessive swelling and weakness of the PC condition after 72 h made it impossible to test at that time point. These results confirmed that the precrosslinked was not sufficient to form stable homogenous gels, while proper gel formation was achieved with the double crosslinking.

[0179] After rheological analyses, it was clear that fully-crosslinked gels (either enzymatic, photocured, and double crosslinked) required high pressure to extrude and that the PC was insufficient to form a stable hydrogel, confirming that the second, enzymatic crosslinking in situ was a required fundamental step to further induce the covalent gelation between polymer chains. Therefore, 3D printing was performed by applying the pre-photo-crosslinking, followed by in situ activation of the enzymatic crosslinking. Firstly, printability test was performed to measure the accuracy in printing pores inside each structure. As visible in Fig. 5A, although the different infill was chosen, the Pr resulted to be very similar between the two conditions. However, it is important to note that not only the Pr was exhibiting values below 1 (1 representing high printing precision), but also that samples exhibited high variability, appreciable from the standard deviations. Moreover, in Fig. 5B some printed structures are shown. For all the printing tests a 22 G and 25 G nozzles was used, demonstrating the suitability and versatility of the pre-crosslinking process for useful extrusion. Smaller diameter nozzles required higher pressures (up to 120 kPa). However, although the Pr discussed before still requires further optimization, the compatibility of the process with printing multilayer structures, or 3D STL models with different resolutions were all feasible.

[0180] To investigate the stability and behavior of the printed PC and DC conditions, material characterization was performed over 14 days by incubating the samples in medium and water. First, it is worth noting that during the printing of the pre-crosslinked gels (PC), macroscopically different gel consistencies were observed, compared to before printing. For this reason, the investigation of silk secondary structure was performed before and after extrusion, and then monitored at days 1, 7, and 14, for each molecular weight. Fig. 6A shows the infrared spectra for samples before and afterextrusion in the range 800-2000 cm1and Fig. 6B and Fig. 6C show the results of deconvolution of the Amide I (1610-1705 cm1) band for both MW samples. All the tested conditions, after extrusion, exhibited a shift from silk I to silk II structure, specifically, in antiparallel P-sheets conformation (Fig. 6B, Table 1).Table 1 Amide 1 deconvolution with secondary structure quantification of 15 mB and 30 mB Sil- Ma, before and after extrusion.

[0181] Regarding the high molecular weight silk (15 mB), the total amount of anti-parallel P- sheets increased from 21.8% to 41.1% after extrusion, slightly increasing over time (Fig. 6C, Fig 6D, Table 2). This transition was less evident but present even in the 30mB Sil-Ma, where the antiparallel P-sheet content reached 34.8%, increasing at day 1 up to 40-48% (Fig. 14, Table 3). The latter effect, the slower transition into crystalline structure for the 30mB silk compared to the 15mB, is likely attributed to a lower effect of shear on the protein chains compared to the higher molecular weight. Indeed, it was reported that after shear stimulus, the rearrangements responsible for the amide I shift, might proceed for several hours, explaining the slower shift into crystalline structures observed in the 30 mB MW. This conformational transition might be attributed to shear stresses applied inside the nozzle, leading to silk rearrangement toward crystalline (P~ sheet) structures, macroscopically responsible of the differences detected in the pre-crosslinked gels after extrusion.Table 2 Amide I deconvolution secondary structure quantification of 15mB Sil-Ma of the pre-photo- crosslinked (PC) and the double crosslinked condition (DC) condition at day 1, 7, and 14 in medium and water.

[0182] Natural silk spinning and artificial extrusion during 3D printing have some similarities. During silk spinning, shear, salt concentration, and pH, play important roles for the fiber formation, thus the solidification of the protein. Specifically, shear might induce silk self-assembly via physical crosslinking. The hypothesis is that in aqueous conditions silk in the form of random coils, thus mainly silk I conformation, is surrounded by a water hydration shell created by hydrogen bonding between silk and water. This situation is energetically favorable until the application of the stress in the nozzle which leads to instability of chain conformation, as result of stretching and alignment with consequent breakage of the H-bonds with water. This results in a dehydration and the generation of inter- and intra-chain interactions and thus new bonds responsible for the crystalline -sheet conformation, the thermodynamically stable form of silk structures.

[0183] Silk fibroin shear-sensitivity, thus aggregation under shear stress, is related to silk fibroin amino acid sequence. Indeed, glycine, alanine, and tyrosine represent 45.9%, 30.3%, and 5.3% of the total amino acidic composition, respectively. They are organized into highly ordered, repeated sequences in form of hexamers along the chains. Particularly, the GX repeated sequence where Xmight be tyrosine, alanine, or serine is mainly responsible for P-sheet structure formation. For instance, tyrosine residues, when present in a central position in the repeat sequence close to glycine, are fundamental for creating inter- and intra-chains H-bonding through the -OH group on its aromatic ring, driving structural transitions. Thus, the silk fibroin sequence composition leads to selfassembly, which under external stimuli as shear can accelerate the transition to silk II conformation.

