Injectable silk fibroin foams and uses thereof

Abstract

To provide compositions, methods, delivery devices and kits for repairing or augmenting a tissue in a subject.SOLUTION: Compositions described herein can be injectable such that they can be placed in a tissue to be treated with a minimally-invasive procedure (e.g., by injection), and comprise a compressed silk fibroin foam, which can expand upon injection into the tissue and have a porosity of at least 50% and retain its original expanded volume within the tissue for a period of time and retain at least 1% of its original expanded volume within the tissue for at least about 2 weeks. The compositions can be used as a filler to replace a tissue void, e.g., for tissue repair and / or augmentation, or as a scaffold to support tissue regeneration and / or reconstruction. In some embodiments, the compositions described herein can be used for soft tissue repair or augmentation.SELECTED DRAWING: Figure 8G

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 USC §119(e) of U.S. Provisional Application No. 61 / 557,610, filed November 9, 2011, the entire contents of which are incorporated herein by reference.

[0002] Government support This invention was made with government support under Grant No. EB002520 awarded by the National Institutes of Health and Grant No. W81XWH-08-2-0032 awarded by the United States Army.

[0003] Technical Field of the Disclosure The invention described herein relates generally to silk fibroin-based materials for biomedical applications, for example, in the repair, augmentation and / or reconstruction of soft tissue. [Background technology]

[0004] background Restoration of soft tissue defects due to trauma, surgical resection, or congenital defects should be initiated by strategies that maintain tissue size and shape near normal dimensions over a long time frame. Current clinical strategies include free fat transfer and artificial fillers. In breast cancer patients who have undergone mastectomy, saline or silicone-filled shells are used to replace the void. This presents the patient with an unnatural look and feel and the risk of capsular contracture, requiring revision surgery. Fat graft and artificial filler options fail to retain volume over time. Thus, they may require a second surgical site, may result in avascular necrosis, and usually do not regenerate the original tissue.

[0005] Bovine and human collagen is widely used as an injectable material for soft tissue augmentation and filling. Collagen, the main extracellular structural protein of the animal body, is used as a graft material to replace or augment connective tissues, such as skin, tendons, cartilage and bone. In addition, collagen has been injected or implanted into the human body for cosmetic purposes for many years. However, the use of collagen in soft tissue augmentation and / or filling is costly and short-lived, e.g., the results often only last for about three months.

[0006] Hyaluronic acid (HA) is a glycosaminoglycan found naturally in the human body and distributed widely throughout connective, epidermal and neural tissues. Non-crosslinked hyaluronic acid compositions tend to degrade within months of injection, thus requiring fairly frequent injections to maintain their soft tissue augmentation effect. More recently, crosslinked hyaluronic acid compositions have been used for soft tissue augmentation. However, such crosslinked compositions contain very large particles of hyaluronic acid, each around 2 mm, suspended in a gel. Larger particles may provide longer lasting effects, but larger particle size may make injection more difficult and may result in an unpleasant experience for the recipient.

[0007] In summary, the main drawbacks of current strategies in regenerating, repairing and / or augmenting soft tissue include the need for large amounts of tissue to be grafted into large tissue defects; donor site morbidity, secondary surgical site, potential for avascular necrosis; loss of scaffold shape and / or size over time; material incompatibility with native tissue; and poor tissue regeneration.Therefore, there is a strong need to develop a strategy or scaffold that can be performed with minimally invasive procedures and that maintains volume recovery for at least three months or more, such as at least six months or at least one year, while the body gradually remodels and regenerates the site to near-normal tissue structure and function. Summary of the Invention

[0008] Abstract Spongy biomaterial scaffolds, e.g., foams, are desirable for tissue engineering, e.g., for regenerating, repairing, and / or augmenting soft tissues, in part because the network of interconnected pores in the sponge-like scaffolds favors cell attachment and also allows for the flow of nutrients and waste products. However, the mechanical properties and / or structure of current biomaterial foams are generally not capable of regenerating tissue or retaining their volume within tissues over long periods of time. In addition, placement of such sponge-like biomaterial scaffolds into tissues can be invasive. Thus, there is an urgent need to develop minimally invasive strategies for repairing or augmenting tissues in individuals, e.g., development of injectable foams that can retain their volume and shape while tissues gradually regenerate and regain their structure and function.

[0009] Embodiments of the various aspects described herein are based, at least in part, on our discovery that silk fibroin-based foams can be injected to fill soft tissue voids, such as tissue loss due to injury or disease - e.g., in trauma, cancer removal surgery, breast reconstruction, breast augmentation, and related needs. In addition, silk fibroin-based foam constructs can also be used for cosmetic purposes, e.g., as a more economical and natural alternative to topical and BOTOX treatments.

[0010] Accordingly, one aspect provided herein is an injectable composition used to repair or augment tissue in a subject, comprising a compressed silk fibroin matrix, where the compressed silk fibroin matrix expands upon injection into tissue and retains at least a portion of its original expanded volume (e.g., at least about 1%, at least about 5%, at least about 10%, at least about 25%, at least about 50% or more) within the tissue to be repaired or augmented for a period of time (e.g., at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, at least about 6 weeks or more).

[0011] Another aspect provided herein relates to a method for repairing or augmenting a tissue of a subject, the method comprising placing a composition comprising a compressed silk fibroin matrix in the tissue to be repaired or augmented, wherein the compressed silk fibroin matrix expands upon placement in the tissue and retains at least a portion of its initial expanded volume (e.g., at least about 1%, at least about 5%, at least about 10%, at least about 25%, at least about 50% or more) within the tissue for a period of time (e.g., at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, at least about 6 weeks or more).In one embodiment, the composition is placed in the tissue to be repaired or augmented by injection.

[0012] In some embodiments, the silk fibroin matrix can be provided in a compressed state for the treatment methods described herein. In some embodiments, the silk fibroin matrix can be provided in an uncompressed state and then compressed to a smaller volume during loading into a delivery applicator (e.g., an injection applicator, such as a needle, cannula, and / or catheter) prior to placement into the tissue to be repaired or augmented.

[0013] In some embodiments of the compositions and methods provided herein, the compressed silk fibroin matrix, after placement (e.g., injection) in tissue, can expand its volume by at least about 50%, at least about 100%, at least about two-fold, at least about three-fold, or more, compared to the compressed volume of the silk fibroin matrix.

[0014] In certain embodiments of the compositions and methods provided herein, the silk fibroin matrix may not include amphiphilic peptides.In other embodiments, the silk fibroin matrix may include amphiphilic peptides.Exemplary amphiphilic peptides may include, for example, RGD motifs.

[0015] In some embodiments of the compositions and methods provided herein, the silk fibroin matrix may retain at least about 50% of its initial expanded volume within the tissue for a period of time, including at least about 60%, at least about 70%, at least about 80% or more of its initial expanded volume.

[0016] In some embodiments of the compositions and methods provided herein, the silk fibroin matrix can retain at least a portion of its initial expanded volume for at least about six weeks, at least about three months, at least about six months or longer.

[0017] The volume retention of the silk fibroin matrix can be controlled, in part, by adjusting the degradation and / or dissolution properties of the silk fibroin matrix. For example, the silk fibroin matrix can be adapted to degrade at a predetermined rate so that the silk fibroin matrix can maintain a desired volume over a predetermined period of time, for example, to promote tissue regeneration or repair. For example, in such an embodiment, the silk fibroin matrix can be adapted to degrade 50% or less of its initial swelling volume, including 30% or less, including 10% or less of its initial swelling volume, for example, in at least about 2 weeks, including at least about 6 weeks, at least about 3 months, at least about 6 months or more.