[0184] After extrusion, no differences were detected over time in protein secondary structure in Sil-Ma 15mB, while, as mentioned earlier, the 30mB rearrangement and stabilization took up to day 1 but then, no significant changes were observed over time. The results for days 1 , 7 and 14 for both the PC and DC related to the 30mB are shown in Fig. 14A and Fig 14B, and the spectra of IR test at day 1 of all the conditions studied are shown in Fig. 14C and Fig. 14D.

[0185] In contrast to the initial design, the IR analyses demonstrated that the pre-crosslinked polymer without the addition of the enzymatic crosslinking, exhibited the same protein structure as the double crosslinking, DC, changing its properties when in combination with extrusion stress. Considering the protein rearrangements and dehydration in the nozzle, swelling tests were performed both in medium and water, monitoring water absorbance over time at days 1, 7 and 14. In agreement with the hypothesis, all the conditions for both molecular weights were highly stable over time, with neither significant water absorption nor contraction detected (Fig. 7). Specifically, swelling / contraction behavior depends on protein concentration, degree of crosslinking, pH, and ionic strength. Moreover, during P-sheet formation, water molecules are removed between the P- strands. Therefore, the combination of dehydration and extensive P-crystallization led to the formation of highly stable hydrogels over time.

[0186] Taken together these results demonstrated the stability of the materials over the tested conditions. After extrusion, both the pre-crosslinking and the double crosslinking were suitable for in situ 3D printing. However, our initial hypothesis was that the pre-crosslinking (PC) condition was unstable compared to the double crosslinking one (DC) since in the latter the HRP-driven gelation led to the formation of stable covalent bonds. For this reason, although the IR and swelling tests did not provide evidence of differences among the conditions, we further characterized the material to verify if our initial hypothesis, the formation of covalent, stable bonds in the double crosslinking condition, was confirmed. The degradation kinetics were assessed both in medium and water, in the presence (Fig. 8 A and Fig. 8B) and absence of enzymes (Fig. 15A and Fig. 15B). For degradation studies, protease XIV was selected since it exhibits 390 cleavages sites along the silk fibroin sequence and, compared to chymotrypsin, is able to digest the crystalline structures as well. In Figure 8, the results after 14 days of digestion are shown. There was a difference between the precrosslinked gels (PC) and the double crosslinked (DC), independent of MW. Specifically, in thepresence of the enzyme, samples prepared with just the pre-photo-crosslinking process exhibited a significantly faster degradation rate compared to the gels prepared via double-crosslinking (DC).

[0187] PC samples at day 14, both the 30mB and 15mB conditions, are missing. Indeed, half of these samples were broken and the others difficult to handle without breaking, thus the measurements were not reliable. Furthermore, from degradation tests in the presence of protease, there was a difference in the degradation kinetics between the pre-crosslinked and double crosslinked conditions. The 15mB condition exhibited similar degradation rates up to day 7 between the PC and the DC, the former rapidly degraded at day 14, whereas the latter was stable with a final residual mass equal to 61% ± 8.15 in water and 61.0% ± 14.74 in medium. For the 30mB Sil-Ma, some differences were evident at day 1 when the PC in medium degraded significantly more compared to the DC in the same conditions. This difference became clearer at day 7 where PC conditions both in medium and water were significantly more degraded compared to the double crosslinked (DC) conditions. The final residual mass of the DC samples was equal to 65% ± 6.29 in water, and 65.83% + 3.14 in medium.

[0188] Degradation kinetics are dependent on structure-related features, as MW, crystallinity, and secondary arrangements. Generally, silk fibroin degradation is slower compared to other biomaterials, due to the P-sheet features, and protease digests the amorphous regions initially and the crystalline structure last, the latter more resistant to enzymatic cleavage. However, the IR analyses demonstrated that no differences in secondary structure were detected over time among all the conditions, for both molecular weights. In contrast, the degradation tests demonstrated a significant difference between the pre-photo-crosslinking and the double crosslinking processes. Therefore, given the same P-sheet content, the main difference between the pre-crosslinked and double crosslinked conditions should be related to the nature of the bonds in the structures. Specifically, silk fibroin undergoes slower degradation kinetics when chemical and physical crosslinking are combined. Indeed, physical crosslinking leads to the formation of -sheets through the formation of several hydrogen bonds, whereas chemical or enzymatic crosslinking leads to the creation of covalent, permanent, and stable bonds. This agrees with our results, since when the HRP is combined with the pre-crosslinking, it induces covalent dityrosine bond formation, synergically working with the already existing physical crosslinking induced by shear stress during extrusion. As result, if the dityrosine bond formation did not affect the overall silk fibroin secondary structure, while at the same time stabilizing these features by forming covalent bonds, thus making the double crosslinked condition (DC) more resistant to the protease cleavage.

[0189] Finally, all the conditions were tested in unconfined compression and the Young’s modulus calculated over the time points, both in medium and water (Fig. 9). The 15 mB Sil-Ma hydrogels(Fig. 9A), did not exhibit significant variations over time, whereas the 30 mB (Fig. 9B) at day 14 showed significant differences between the PC and DC, the latter being stiffer compared to the PC. This might be correlated to the presence of weaker bonds in the pre-crosslinked condition, which under compression could easily break and reform in comparison to the covalently bound structures, as in DC. Interestingly, the 15 mB and 30 mB exhibited similar moduli over time, which might be unexpected due to the different molecular weight. However, it is worth noting, that when silk is regenerated, each molecular weight is polydisperse. Specifically, since degumming is a random process, the longer the degumming time, the higher the poly dispersity. The 30 mB did not exhibit many differences compared to the 15 mB, whereas they both exhibited differences with much longer degumming times (i.e., 60 min). This explains the absence of differences between the two molecular weights, as they are perhaps too similar in degumming time. This is also visible in the electron microscopy images of DC 15mB and 30mB Sil-Ma shown in Fig. 16.

[0190] After the material characterization, a preliminary biological evaluation was performed to investigate cell viability on the hydrogels, printed as acellular scaffolds. hBM-MSCs were seeded on samples and metabolic activity and cell adhesion were studied by AlamarBlue Assay and confocal imaging, respectively (Fig. 10). All the tested conditions supported cell viability over the 7 days. A significant increase in cell metabolic activity was detected from day 1 to 7 (Fig. 10A), without any significant differences among the tested conditions and the different molecular weights.

[0191] Confocal imaging was performed to evaluate cell adhesion on substrates. In Fig. 10B - Fig. 10L, some representative images of the results obtained are shown. At day 1, cells tended to interact one each other forming clusters (Fig. 10B, Fig. 10C, Fig. 10D), and some protrusions (Fig. 10B) were observed. After 7 days of culture the cells exhibited an elongated, stretched shape over all the tested conditions (Fig. 10E, Fig. 10F, Fig. 10G, Fig. 10H, Fig. 101, Fig. 10L). This different behavior between days 1 and 7 can be explained considering that mulberry silk fibroin (Bombyx mori) lacks RGD sequences, important for cell adhesion, thus leading to longer times for cells to elongate and spread. However, silk fibroin amino acid composition as well as several factors as topography, porosity, stimuli, and stiffness are crucial in dictating cell behavior on 3D materials. Among these, stiffness and gel topography might have positively impacted cell adhesion, leading to the elongation, stretching, and cell-material interactions, as observed at day 7. Additionally, the absence of any differences in terms of cell adhesion and cell shape between the pre-photo-crosslinked (PC) condition and the double crosslinked (DC) suggests that the driving force for cell interactions with the material might be protein rearrangements, not significantly changing among all the conditions and over time, as demonstrated through IR analyses.

[0192] The aim of this disclosure was to assess cell viability. While only a single seeding density was explored herein, a skilled artisan will appreciate how to explore the use of higher cell density.

[0193] Finally, with a view toward in vivo applications, 3D printing of the double crosslinked condition was performed on fresh chicken breast (Fig. 11 A, Fig. 1 IB) to investigate if the extruded ink adhered to the substrate. As for all the other tests, the temperature of the printing bed was set at 37 °C. Although this assessment was a preliminary evaluation, the Sil-Ma based ink attached to the chicken breast, without detaching when inverting the substrate (Fig. 11 C). Indeed, silk fibroin exhibits adhesive properties due to its amino acidic composition, such as the serine polar side chains which play a key role in interacting with substrates via the formation of hydrogens bonds. These interactions may also be attributed to the incomplete gelation in the pre-crosslinking phase, leading to a viscous pre-gel which, by completing its gelation in situ, allowed the physical interlocking of the gel within the tissue.

[0194] Taken together, the results demonstrated that silk fibroin can be tuned to match in situ 3D printing requirements by combining two different crosslinking processes including photo- and enzymatic-. Moreover, the characterization of the hydrogels after 3D printing provides useful data for further optimization of the printed shapes to better mimic tissue complexity.