[0018] In some embodiments, the silk fibroin matrix can be adapted to degrade at a predetermined rate such that the volume of the silk fibroin matrix gradually decreases (while still providing sufficient support) as the tissue in which it is placed begins to regenerate. In such embodiments, the silk fibroin matrix can be adapted to degrade at least about 5% of its initial expanded volume, including at least about 10%, at least about 20%, at least about 30% or more of its initial expanded volume, over a period of at least about 2 weeks, including at least about 6 weeks, at least about 3 months, at least about 6 months or more.

[0019] Depending on the size of the tissue defect and / or the desired properties of the silk fibroin matrix, the silk fibroin matrix (before compression) can be adapted to be any size. In some embodiments, the silk fibroin matrix (before compression) can have a size of about 1 mm to about 5 mm in diameter. In some embodiments, the silk fibroin matrix (before compression) can have a size larger than 5 mm in diameter. Because the silk fibroin matrix is ​​compressible, the size of the silk fibroin matrix can be larger to be suitable for filling larger size defects, as long as the size of the compressed silk fibroin matrix is ​​suitable for injection into tissue.

[0020] The silk fibroin matrix can be adapted to mimic the structural morphology of native tissue and / or deliver active agents to localized regions of tissue. For example, the silk fibroin matrix can be porous. In some embodiments, the porosity of the silk fibroin matrix can be adapted to mimic the structural morphology and / or cell density gradient found in native tissue. In some embodiments, the porosity of the silk fibroin matrix can be adapted to deliver active agents to tissue with a predetermined release profile. In some embodiments, the porosity of the silk fibroin matrix can be adapted to retain at least a portion of its initial expanded volume for a period of time. For example, the porous silk fibroin matrix can have a porosity of at least about 1%, including, for example, at least about 3%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 70%, at least about 80%, at least about 90% or more. The pore size of such a porous silk fibroin matrix can range from about 1 μm to about 1500 μm, from about 50 μm to about 650 μm, or from about 100 μm to about 600 μm.

[0021] In one embodiment, the silk fibroin matrix can be fabricated by freezing a silk fibroin solution. In some embodiments, the silk fibroin solution can have a concentration of about 0.5% w / v to about 10% w / v or about 1% w / v to about 6% w / v.

[0022] In some embodiments of any aspect described herein, the silk fibroin matrix can be a silk fibroin foam.

[0023] In some embodiments, the silk fibroin matrices or compositions described herein may further comprise a hydrogel material.

[0024] The injectable composition described herein comprising a silk fibroin matrix may further comprise at least one active agent. In some embodiments, the silk fibroin matrix of the composition described herein may further comprise at least one active agent. Non-limiting examples of active agents may include biologically active agents, cosmetically active agents, cell adhesives, dermal filling materials, and any combination thereof. In some embodiments, the active agent may be cells, such as, but not limited to, stem cells. In some embodiments, the active agent may be adipose-derived stem cells. In some embodiments, the active agent may be a biological fluid or concentrate, such as, but not limited to, aspirated lipoprotein or aspirated bone marrow. In some embodiments, the active agent may be a therapeutic agent. In some embodiments, the active agent may be a cosmetically active agent. In some embodiments, the active agent may be a dermal filling material.

[0025] Various embodiments of the compositions described herein can be injected into the tissue that is to be repaired or augmented by any method known in the art, for example, subcutaneously, submuscularly or intramuscularly.Some embodiments of the compositions can be at least partially dry when injected into tissue.Alternatively, the compositions can be at least partially hydrated.In some embodiments, the compositions can further comprise a carrier, for example, a buffer solution and / or a biological fluid or concentrate (e.g., lipoaspirate), when injected into tissue.

[0026] The tissue that is repaired or augmented by the compositions and / or methods described herein can be soft tissue.Exemplary examples of soft tissue include, but are not limited to, tendon, ligament, skin, breast tissue, fibrous tissue, connective tissue, muscle, and any combination thereof.In certain embodiments, the soft tissue is skin.In other embodiments, the soft tissue is breast tissue.

[0027] Also provided herein is a delivery device comprising one embodiment of the injectable composition and / or silk fibroin matrix. The delivery device may include an injection device (e.g., in the form of a syringe or injection gun) and / or any minimally invasive administration device. Thus, in some embodiments, the present disclosure relates to an injection device comprising the injectable composition described herein. The delivery or injection device may further comprise a tubular structure for introducing the silk fibroin matrix or composition described herein into a tissue that one wishes to repair or augment. The tubular structure may be tapered (e.g., comprising a conical internal space), for example, to facilitate loading the compressed silk fibroin matrix therein. In some embodiments, the tubular structure may be one that can compress the silk fibroin matrix to a predetermined volume (e.g., the internal volume of the tubular structure) while loading the silk fibroin matrix therein. Examples of tubular structures include, but are not limited to, a needle, a cannula, a catheter, or any combination thereof. In some embodiments, the tubular structure may be one that is preloaded with silk fibroin matrix. In this embodiment, the silk fibroin matrix may be in a compressed state within the tubular structure. In some embodiments, the delivery or injection device may further include a mechanical element (e.g., an elongated rod-like structure) that facilitates the ejection of the compressed silk fibroin matrix through the tubular structure. In some embodiments, the delivery or injection device may further include a carrier for injection. In some embodiments, the delivery or injection device may further include a local anesthetic.

[0028] In some embodiments of any aspect described herein, the composition and / or delivery device may be stored or transported at a temperature between about 0° C. and about 60° C., such as between about 10° C. and about 60° C. or between about 15° C. and about 60° C. Under such temperatures, the biological activity of an active agent embedded or distributed within the silk fibroin matrix may be stabilized for a period of time. [Brief description of the drawings]