[0195] Silk fibroin-based inks were designed for in situ printing. The versatility of the process was demonstrated on two different molecular weights, and by adapting the fabrication process (i.e., light exposure time), in situ 3D printing was achievable. A double crosslinking process was developed, combining pre-gelation via incomplete photo-crosslinking with in situ HRP-driven gelation. This led to hydrogels stable over time both in medium and water, with good degradation kinetics over two weeks in the presence of protease type XIV. Additionally, the fundamental role of physical crosslinking induced by extrusion stress in the nozzle led to fast transitions of random coils and P- tum structures toward -sheets, providing stability in water and medium in the swelling study, but more importantly, dictating the degradation kinetics. Although both the PC and DC were good candidates for in situ gel formation, the main difference between them was the synergistic effect of the enzymatic, covalent crosslinking in the latter. If on one side it did not affect silk secondary structure conformation, on the other the covalent bonds induced hydrogels more resistant to degradation in presence of protease, compared to the pre-crosslinked counterpart. This demonstrated that silk fibroin ink properties can be tuned according to the final application.

[0196] It is important to note that starting solutions with low concentrations were used. Nevertheless, by combining the covalent crosslinks with the physical ones, hydrogels were obtained with compressive moduli and swelling behavior different from gels prepared without printing. Thissimplifies the fabrication process, avoiding challenging steps such as silk fibroin concentration, to improve consistency in gel outcomes.

[0197] Materials and Methods

[0198] Preparation of aqueous silk fibroin solutions

[0199] Silk fibroin was extracted and regenerated as described (D. N. Rockwood, R. C. Preda, T. Yiicel, X. Wang, M. L. Lovett and D. L. and Kaplan, Nat. Protoc., 2011, 6, 1612-1631). Briefly, the degumming was performed by pouring 5 g of cut silk fibroin cocoons in 2 L of boiling water with 0.2 M of Na2COa (Merck Sigma Aldrich, Darmstadt, Germany). To obtain two different molecular weights of silk fibroin, the silk was boiled for 15 minutes (Silk 15mB = minutes boiled) or 30 minutes (30 mB), equal to ca. 300 kDa and 200 kDa, respectively. After boiling, silk was washed three times in DI water at room temperature for 20 minutes and later dried under a fume hood for 24 h. Dissolution was performed by dissolving the silk in 9.3 M aqueous solution of lithium bromide (LiBr, Merck Sigma Aldrich, Darmstadt, Germany) at a concentration of 20% (w / V). The solution was incubated for 2 hours at 60°C. Then, silk fibroin was chemically modified by performing a methacrylation reaction (Bucciarelli, M. Petretta, B. Grigolo, L. Gambari, A. M. Bossi, F. Grass! and D. Maniglio, Gels, 2022, 8, 833). Specifically, glycidyl methacrylate (GMA - Merck Sigma Aldrich, Darmstadt, Germany) was added dropwise into the silk solution and reacted for 3 hours under stirring at 60 °C. This is a nucleophilic addition reaction, leading to the opening of the epoxy ring of the GMA that reacts with silk primary amines, forming di-0-hydroxyamide. Fully dissolved, methacrylate silk (Sil-Ma) was later dialyzed against DI water for three days with regular water changes to remove LiBr and unreacted GMA. Finally, the solution was centrifuged twice for 20 minutes at 9,000 rpm at 4 °C to remove impurities from the final solution. All the conditions were sterilized via filtration (Primo Vacuum Filter Systems, 0.45 pm, PES - Euroclone, Italy). The concentration was calculated from the dry weight. Unless otherwise stated, both Sil-Ma 15 mB and 30 mB were used at a concentration of 5% w / V.

[0200] UV spectroscopy - 2,4,6-trinitrobenzene sulfonic acid (TNBS) assay

[0201] TNBS assay was performed (F. Agostinacchio, D. Maniglio, E. Callone, C. Migliaresi, S. Dire and A. Motta, Soft Matter, 2021, 17, 6863-6872) to measure the methacrylation degree on Sil- Ma solution. Briefly, since the GMA reacts with free amines along silk sequence, the quantification of free amine on methacrylate silk in comparison with unmodified silk was investigated. In detail, both unmodified silk (SF) and methacrylate silk (Sil-Ma) were diluted to a final concentration of 1 mg / mL. The test was carried out at pH 8.5, and for this reason, all the solution were dialyzed against NaHCO? to reach the desired pH. Calibration curve was prepared using as standard 0-alanine (MW 89.09 Da) at known concentrations (Table 3). After this, 300 pL of TNBS solution (Merck SigmaAldrich, Darmstadt, Germany) at 0.02 (w / V) at pH 8.5 were added to 150 |iL of all the standards and samples and incubated for 2 hours at 40 °C ± 1 °C. After the incubation, the color of each sample shift to yellow-orange color according to free amine concentration. All the samples were transferred into a 96-well plate and absorbance was measured at 418 nm with a microplate reader (Tecan Infinite M200). Three replicates per condition were tested. The degree of substitution (DS) was calculated with the following formula: DS % = [l-(free amine concentration on Sil-Ma / free amine concentration on SF )]* 100.Table 3 Calibration curve with known concentration of P-alanine.

[0202] Sil-Ma hydrogels crosslinking process

[0203] Sil-Ma (5% w / V) was gently mixed with 0.02% (w / V) lithium phenyl-2,4,6- trimethylbenzoylphosphinate (LAP) (Merck Sigma Aldrich, Darmstadt, Germany) as photo-initiator, 0.01% (v / v) H2O2, and 10 U / mL of type VI horseradish peroxidase (HRP) (Merck Sigma Aldrich, Darmstadt, Germany). Next, the following steps were followed: (a) Pre-crosslinked gel formation: The solution was poured into a glass beaker and pre -photo-crosslinked by irradiation with a UV light source at 365 nm (I = 90 W / m2) for 40 s (for Sil-Ma 30mB, 60 s). This step led to the formation of a pre-photo-crosslinked gel, called PC (pre-crosslinked). Photo-crosslinking reaction is achieved thank to the formation of free radicals on silk fibroin chain after the activation of the LAP, under light exposure, (b) Double crosslinked gel: after the pre-photo-crosslinking, HRP activation led to full gelation and this condition was named double crosslinked (DC). Samples without enzymatic crosslinking, but only subjected to the pre -photo-gelation (PC) process, represented controls in the study.

[0204] Rheological analyses

[0205] All rheological analyses were performed with an HR2 Rheometer (TA Instruments, Delaware). Viscoelastic tests were carried out to verify the presence of a flow point in the different crosslinking phases, thus the printability of the materials, and to compare the behavior over time of PC and DC conditions. The flow point was verified by performing an oscillation amplitude test at constant frequency (1 Hz) and with increasing shear stress (up to 1,000 Pa). The storage modulus G' over 24 h time frame was measured in the LVE region in oscillation time mode, at 1 Hz and 3 Pa for3 minutes. As additional controls and to better characterize the materials, oscillation amplitude tests were performed on gels whose gelation was completed before printing, achieved via full photocrosslinking (FC), and complete enzymatic crosslinking (EC) as described in Table 4.Table 4 List of tested conditions and descriptions.

[0206] After photo-crosslinking, the pre-gel was transferred into a 3D printing syringe (3cc - Cellink, Sweden). 3D printing was performed with an extrusion 3D bioprinter, BioX (Cellink, Sweden). The printing head temperature was fixed at 20°C to avoid premature activation of the HRP, thus leading to heterogeneous gelation. The printing bed temperature was 37 °C, to enhance optimal HRP activation conditions and to resemble physiological temperature. Sil-Ma-based inks were printed with a 22 G nozzle, at a print speed of 8-9 mm / s, and a pressure of 50-80 kPa. After extrusion, the printed structures were incubated at 37°C for 1 hour to complete the enzymatic crosslinking.

[0207] Printability

[0208] Printability index (Pr) represents a key parameter to measure the accuracy of printed pores. For this experiment, 20 mm grids were printed with two different grid infill, 10% and 15%. All the samples were printed with a 25G nozzle. After printing pictures were acquired and analyzed with ImageJ to measure the area and the perimeter of the squares inside each structure. After this, all the measures were averaged and printability index calculated according to the formula: p2 / 16A, where p represents the perimeter of the pore and A the area. At least 3 replicates per condition were printed. High printing accuracy, thus high precision result in a Pr =1, while values lower or higher indicate round and irregular geometries.

[0209] Infrared spectroscopy

[0210] Infrared spectroscopy was performed to evaluate the secondary structure of Sil-Ma inks before printing and the effect of shear stress in the nozzle on protein structure. Additionally, after printing both the PC and DC were incubated for 2 weeks in DI water and medium at 37 °C (DMEM high glucose - Euroclone, Italy) and the protein conformation over time was studied at days 1 , 7, and 14, never drying the samples. Tests were performed with PerkinElmer Spectrum OneSpectrophotometer (PerkinElmer, Waltham, MA, USA) in attenuated total reflectance (ATR-FTIR) mode. Prior to the test, to remove any interference of medium and water, all gels were washed three times in deuterated water (D2O) (Merch Sigma Aldrich, Darmstadt, Germany). Spectra were acquired with 16 scans at 4 cm1resolution, in the wavenumber range of 550-4000- cm'1to detect silk fibroin Amide I, II, and III at 1641, 1513, and 1233 cm1, respectively. To investigate silk secondary structure rearrangement, analysis of amide I peaks (1610-1705 cm1) was run with Fourier self-deconvolution.