[0029] [Figure 1] 1A-1B show images of a silk fibroin-based injectable foam disk according to one or more embodiments described herein. Fig. 1A shows an image of a silk fibroin-based injectable foam disk being cut, for example, using a 4 mm biopsy punch. Fig. 1B shows an image of the silk fibroin-based injectable foam disk after cutting. [Diagram 2] 2A-2B show images of an exemplary method of placing a silk fibroin-based injectable foam disk into an injectable location inside a syringe tip (e.g., a pipette tip). FIG. 2A shows an image of a silk fibroin-based injectable foam disk being loaded into a pipette tip. FIG. 2B shows an image of a silk fibroin-based injectable foam disk being packed into an injection location inside a pipette tip, e.g., using a stiff wire. [Diagram 3] 3A-3D show images of an exemplary method of injecting silk fibroin-based injectable foam discs into tissue. FIG. 3A shows an image of creating a hole in chicken thigh tissue, e.g., using a straight 14-gauge needle. FIG. 3B shows an image of inserting a pipette tip containing a silk fibroin-based injectable foam disc (e.g., as shown in FIG. 2B) into the hole. FIG. 3C shows an image of ejecting a silk fibroin-based foam disc into tissue, e.g., using a stiff wire, while slowly withdrawing the pipette tip. FIG. 3D shows an image of an injectable silk fibroin-based foam disc placed in a live chicken thigh (black arrow points to silk fibroin-based foam disc injected into tissue). [Figure 4]4A-4F show images of an exemplary method of extracting injected silk fibroin-based foam from tissue. FIG. 4A shows an image of palpating tissue (e.g., live chicken tissue) for injected silk fibroin-based foam. FIG. 4B shows an image of cutting (e.g., with a razor blade) near where the embedded silk fibroin-based foam is located. FIG. 4C shows an image of the embedded silk fibroin-based foam exposed after cutting. FIG. 4D shows a different view of the cross section of the silk fibroin-based foam of FIG. 4C. FIG. 4E shows an image of removing the exposed silk fibroin-based foam (e.g., with a pair of tweezers). FIG. 4F shows an image of the silk fibroin-based foam extracted from the tissue. [Diagram 5] 5 shows an exemplary method of using one or more embodiments of the injectable composition described herein. To form an exemplary injectable composition, a porous silk fibroin-based foam (e.g., formed by freezer treatment) can be mixed with aspirated lipoprotein as a carrier, optionally including adipose-derived stem cells (ASCs). The injectable composition can then be injected into a subject, e.g., an animal model. [Figure 6A] 6A-6C show images of another exemplary method of injecting silk fibroin-based injectable foam discs into tissue-like materials. Figure 6A is a collection of images showing the steps of preparing a catheter for injecting silk fibroin-based foam into tissue. A hole is drilled in the target area of ​​the tissue through which the catheter can be inserted. [Figure 6B] Figures 6A-6C show images of another exemplary method of injecting silk fibroin-based injectable foam disks into tissue-like materials. Figure 6B is a collection of images showing the steps of loading silk fibroin-based foam into a catheter inserted into tissue. After inserting the catheter into tissue, the silk fibroin-based foam is loaded into the catheter, for example, via an adapter. [Figure 6C]6A-6C show images of another exemplary method of injecting silk fibroin-based injectable foam disks into tissue-like materials. Figure 6C is a collection of images showing the process of pushing the silk fibroin-based foam through a catheter. After loading the silk fibroin-based foam into an adapter with a catheter, a rod is used to push the foam through the catheter and into the tissue. [Figure 7A] 7A-7B show images of an exemplary method of injecting silk fibroin-based foam into tissue in vivo. Figure 7A shows an exemplary custom modified injection gun used to facilitate the injection of silk fibroin-based foam into tissue in vivo. A foam ramrod is mounted on the injection gun. [Figure 7B] 7A-7B show images of an exemplary method of injecting silk fibroin-based foam into tissue in vivo. FIG. 7B is a collection of images showing an exemplary step of injecting silk fibroin-based foam into tissue in a rat or mouse model. A catheter (e.g., 14G gauge) is inserted into the target tissue area, after which silk fibroin-based foam is loaded into the catheter. The catheter is then connected to a foam syringe (e.g., an injection gun shown in FIG. 7A) so that the silk fibroin-based foam can be slowly injected through the catheter into the target tissue area (e.g., subcutaneous area). [Figure 8A]8A-8G show images and results of several embodiments of silk fibroin-based foams injected into a rat model in vivo. FIG. 8A-8C show images of silk fibroin-based foams injected into a rat model in vivo after removal of the rat skin. Silk fibroin-based foams made from different concentrations of silk fibroin solutions (e.g., 1%, 3%, 6% silk fibroin) and cocoon sources (Japan: JP vs. Taiwan: TW) were evaluated after injection for 1 day (FIG. 8A), 14 days (FIG. 8B), and 30 days (FIG. 8C). FIG. 8A shows that the injected silk fibroin-based foams maintained transparency one day after injection unless stained by blood from puncturing a blood vessel (e.g., TW3). [Figure 8B] 8A-8G show images and results of several embodiments of silk fibroin-based foams injected into a rat model in vivo. FIG. 8A-8C show images of silk fibroin-based foams injected into a rat model in vivo after removal of the rat skin. Silk fibroin-based foams made from different concentrations of silk fibroin solutions (e.g., 1%, 3%, 6% silk fibroin) and cocoon sources (Japan: JP vs. Taiwan: TW) were evaluated after injection for 1 day (FIG. 8A), 14 days (FIG. 8B), and 30 days (FIG. 8C). FIG. 8B-8C show that the injected silk fibroin-based foams turned reddish hue about 14 days and about 30 days after injection, respectively. However, no significant changes were observed in terms of vascularization into the injected foams. [Figure 8C] Figures 8A-8G show images and results of several embodiments of silk fibroin-based foams injected into an in vivo rat model, with details as described in the legend to Figure 8B. [Figure 8D] Figures 8A-8G show images and results of several embodiments of silk fibroin-based foams injected into a rat model in vivo, and Figure 8D shows an image of the injected foam as seen from the outside of the rat's skin. [Figure 8E] Figures 8A-8G show images and results of several embodiments of silk fibroin-based foams injected into a rat model in vivo. Figure 8E shows a set of images showing the overall morphology of several embodiments of silk fibroin-based injectable foams (corresponding to those of Figures 8A-8C) explanted after the indicated post-injection periods (e.g., 1 day, 14 days, and 30 days post-injection). There is no observable visual difference in the overall morphology at the indicated time points. Silk fibroin foams become consistently stiffer with increasing weight percent of silk. All explants are soft to the touch and return to their original shape after deformation. [Figure 8F] Figures 8A-8G show images and results of several embodiments of silk fibroin-based foams injected into a rat model in vivo. Figure 8F shows the volume retention results of silk fibroin-based foams after injection into tissue for 1 day or 14 days. The results in Figure 8F are expressed as a percentage of volume retention relative to its initial volume. [Figure 8G] Figures 8A-8G show images and results of several embodiments of silk fibroin-based foams injected into a rat model in vivo. Figure 8G shows the volume retention results of silk fibroin-based foams after injection into tissue for 14, 30 or 60 days. The results in Figure 8G are expressed as a percentage of volume retention relative to its initial volume. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] Detailed Description of the Invention Described herein are methods, compositions, delivery devices and kits for repairing or augmenting tissue in a subject. According to various aspects described herein, a reversibly deformable and / or injectable silk fibroin scaffold (e.g., silk fibroin foam) can be compressed (e.g., to a smaller volume) and placed (e.g., by injection) in the tissue to be repaired or augmented. Once placed (e.g., injected) in the tissue, the compressed silk fibroin matrix expands in volume (e.g., increases in volume by at least about 10% of its compressed volume) and retains at least a portion of its original expanded volume (e.g., at least about 50% or more of its original expanded volume) within the tissue to be repaired or augmented for a period of time (e.g., at least about 2 weeks or more). Such reversibly deformable and / or injectable silk fibroin matrix can be introduced into the defect site through a minimally invasive procedure.

[0031] Silk Fibroin Matrix The silk fibroin matrix described herein is deformable. The silk fibroin matrix can be compressed prior to and / or during placement (e.g., injection) into tissue to be repaired or augmented. Once placed (e.g., injected) into tissue, the compressed silk fibroin matrix can expand within the tissue and retain its initial expanded volume within the tissue for a period of time.

[0032] As used herein, the term "deformable" generally refers to the ability of an object to change size (e.g., volume) and / or shape in response to an external pressure / force while maintaining the integrity of the object (i.e., the object remains generally intact during deformation without breaking into pieces and has the ability to recover at least a portion of its original size and shape). In the case of a deformable silk fibroin matrix, when compressed by an applied force, the silk fibroin matrix can reduce its volume and / or change its shape such that it becomes small enough (but remains intact) to be loaded into an injection applicator (e.g., a needle, cannula, or catheter) having dimensions much smaller than those of the uncompressed silk fibroin matrix, and / or such that the silk fibroin matrix is ​​compressed to conform to the cross-sectional shape of the injection applicator.