[0211] Swelling

[0212] Swelling behavior of DC and PC conditions was monitored over 14 days and studied at days 1, 7 and 14, in medium and DI water. Each sample was extruded for 20 s (ca. 200 pL of gel) into a silicone mold (n = 5) and the initial weight was measured (Wo). At each time point, the sample was blotted on paper to remove excess solvent and the final weight (Wi) was measured. The swelling percentage was calculated as (W1 / W0) x 100. During the experiment, all the samples were incubated in an incubator at 37 °C.

[0213] Degradation

[0214] Samples were extruded as described in the previous paragraph and incubated in 2 mL of medium and water, and degradation was studied in the presence and absence of 0.001 U / mL Protease type XIV from Streptomyces griseus (Merck Sigma Aldrich Darmstadt, Germany). All the samples were incubated at 37 °C. Fresh enzyme was added every two days. At each time point (days 1, 7, and 14) medium or water was removed, the samples were washed overnight in ultrapure DI water, frozen at -80 °C, and then lyophilized. The dry weight was measured, and degradation reported as the residual mass percentage measured at each time point compared to sample weight at time zero (to) (n= 4).

[0215] Mechanical compression

[0216] Unconfined compression was performed on DC and PC samples, incubated in both medium and DI water for 14 days. The test was carried out with a Bose Electroforce 3200 machine (TA Instruments, DE, USA) equipped with a 200 N load cell (sensitivity 0.05N). A preload of 0.5 N and a compression rate of 0.5 mm / m were applied to all samples. The diameter and height of each sample was measured with a caliper prior to the test. The Young’s modulus was calculated from the slope of the initial (linear) part of the curve.

[0217] Biological evaluation

[0218] For in vitro experiments, human bone marrow-derived mesenchymal stem cells (hBM- MSCs) from the American Type Culture Collection (ATCC) were used. The cells were cultured in expansion medium composed of DMEM High Glucose medium (Euroclone, Italy), supplementedwith Fetal Bovine Serum (FBS, Euroclone, Italy) 15%, and antibiotic / antimycotic (Euroclone, Italy) 1%. The seeding density was 2*105cells / ml. Cells were cultured for 7 days and the medium was replaced every 3 days.

[0219] Confocal analysis

[0220] Confocal imaging (Nikon Al Laser Microscope - Japan) was performed to study cell adhesion. At each time point (days 1, 7), two replicates for each condition were analyzed. The samples were fixed with paraformaldehyde (PFA - Sigma Aldrich, Darmstadt, Germany) 4% and incubated at room temperature (RT) for 30 minutes. Subsequently, the samples were washed twice with PBS (Euroclone, Italy). Nuclei and cytoskeleton staining was performed by adding 4', 6- diamidino-2-phenylindole (DAPI - Sigma Aldrich, Darmstadt, Germany) and rhodamine phalloidin (Invitrogen - ThermoFisher - USA) according to the manufacturers’ instructions, respectively. The samples were incubated at RT for 20 minutes, protected from light. Finally, two washes in PBS were performed and all the samples were observed by confocal microscopy.

[0221] AlamarBlue assay

[0222] Cell metabolism was measured by AlamarBlue assay. At each time point, four replicates for each condition and the blank controls (gels without cells) were incubated with resazurin reagent (Chemodex Ltd, Switzerland) 10% in complete medium at 37°C for 3 hours. Then 100 pL of the supernatant of each sample was transferred to a black 96-well plate in duplicate; the fluorescence intensity with excitation wavelength at 535 nm and emission at 590 nm were measured with a microplate reader (Tecan Infinite M200) using complete medium and the Alamar reagent as negative controls. Since the test was not disruptive, the sponges were later washed, new medium was added, and the plate was incubated at 37°C until the next time point.

[0223] Statistical analysis

[0224] For AlamarBlue, GraphPad Prism 9.0 was used and samples in each condition were compared among the different time points with two-way Anova test and Tukey’s multiple comparison test. Significance was attributed when p<0.05 (*), p<0.01(**), p<0.001(***).

[0225] EQUIVALENTS AND SCOPE

[0226] The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combinations (or subcombinations) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the presentinvention is not intended to be limited to the above Description, but rather is as set forth in the following claims:

Claims

CLAIMSWhat is claimed is:

1. A non-crosslinked composition having capacity for double crosslinking, the non-crosslinked composition comprising: a population of methacrylate-modified silk fibroin fragments, each of the methacrylate- modified silk fibroin fragments comprising methacrylate side chains; a photo-crosslinker; an enzymatic crosslinker and optionally one or more enzymatic crosslinker co-reactants; and water.