[0033] As used herein, the term "compression" generally refers to the reduction of the volume of silk fibroin matrix. In some embodiments, the reduction of the volume of silk fibroin matrix may also result in the change of one or more physical properties of silk fibroin matrix, such as an increase in the initial density (before compression) of silk fibroin matrix, a decrease in the initial pore size (before compression) and / or the initial porosity (before compression) of silk fibroin matrix. The compression of silk fibroin matrix to a predetermined volume can be performed by any method known in the art, such as by physically loading silk fibroin matrix into the inner space of a delivery applicator (e.g., needle, cannula or catheter) or by vacuum.

[0034] In some embodiments, the silk fibroin matrix can be compressed to a volume of 80% or less of its original volume (i.e., the volume of the silk fibroin matrix before compression), including 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, or less than its original volume (i.e., the volume of the silk fibroin matrix before compression). In some embodiments, the silk fibroin matrix can be compressed to a volume of at least about 10% of its original volume (i.e., the volume of the silk fibroin matrix before compression), including at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or more, but not 100% of its original volume (i.e., the volume of the silk fibroin matrix before compression). In some embodiments, silk fibroin matrices, such as silk fibroin foams, fabricated from silk fibroin solutions with concentrations of about 1% to about 6% can be compressed to approximately 20% to 30% of their original volume. The amount of compression that can be achieved without destruction or permanent deformation of the silk fibroin matrix depends, for example, on the material properties, silk fibroin concentration, and / or the fabrication / processing method and parameters. In some embodiments, higher molecular weight matrices or other improved processing can result in higher levels of compression of silk fibroin-based matrices, such as silk fibroin-based matrices compressed to less than 20% of their original volume.

[0035] In some embodiments, the silk fibroin matrix can be compressed to a volume that increases the original density (e.g., ratio of weight to uncompressed volume) of the silk fibroin matrix by at least about 10%, including, for example, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold or more.

[0036] After the compressed silk fibroin matrix is ​​released from the delivery applicator (e.g., injection applicator) and placed (e.g., injected) into tissue, the silk fibroin matrix may expand depending on the removal of compression stress, the size of the tissue void, the mechanical properties of the tissue and the silk fibroin matrix, and any combination thereof. In some embodiments, the compressed silk fibroin matrix may expand and recover to its original volume (i.e., the volume of the silk fibroin matrix before compression). In some embodiments, the compressed silk fibroin matrix may expand to a size sufficient to fill the void of the tissue to be repaired or augmented. In some embodiments, the silk fibroin matrix may expand to a size such that the silk fibroin matrix and the tissue surrounding the silk fibroin matrix press against each other with equal force. In certain embodiments, the compressed silk fibroin matrix, when placed (e.g., injected) into a tissue to be repaired or augmented, can expand to a size of at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or up to 100% of its original volume (i.e., the volume of the silk fibroin matrix before compression). In some embodiments, the compressed silk fibroin matrix, when placed (e.g., injected) into a tissue to be repaired or augmented, can expand its volume by at least about 1-fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, or more, compared to its compressed volume (e.g., the volume of the silk fibroin matrix compressed within a delivery applicator, e.g., an injection applicator, e.g., a needle, cannula, or catheter). In such embodiments, without wishing to be bound by theory, the silk fibroin matrix may expand beyond its original volume, in part, by the silk fibroin matrix absorbing moisture or water from the surrounding tissue and causing itself to swell.

[0037] In some embodiments, viewed alternatively, the compressed silk fibroin matrix may expand its volume by at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 1-fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold or more relative to the volume of the compressed silk fibroin matrix.

[0038] The expansion of silk fibroin matrix in tissue to reach plateau volume can be spontaneous (e.g., within 5 seconds or less) or occur over a period of time, e.g., seconds, minutes, and hours. In some embodiments, at least about 50% increase in silk fibroin matrix volume can occur within 3 hours, 2 hours, 1 hour, 30 minutes, 20 minutes, 10 minutes, 5 minutes, or less, of silk fibroin matrix injection into tissue, and the remaining increase in silk fibroin matrix volume can occur over a much longer time scale. The rate of expansion of silk fibroin matrix in tissue can depend on several factors, including, but not limited to, hydration state, pressure, void volume, and material properties of silk, structural properties of foam (including porosity), and interactions between fluid and structure. For example, when silk fibroin matrix (e.g., silk fibroin foam) is injected into a fluid-filled void, its expansion can be rapid. When a dry silk fibroin matrix (eg, a dry silk fibroin foam) is injected, hydration and swelling is believed to occur much slower, for example, minutes to hours.

[0039] After the silk fibroin matrix expands upon injection into tissue, the silk fibroin matrix can retain at least a portion of its initial expanded volume within the tissue (e.g., at least about 50% or more) for at least a period of time (e.g., at least about 2 weeks, at least about 4 weeks, at least about 6 weeks or more).

[0040] "Initial swollen volume" with reference to the silk fibroin matrices described herein generally refers to the volume of the silk fibroin matrix measured after swelling upon injection into a tissue to be repaired or augmented, or the corresponding increase in tissue volume measured after swelling of the silk fibroin matrix upon injection. For example, the initial swollen volume of the silk fibroin matrix can be measured immediately, e.g., when there is no significant increase in the volume of the silk fibroin matrix for at least about 72 hours, at least about 48 hours, at least about 24 hours, at least about 12 hours, at least about 6 hours, at least about 3 hours or less, after injection of the silk fibroin matrix or injectable composition into the tissue. Alternatively, the initial swelling volume of the silk fibroin matrix may be determined by measuring the corresponding increase in tissue volume (attributed to swelling of the silk fibroin matrix) immediately after injection of the silk fibroin matrix or injectable composition into the tissue when there is no significant increase in tissue volume for at least about 72 hours, at least about 48 hours, at least about 24 hours, at least about 12 hours, at least about 6 hours, at least about 3 hours or less. In some embodiments, the initial swelling volume may represent the initial volume of the silk fibroin matrix (i.e., the volume of the silk fibroin matrix before compression).

[0041] As used herein, the term "retention" refers to the maintenance of the volume (e.g., size and / or shape) of the silk fibroin matrix described herein over a period of time. In some embodiments, the silk fibroin matrix can retain at least about 20% of its initial expanded volume over a period of time, including, for example, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or more of its initial expanded volume. In some embodiments, the silk fibroin matrix can retain at least about 1% of its initial expanded volume over a period of time, including, for example, at least about 3%, at least about 5%, at least about 10%, at least about 15%, at least about 20% or more of its initial expanded volume. In some embodiments, the silk fibroin matrix can retain 100% of its initial expanded volume, e.g., no detectable change in volume, in the tissue to be repaired or augmented over a period of time. In one embodiment, the silk fibroin matrix may retain at least about 1% of its initial swelling volume within the tissue to be repaired or augmented for a period of time. In one embodiment, the silk fibroin matrix may retain at least about 50% of its initial swelling volume within the tissue to be repaired or augmented for a period of time. In one embodiment, the silk fibroin matrix may retain at least about 60% of its initial swelling volume within the tissue to be repaired or augmented for a period of time. In one embodiment, the silk fibroin matrix may retain at least about 70% of its initial swelling volume within the tissue to be repaired or augmented for a period of time. In one embodiment, the silk fibroin matrix may retain at least about 80% of its initial swelling volume within the tissue to be repaired or augmented for a period of time. The volume of the silk fibroin matrix placed in the tissue may be determined or indicated by a change in at least one of the tissue properties, such as tissue volume, tissue elasticity, and / or tissue hardness. In some embodiments, the volume of silk fibroin matrix placed in tissue can be determined from the explants.

[0042] The silk fibroin matrix can retain at least a portion of its original expanded volume for any period of time, such as weeks, months, or years. In some embodiments, the silk fibroin matrix may retain at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, at least about 6 weeks, at least about 7 weeks, at least about 8 weeks, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, at least about 1 year, at least about 2 years, at least about 3 years, at least about 4 years, at least 5 years or more, such as at least about 1% of its initial expanded volume (including, for example, at least about 3%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80% or more of their initial volume). In certain embodiments, the silk fibroin matrix may retain at least about 3 months or more, e.g., at least about 70% or more of its initial expanded volume. In other embodiments, there may be no significant change in the volume of the silk fibroin matrix or a corresponding increase in tissue volume after placement in the tissue desired to be repaired or augmented for at least about 3 months or more. In some embodiments, the silk fibroin matrix may retain at least about 6 months or more, e.g., at least about 9 months, at least about 12 months, at least about 18 months or more, e.g., at least about 70% or more of its initial expanded volume. In other embodiments, there may be no significant change in the volume of the silk fibroin matrix or a corresponding increase in tissue volume after placement in the tissue desired to be repaired or augmented for at least about 6 months or more.In certain embodiments, the silk fibroin matrix may retain at least about 20% or more of its initial expanded volume for at least about 1 year or more (including, for example, at least about 2 years, at least about 3 years, at least about 4 years, at least about 5 years or more). In some embodiments, the silk fibroin matrix may retain at least about 50% or more of its initial expanded volume for at least about 1 year or more (including, for example, at least about 2 years, at least about 3 years, at least about 4 years, at least about 5 years or more). In some embodiments, the silk fibroin matrix may retain at least about 1% or more of its initial expanded volume for at least about 1 year or more (including, for example, at least about 2 years, at least about 3 years, at least about 4 years, at least about 5 years or more).

[0043] The volume retention of the silk fibroin matrix can also be characterized, for example, by the degradation of the silk fibroin matrix. Generally, the slower the degradation of the silk fibroin matrix, the longer the silk fibroin matrix can retain its original expanded volume in the tissue. Thus, some embodiments provided herein are directed to an injectable composition for use in repairing or augmenting tissue of a subject, comprising a compressed silk fibroin matrix, wherein the compressed silk fibroin matrix expands upon injection into the tissue, and the silk fibroin matrix is ​​adapted to degrade within the tissue to be repaired or augmented over a period of time.

[0044] The term "degrade" or "degradation" when used in reference to the silk fibroin matrix described herein refers to a decrease in the volume or size of the silk fibroin matrix. The degradation of the silk fibroin matrix can occur through the cleavage of the silk fibroin matrix into smaller fragments and / or dissolution of the silk fibroin matrix or its fragments. In some embodiments, the silk fibroin matrix can be adapted to degrade 80% or less of its initial swelling volume, including, for example, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, 10% or less or less of its initial swelling volume. In some embodiments, the silk fibroin matrix can not show significant degradation (e.g., no detectable change in volume) in the tissue to be repaired or augmented. In one embodiment, the silk fibroin matrix can be adapted to degrade 50% or less of its initial swelling volume in the tissue to be repaired or augmented over a period of time. In one embodiment, the silk fibroin matrix can be adapted to degrade 40% or less of its initial swelling volume within the tissue to be repaired or augmented over a period of time. In one embodiment, the silk fibroin matrix can be adapted to degrade 30% or less of its initial swelling volume within the tissue to be repaired or augmented over a period of time. In one embodiment, the silk fibroin matrix can be adapted to degrade 20% or less of its initial swelling volume within the tissue to be repaired or augmented over a period of time. In one embodiment, the silk fibroin matrix can be adapted to degrade 10% or less of its initial swelling volume within the tissue to be repaired or augmented over a period of time.

[0045] In some embodiments, the silk fibroin matrix can be adapted to degrade at a predetermined rate such that the initial swelling volume of the silk fibroin matrix gradually decreases (while still providing sufficient support) as the tissue in which the silk fibroin matrix is ​​placed begins to regenerate. In such embodiments, the silk fibroin matrix can be adapted to degrade at least about 5% of its initial swelling volume, including at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95% or more of its initial swelling volume over a predetermined period of time (e.g., at least about 2 weeks, including at least about 6 weeks, at least about 3 months, at least about 6 months or more).

[0046] The silk fibroin matrix can be adapted to degrade at any rate. In some embodiments, the silk fibroin matrix can be adapted to degrade at least a portion of its initial expansion volume over any period of time, for example, weeks, months, or years. In some embodiments, the silk fibroin matrix can be adapted to degrade at least a portion of its initial expansion volume, for example, 50% or less of its initial expansion volume (including, for example, 40% or less, 30% or less, 20% or less of its initial expansion volume) over at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, at least about 6 weeks, at least about 7 weeks, at least about 8 weeks, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, at least about 1 year, at least about 2 years, at least about 3 years, at least about 4 years, at least about 5 years or more. In certain embodiments, the silk fibroin matrix may be adapted to degrade, for example, 30% or less of its initial swelling volume, for at least about 3 months or more. In other embodiments, no significant degradation may occur (i.e., no detectable change in the volume of the silk fibroin matrix) after placement in the tissue to be repaired or augmented for at least about 3 months or more. In some embodiments, the silk fibroin matrix may be adapted to degrade, for example, 30% or less of its initial swelling volume, for at least about 6 months or more (including, for example, at least about 9 months, at least about 12 months, at least about 18 months or more). In other embodiments, no significant degradation may occur (i.e., no detectable change in the volume of the silk fibroin matrix) after placement in the tissue to be repaired or augmented for at least about 6 months or more.In certain embodiments, the silk fibroin matrix can be adapted to degrade 80% or less of its initial expanded volume in at least about one year or more (including, for example, at least about two years, at least about three years, at least about four years, at least about five years or more). In some embodiments, the silk fibroin matrix can be adapted to degrade 50% or less of its initial expanded volume in at least about one year or more.

[0047] The same or similar formulations of silk fibroin matrix or injectable composition may respond differently in subjects. By way of example only, the volume retention or degradation rate of silk fibroin matrix in tissue may vary from subject to subject due to, for example, differences in the tissue microenvironment, such as the types and / or levels of various proteins or enzymes (e.g., proteolytic enzymes) present in the tissue.

[0048] In some embodiments, the silk fibroin matrix can be adapted to maintain a constant volume retention and / or degradation rate over a period of time. In some embodiments, the silk fibroin matrix can be adapted to have a volume retention or degradation rate that changes over time. For example, the silk fibroin matrix can be coated with a polymeric or biomaterial, such as a different concentration of silk fibroin and / or a different biodegradable and biocompatible polymer. Such coatings can have different functions and / or different degradation rates than that of the silk fibroin matrix core. By way of example only, the coating of the silk fibroin matrix can include at least one active agent and be adapted to degrade at a different rate (e.g., faster rate) than that of the silk fibroin matrix core. Thus, upon placement of the silk fibroin matrix in tissue, the coating of the silk fibroin matrix can be adapted to degrade faster to release an active agent, for example, to relieve pain and / or promote wound healing, while the silk fibroin matrix core can retain their volume for a longer period of time.