2. The non-crosslinked composition of claim 1, the composition comprising the one or more enzymatic crosslinker co-reactants.

3. The non-crosslinked composition of claim 2, wherein the one or more enzymatic crosslinker co-reactants is hydrogen peroxide.

4. A non-crosslinked composition having capacity for double crosslinking, the non-crosslinked composition comprising: a population of methacrylate-modified silk fibroin fragments, each of the methacrylate- modified silk fibroin fragments comprising methacrylate side chains; a photo-crosslinker; one or more enzymatic crosslinker co-reactants and optionally an enzymatic crosslinker; and water.

5. The non-crosslinked composition of claim 4, the composition comprising the enzymatic crosslinker.

6. The non-crosslinked composition of claim 4, wherein the one or more enzymatic crosslinker co-reactants is hydrogen peroxide.

7. A pre-crosslinked composition that is partly photo-crosslinked and has capacity for further photo-crosslinking and an additional enzymatic crosslinking to become double-crosslinked, the precrosslinked composition comprising the non-crosslinked composition of any one of the preceding claims having partial photo-crosslinking between methacrylate-modified silk fibroin fragments of the population of methacrylate-modified silk fibroin fragments, the partial photo-crosslinking producing an ink that one or more of: exhibits shape fidelity after extrusion, forms a homogenous and uniform filament that can be deposited on a printing bed keeping its shape and avoids the layers collapsingwhen more than one is printed, storage modulus G’ is higher than G”, the crossover point is at most in the range of 300Pa of stress, or the ink can be extruded with an extrusion pressure that does not exceed 400 kPa.

8. The pre-crosslinked composition of the immediately preceding claim, wherein the partial photo-crosslinking involves between 1 % and 50%, between 40% and 70%, between 1 % and 99%, or between 50% and 99.9% of the methacrylate side chains.

9. The pre-crosslinked composition of any one of claims 7 to the immediately preceding claim, wherein the pre-crosslinked composition is adhesive to mammalian tissue or an anatomical surface.

10. The pre-crosslinked composition of the immediately preceding claim, wherein the precrosslinked composition has an adhesive strength of at least at least 200 kPa, 500 kPa, at least 750 kPa, at least IMPa, at least 2 MPa, at least 3 MPa, or at least 5 MPa.

11. A double-crosslinked composition comprising the pre-crosslinked composition of any one of claims 7 to the immediately preceding claim, wherein the additional enzymatic crosslinking has generated enzymatic crosslinks between the methacrylate-modified silk fibroin fragments of the population of methacrylate-modified silk fibroin fragments.

12. The double-crosslinked composition of the immediately preceding claim, wherein the enzymatic crosslinks are dityrosine bonds.

13. A seeded, double-crosslinked composition comprising the double-crosslinked composition of any one of claims 11 to the immediately preceding claim and a plurality of cells seeded on the double-crosslinked composition.

14. The seeded, double-crosslinked composition of the immediately preceding claim, wherein the plurality of cells have a seeding density of at least IxlO5cells / mL.

15. A method of making a non-crosslinked composition, the method comprising: combining a population of methacrylate-modified silk fibroin fragments, a photo-crosslinker, an enzymatic crosslinker, optionally one or more enzymatic crosslinker co-reactants, and water to form the noncrosslinked composition.

16. A method of using a non-crosslinked composition, the method comprising: pre-crosslinking a non-crosslinked composition, thereby converting the non-crosslinked composition into a precrosslinked composition, wherein the non-crosslinked composition is optionally the non-crosslinked composition of any one of claims 4 to 6.

17. The method of the immediately preceding claim, the method further comprising: printing the pre-crosslinked composition in a predefined shape to produce a printed, pre-crosslinked article.

18. The method of the immediately preceding claim, the method further comprising enzymatically crosslinking the pre-crosslinked article.

19. The method of any one of claims 17 to the immediately preceding claim, wherein the printing is in vivo printing in a mammalian subject, optionally a human subject.

20. The method of any one of claims 17 to the immediately preceding claim, wherein the predefined shape includes internal structural features.

21. The composition or method of any one of the preceding claims, wherein the methacrylate side chains are present on the methacrylate-modified silk fibroin in an amount by weight of between 0.3% mol and 4% mol of the silk fibroin.