[0049] Silk fibroin is particularly attractive as a biopolymer candidate for use in the embodiments described herein due to its versatile processing, e.g., in fully aqueous systems (Sofia et al., 54 J. Biomed. Mater. Res. 139 (2001); Perry et al., 20 Adv. Mater. 3070-72 (2008)), relatively easy functionalization (Murphy et al., 29 Biomat. 2829-38 (2008)), and biocompatibility (Santin et al., 46 J. Biomed. Mater. Res. 382-9 (1999)). For example, silk has been approved by the US Food and Drug Administration as a tissue engineering scaffold in human implants. See Altman et al., 24 Biomaterials: 401 (2003).

[0050] As used herein, the term "silk fibroin" includes silkworm fibroin and insect or spider silk proteins. See, for example, Lucas et al., 13 Adv. Protein Chem. 107 (1958). Any type of silk fibroin may be used in the various embodiments described herein. Silk fibroin produced by silkworms, such as Bombyx mori, is the most common and represents a green, renewable source. For example, silk fibroin used in silk films may be obtained by extracting sericin from B. mori cocoons. Organically grown silkworm cocoons are also commercially available. However, many different silks can be used, including spider silk (e.g., from Nephila clavipes), transgenic silk, genetically engineered silk, such as silk from bacteria, yeast, mammalian cells, transgenic animals, or transgenic plants (see, e.g., WO 97 / 08315; U.S. Patent No. 5,245,012), and variants thereof. In some embodiments, silk fibroin can be derived from other sources, such as spiders, other silkworms, bees, and bioengineered variants thereof.

[0051] In various embodiments, silk fibroin can be modified for various applications and / or desired mechanical or chemical properties (e.g., to facilitate the formation of a gradient of an active agent within the silk fibroin matrix). Those skilled in the art can select an appropriate method for modifying silk fibroin, for example, depending on the side groups of the silk fibroin, the desired reactivity of the silk fibroin, and / or the desired charge density of the silk fibroin. In one embodiment, the modification of silk fibroin can be using amino acid side chain chemistry, for example, chemical modification through covalent bonds or modification through charge-charge interactions. Exemplary chemical modification methods include, but are not limited to, carbodiimide coupling reactions (see, e.g., U.S. Patent Application No. US 2007 / 0212730), diazonium coupling reactions (see, e.g., U.S. Patent Application No. US 2009 / 0232963), avidin-biotin interactions (see, e.g., International Application No. WO 2011 / 011347), and PEGylation with chemically active or activated derivatives of PEG polymers (see, e.g., International Application No. WO 2010 / 057142). Silk fibroin can also be modified through genetic modification to alter the functionality of the silk protein (see, e.g., International Application No. WO 2011 / 006133). For example, silk fibroin can be genetically modified to provide further modifications of silk, such as the incorporation of fusion polypeptides containing fibrous protein domains and mineralization domains that can be used to form organic-inorganic composites. See WO 2006 / 076711. Additionally, silk fibroin matrices can be combined with compounds that affect the flexibility of the matrix, such as glycerol. See, e.g., WO 2010 / 042798, Modified Silk Films Containing Glycerol.

[0052] As used interchangeably herein, the phrase "silk fibroin matrix" or "silk fibroin-based matrix" generally refers to a matrix that includes silk fibroin. In some embodiments, the phrase "silk fibroin matrix" and "silk fibroin-based matrix" refer to a matrix in which silk fibroin constitutes at least about 30% of the total composition, including at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95% or more of the total composition. In certain embodiments, the silk fibroin matrix or silk fibroin-based matrix can be formed substantially from silk fibroin. In various embodiments, the silk fibroin matrix or silk fibroin-based matrix can be formed substantially from silk fibroin containing at least one active agent. In some embodiments, the silk fibroin matrix or silk fibroin-based matrix can refer to a silk fibroin foam or silk fibroin-based foam.

[0053] The silk fibroin matrix described herein can be adapted to be any shape, such as a sphere, polygon, ellipse, cylinder, tube, or any shape known in the art. The size of the silk fibroin matrix can vary depending on many factors, including, but not limited to, the size of the tissue to be repaired or augmented and / or the desired properties of the silk fibroin matrix, such as volume retention or degradation profile. In some embodiments, the silk fibroin matrix (before compression) can have a diameter of about 1 mm to about 5 mm in size. In some embodiments, the silk fibroin matrix (before compression) can have a diameter of greater than 5 mm in size. Because the silk fibroin matrix is ​​compressible or deformable, the size of the silk fibroin matrix can be made larger to be suitable for filling larger size defects, as long as the size of the compressed silk fibroin matrix is ​​suitable for injection into tissue.

[0054] Silk fibroin matrices can be produced from water-based or organic solvent-based silk fibroin solutions. In some embodiments, silk fibroin matrices produced from organic solvent-based silk fibroin solutions can retain their original volume for a longer period of time than water-based silk fibroin matrices. The water- or organic solvent-based silk fibroin solutions used to produce the silk fibroin matrices described herein can be prepared using any technique known in the art. The concentration of silk fibroin in the solutions used for soft tissue repair or augmentation can be adapted to the individual volume retention requirements, for example, when a higher concentration silk fibroin solution can be used if a longer volume retention of the silk fibroin matrix is ​​desired when injected into the tissue to be repaired or augmented. In some embodiments, silk fibroin solutions for producing the silk fibroin matrices described herein can vary from about 0.1% (w / v) to about 30% (w / v), inclusive. In some embodiments, silk fibroin solutions can vary from about 0.5% (w / v) to about 10% (w / v). In some embodiments, silk fibroin solution can vary from about 1% (w / v) to about 6% (w / v).Suitable processes for preparing silk fibroin solution are disclosed in, for example, United States Patent US 7635755; and International Application Nos. WO / 2005 / 012606 and WO / 2008 / 127401.Microfiltration steps can be used therein.For example, the prepared silk fibroin solution can be further processed, for example, by centrifugation and / or syringe-based microfiltration, before further processing to become silk fibroin matrix as described herein.

[0055] In some embodiments, silk fibroin can also be mixed with other biocompatible and / or biodegradable polymers to form a mixed polymer matrix containing silk fibroin. One or more biocompatible and / or biodegradable polymers (e.g., two or more biocompatible polymers) can be added to the silk fibroin solution. Biocompatible polymers that can be used herein include, but are not limited to, polyethylene oxide (PEO), polyethylene glycol (PEG), collagen, fibronectin, keratin, polyaspartic acid, polylysine, alginate, chitosan, chitin, hyaluronic acid, pectin, polycaprolactone, polylactic acid, polyglycolic acid, polyhydroxyalkanoate, dextran, polyanhydride, polymer, PLA-PGA, polyanhydride, polyorthoester, polycaprolactone, polyfumarate, collagen, chitosan, alginate, hyaluronic acid, and other biocompatible and / or biodegradable polymers. See, e.g., International Application Nos. WO 04 / 062697; WO 05 / 012606.

[0056] In some embodiments, at least one active agent described herein can be added to the silk fibroin solution before further processing into the silk fibroin matrix described herein. In some embodiments, the active agent can be homogeneously or heterogeneously dispersed or gradiently dispersed within the silk fibroin, for example, using the carbodiimide-mediated modification method described in U.S. Patent Application No. US 2007 / 0212730.

[0057] In some embodiments, the silk fibroin matrix can be formed first and then contacted (e.g., immersed) with at least one active agent such that open surfaces of the matrix can be coated with the at least one active agent.

[0058] In some embodiments, the silk fibroin matrices described herein may include a porous structure, for example, to mimic the structural morphology of native tissue, to tailor the degradation rate / volume retention of the silk fibroin matrix, and / or to tailor the release profile of active agents embedded therein, if any. As used herein, the terms "porous" and "porosity" are used to generally describe a structure having an interconnected network of holes or voids (which may be, for example, openings, intermediate spaces, or other channels) throughout its volume. The term "porosity" is a measure of the voids in a material, and is the fraction of the void volume relative to the total volume, expressed as a percentage from 0 to 100% (or 0 to 1).

[0059] In some embodiments, the porous silk fibroin matrix can be configured to have any porosity depending on the desired properties. For example, in some embodiments, the porous silk fibroin matrix can have a porosity of at least about 1%, at least about 3%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or more. In some embodiments, the porosity can range from about 70% to about 99% or from about 80% to about 98%. The pore size and total porosity values ​​can be quantified using conventional methods and models known to those skilled in the art. For example, the pore size and porosity can be measured using standardized techniques, such as mercury porosimetry and nitrogen absorption. Those skilled in the art can determine the porosity of the silk fibroin matrix that is optimal for various purposes. For example, the porosity and / or pore size of the silk fibroin matrix can be optimized based on the desired degradation rate or volume retention of the silk fibroin matrix, the release profile of the active agent from the silk fibroin matrix, and / or the structural morphology of the tissue to be repaired or augmented.

[0060] The pores may be adapted to have any shape, for example, circular, elliptical or polygonal. The porous silk fibroin matrix may be adapted to have a pore size of about 1 μm to about 1500 μm, about 10 μm to about 1000 μm, about 25 μm to about 800 μm, about 50 μm to about 650 μm, or about 100 μm to about 600 μm. In some embodiments, the pores may have a size of about 100 μm to about 600 μm. In some embodiments, the silk fibroin matrix may have a pore size of less than 1 μm. In other embodiments, the silk fibroin matrix need not be porous. In such embodiments, the pore size of the silk fibroin matrix may be less than 10 nm or undetectable. The term "pore size" as used herein refers to the dimensions of the pores. In some embodiments, the pore size may refer to the longest dimension of the pore, for example, the diameter of a pore having a circular cross section or the length of the longest cross-sectional chord that may be formed across a pore having a non-circular cross section. In other embodiments, the pore size may refer to the shortest dimension of the pore.

[0061] Methods for generating porous structures within silk fibroin matrices, such as freeze-drying, porogen leaching (e.g., salt leaching), and air bubble methods, are well known in the art and are described, for example, in U.S. Pat. No. US 7,842,780; and U.S. Application Nos. US 2010 / 0279112; and US 2010 / 0279112, the contents of which are incorporated herein by reference in their entireties.

[0062] In some embodiments, the porous silk fibroin matrix is ​​not produced by a porogen leaching method (eg, a salt leaching method), such as described in US 7842780; and US 2010 / 0279112.

[0063] In some embodiments, the porous silk fibroin matrix can be produced by freeze-drying. See, for example, US 7842780 and US 2010 / 0279112. In such embodiments, the silk fibroin solution in a non-adhesive container can be frozen at subzero temperatures, for example, at about -80°C to about -20°C, for at least about 12 hours, at least about 24 hours or more, and then freeze-dried. In one embodiment, the silk fibroin solution can be frozen unidirectionally. In some embodiments, the silk fibroin solution can be salt-free. In some embodiments, alcohol, for example, 15% to 25% methanol or propanol, can be added to the silk fibroin solution.

[0064] In certain embodiments, porous silk fibroin matrices can be produced by freezing a silk fibroin solution at a temperature ranging between about -1°C and about -20°C or between about -5°C and -10°C for at least about 2 days, at least about 3 days, or more, followed by freeze-drying for at least about 2 days, at least about 3 days, or more. See, e.g., US 61 / 477,486. The temperature and / or duration of freezing and / or the duration of freeze-drying can be adjusted to generate silk fibroin matrices of different porous structures and / or mechanical properties.

[0065] In some embodiments, the silk fibroin solution can be exposed to an electric field, for example by applying a voltage to the silk fibroin solution.The silk fibroin solution that does not turn into a gel after being exposed to an electric field can then be placed in a freezer for an extended period of time, for example, at least about 1 day, at least about 2 days, at least about 3 days, at least about 5 days, at least about 6 days or more.The frozen silk fibroin matrix can then be removed from the freezer and stored at about room temperature, thereby generating silk fibroin matrices with various porous structures and / or properties.

[0066] In some embodiments, the silk fibroin matrix described herein may be subjected to a post-treatment that may affect at least one silk fibroin property. For example, the post-treatment of the silk fibroin matrix may affect the silk fibroin properties, including β-sheet content, solubility, active agent loading capacity, degradation time, drug permeability, or any combination thereof. Post-treatment options for silk include controlled slow drying (Lu et al., 10 Biomacromolecules 1032 (2009)), underwater annealing (Jin et al., Water-Stable Silk Films with Reduced β-Sheet Content, 15 Adv. Funct. Mats. 1241 (2005)), stretching (Demura & Asakura, Immobilization of glucose oxidase with Bombyx mori silk fibroin by only stretching treatment and its application to glucose sensor, 33 Biotech & Bioengin. 598 (1989)), compression, as well as immersion in methanol (Hofmann et al., 2006), ethanol (Miyairi et al., 1978), glutaraldehyde (Acharya et al., 2008), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (Bayraktar et al., (2005) including solvent immersion.

[0067] In some embodiments, post-treatment of the silk fibroin matrix, such as annealing in water or soaking in a solvent, can achieve controlled release of active agents from the silk fibroin matrix. In some embodiments, post-treatment of the silk fibroin matrix, such as annealing in water or soaking in a solvent, can allow for tuning of the degradation or dissolution properties of the silk fibroin matrix used in the methods described herein. In some embodiments, post-treatment of the silk fibroin matrix, such as annealing in water or soaking in a solvent, can allow for tuning of the volume retention properties of the silk fibroin matrix used in the methods described herein.

[0068] In some embodiments, the silk fibroin matrix described herein can be coated with at least one layer of a biocompatible and / or biodegradable polymer described herein, for example, to adjust the degradation and / or volume retention properties of the silk fibroin matrix upon injection into a tissue to be treated and / or to adjust the rate at which an active agent is released from the silk fibroin matrix. In such embodiments, the biocompatible and / or biodegradable polymer can include at least one active agent.

[0069] In some embodiments, the silk fibroin matrices described herein can be coated with cell adhesion molecules, such as, but not limited to, fibronectin, vitronectin, laminin, collagen, any extracellular matrix molecule known in the art, and any combination thereof.

[0070] In some embodiments, the silk fibroin matrix described herein can be sterilized.The sterilization methods of biomedical devices are well known in the art, including but not limited to gamma or ultraviolet irradiation, autoclave (e.g., heat / steam); alcohol sterilization (e.g., ethanol and methanol); and gas sterilization (e.g., ethylene oxide sterilization).