22. The composition or method of any one of the preceding claims, wherein the methacrylate- modified silk fibroin has a silk fibroin backbone with an average molecular weight of between 25 kDa and 500 kDa, between 50 kDa and 400 kDa, between 100 kDa and 300 Kda, or between 150 kDa and 350 kDa23. The composition or method of any one of the preceding claims, wherein the methacrylate- modified silk fibroin has a silk fibroin backbone having an average molecular weight of greater than 150 kDa.

24. The composition or method of any one of the preceding claims, wherein the methacrylate- modified silk fibroin includes a silk fibroin obtained by boiling for at least 5 minutes, at least 15 minutes, at least 20 minutes, at least 30 minutes, at least 45 minutes, or at least 60 minutes, and at least 15 minutes, at least 30 minutes, at least 60 minutes, or at least 120 minutes.

25. The composition or method of any one of the preceding claims, wherein at least 50% by weight of the methacrylate-modified silk fibroin has a silk fibroin backbone with a molecular weight of at least 25 kDa.

26. The composition or method of any one of the preceding claims, wherein at least 50% by weight of the methacrylate-modified silk fibroin has a silk fibroin backbone with a molecular weight of at least 50 kDa.

27. The composition or method of any one of the preceding claims, wherein at least 50% by weight of the methacrylate-modified silk fibroin has a silk fibroin backbone with a molecular weight of at least 100 kDa.

28. The composition or method of any one of the preceding claims, wherein at least 50% by weight of the methacrylate-modified silk fibroin has a silk fibroin backbone with a molecular weight of at least 250 kDa.

29. The composition or method of any one of the preceding claims, wherein at least 50% by weight of the methacrylate-modified silk fibroin has a silk fibroin backbone with a molecular weight of at least 450 kDa.

30. The composition or method of any one of the preceding claims, wherein the methacrylate- modified silk fibroin is present in the non-crosslinked composition in an amount by weight of between 2% and 10%.

31. The composition or method of the immediately preceding claim, wherein the methacrylate- modified silk fibroin is present in the non-crosslinked composition in an amount by weight of at least 2%, at least 3%, at least 4%, or at least 5% and at most 10%, at most 9%, at most 8%, at most 7%, at most 6%, or at most 5%.

32. The composition or method of any one of the preceding claims, wherein the photo-crosslinker is present in the non-crosslinked composition in an amount by weight of between 0.001% and 0.1%.

33. The composition or method of the immediately preceding claim, wherein the photocrosslinker is present in the non-crosslinked composition in an amount by weight of at least 0.001%, at least 0.005%, or at least 0.01% and at most 0.5%, at most 0.1%, at most 0.08%, or at most 0.05%.

34. The composition or method of any one of the preceding claims, wherein the enzymatic crosslinker is present in the non-crosslinked composition in an amount providing enzymatic activity of between 5 U / mL and 20 U / mL.

35. The composition or method of the immediately preceding claim, wherein the enzymatic crosslinker is present in the non-crosslinked composition in an amount providing enzymatic activity of at least 2 U / mL, at least 5 U / mL, or at least 10 U / mL and at most 20 U / mL, at most 15 U / mL, or at most 10 U / mL.

36. The composition or method of any one of the preceding claims, wherein the photo-crosslinker is lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP).

37. The composition or method of any one of the preceding claims, wherein the water is present in the non-crosslinked composition in an amount by weight of between 85% and 95%, including but not limited to, between 90% and 95%.

38. A method comprising: photo-crosslinking the pre-crosslinked composition of any one of claims 7 to 10 or 21 to 37 to establish a desired printing viscosity; printing the photo-crosslinked composition; and enzymatically crosslinking the printed composition to establish a final mechanical property.

39. The method of claim 38, wherein the desired printing viscosity is between 30 mPa*s and 6xl07mPa*s, including but not limited to at least 30 mPa*s, at least 300 mPa*s, at least 3,000 mPa*s, at least 3xl04mPa*s, at least 3xl05mPa*s, at least 3xl06mPa*s, or at least 3xl07mPa*s, and at most 6xl06mPa*s, at most 6xl05mPa*s, at most 6xl04mPa*s, at most 6000 mPa*s, at most 600 mPa*s, or at most 60 mPa*s.

40. The method of claim 38 or 39, wherein the final mechanical property is a storage modulus of at least 300 Pa, at least 400 Pa, at least 500 Pa, at least 600 Pa, at least 700 Pa, at least 800 Pa, at least 900 Pa, at least 1 kPa, at least 1.1 kPa, at least 1.2 kPa, at least 1.3 kPa, at least 1.4 kPa, or at least 1.5 kPa.

41. The composition or method of any of the preceding claims, wherein the degree of substitution of the methacrylate side chains present on the methacry late-modified silk fibroin is between 0.1% to 99.9%, between 30% and 80%, between 50% and 75%, or between 60% and 70%.

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