[0071] Additionally, the silk fibroin matrices described herein can take advantage of the many techniques that have been developed to functionalize silk fibroin (e.g., active agents such as dyes and sensors). For example, US Patent No. 6,287,340, Bioengineered anterior cruciate ligament; WO 2004 / 000915, Silk Biomaterials & Methods of Use Thereof; WO 2004 / 001103, Silk Biomaterials & Methods of Use Thereof; WO 2004 / 062697, Silk Fibroin Materials & Use Thereof; WO 2005 / 000483, Method for Forming inorganic Coatings; WO 2005 / 012606, Concentrated Aqueous Silk Fibroin Solution & Use Thereof; WO 2011 / 005381, Vortex-Induced Silk fibroin Gelation for Encapsulation & Delivery; WO 2005 / 123114, Silk-Based Drug Delivery System; WO 2006 / 076711, Fibrous Protein Fusions & Uses Thereof in the Formation of Advanced Organic / Inorganic Composite Materials; U.S. Patent Application Publication No. 2007 / 0212730, Covalently immobilized protein gradients in three-dimensional porous scaffolds; WO 2006 / 042287, Method for Producing Biomaterial Scaffolds; WO 2007 / 016524, Method for Stepwise Deposition of Silk Fibroin Coatings;WO 2008 / 085904, Biodegradable Electronic Devices; WO 2008 / 118133, Silk Microspheres for Encapsulation & Controlled Release; WO 2008 / 108838, Microfluidic Devices & Methods for Fabricating Same, WO 2008 / 127404, Nanopatterned Biopolymer Device & Method of Manufacturing Same; WO 2008 / 118211, Biopolymer Photonic Crystals & Method of Manufacturing Same; WO 2008 / 127402, Biopolymer Sensor & Method of Manufacturing Same; WO 2008 / 127403, Biopolymer Optofluidic Device & Method of Manufacturing the Same; WO 2008 / 127401, Biopolymer Optical Wave Guide & Method of Manufacturing Same; WO 2008 / 140562, Biopolymer Sensor & Method of Manufacturing Same; WO 2008 / 127405, Microfluidic Device with Cylindrical Microchannel & Method for Fabricating Same; WO 2008 / 106485, Tissue-Engineered Silk Organs; WO 2008 / 140562, Electroactive Biopolymer Optical & Electro-Optical Devices & Method of Manufacturing Same; WO 2008 / 150861, Method for Silk Fibroin Gelation Using Sonication;WO 2007 / 103442, Biocompatible Scaffolds & Adipose-Derived Stem Cells; WO 2009 / 155397, Edible Holographic Silk Products; WO 2009 / 100280, 3-Dimensional Silk Hydroxyapatite Compositions; WO 2009 / 061823, Fabrication of Silk Fibroin Photonic Structures by Nanocontact Imprinting; see WO 2009 / 126689, System & Method for Making Biomaterial Structures. ;

[0072] In an alternative embodiment, the silk fibroin matrix may contain plasmonic nanoparticles that form photothermal elements. This approach takes advantage of the excellent doping performance of silk fibroin. Hyperthermia is known to aid in the delivery of various drugs. See Park et al., Effect of Heat on Skin Permeability 359 Intl. J. Pharm. 94 (2008). In one embodiment, a short heat release in a very limited area may be used to maximize permeability while minimizing harmful effects on the surrounding tissue. Thus, a silk fibroin matrix doped with plasmonic particles can provide specificity to hyperthermia by concentrating light and generating heat locally only through the silk fibroin matrix. In some embodiments, the silk fibroin matrix may contain photothermal agents, such as gold nanoparticles.

[0073] In some embodiments, the silk fibroin matrix used in the methods described herein may include an amphipathic peptide. In other embodiments, the silk fibroin matrix used in the methods described herein may be free of an amphipathic peptide. An "amphipathic peptide" has both hydrophilic and hydrophobic properties. An amphipathic molecule can interact with a biological membrane, usually by inserting its hydrophobic portion into a lipid membrane while exposing its hydrophilic portion to an aqueous environment. In some embodiments, the amphipathic peptide may include an RGD motif. An example of an amphipathic peptide has the amino acid sequence: and 23RGD peptide having TIFF2025071260000002.tif13128. Other examples of amphipathic peptides include those disclosed in U.S. Patent Application No. US 2011 / 0008406.

[0074] Injectable Compositions Comprising Silk Fibroin Matrices - Patent application In another aspect, provided herein is an injectable composition for use in repairing or augmenting tissue in a subject, comprising a compressed silk fibroin matrix, wherein the compressed silk fibroin matrix expands upon injection into tissue and retains its original expanded volume (e.g., at least about 50% or more) within the tissue to be repaired or augmented for a period of time (e.g., at least about 2 weeks, at least about 4 weeks, at least about 6 weeks or more).

[0075] As used herein, the term "injectable composition" generally refers to a composition that can be delivered or administered to tissues through a minimally invasive procedure. The term "minimally invasive procedure" refers to a procedure that is performed by entering the subject's body through the skin or through a body cavity or anatomical opening with as little damage as possible (e.g., small incision, injection). In some embodiments, the injectable composition can be administered or delivered to tissues by injection. In some embodiments, the injectable composition can be delivered to tissues through making a small incision on the skin and inserting a needle, cannula and / or tube, such as a catheter, therein. Without wishing to be limited, the injectable composition can be administered or placed in tissues by surgery, such as implantation.

[0076] In some embodiments, the injectable composition may comprise at least one active agent described herein.

[0077] In some embodiments, the injectable composition may include at least one cell. The term "cell" as used herein refers to any cell of a prokaryotic or eukaryotic organism, including plants, yeast, worms, insects, and mammals. In some embodiments, the cell may be a mammalian cell. Mammalian cells include, but are not limited to, cells from primates, humans, and any animal of interest, including, but not limited to, mice, rats, hamsters, rabbits, dogs, cats, livestock animals, such as horses, cows, murines, sheep, canines, felines, etc. The cells may be from a wide range of tissue types, including, but not limited to, hematopoietic cells, neuronal cells, mesenchymal cells, skin cells, mucosal cells, stromal cells, muscle cells, splenocytes, reticuloendothelial cells, epidermal cells, endothelial cells, liver cells, kidney cells, gastrointestinal cells, lung cells, T cells, etc. Also included are stem cells, embryonic stem (ES) cells, ES-derived cells and stem cell precursors, including, but not limited to, hematopoietic stem cells, neural stem cells, stromal stem cells, muscle stem cells, cardiovascular stem cells, hepatic stem cells, pulmonary stem cells and gastrointestinal stem cells, as well as adipose-derived stem cells. In one embodiment, the cells are adipose-derived stem cells. In some embodiments, the cells can be ex vivo or cultured cells, e.g., in vitro. For example, in the case of ex vivo cells, the cells can be obtained from healthy and / or diseased subjects. The cells can be obtained by, as a non-limiting example, biopsy or other surgical means known to those skilled in the art. In some embodiments, fat cells can be collected from a subject by conventional fat suction or aspiration techniques. In such embodiments, the cells can be derived from lipoaspirate. In other embodiments, the cells can be derived from bone marrow aspirate. Depending on the type of tissue to be repaired or augmented, the cells can be derived from any biological fluid or concentrate, e.g., lipoaspirate or bone marrow lipoaspirate. In some embodiments, the injectable composition or silk fibroin matrix can be delivered directly with a biological fluid or concentrate, such as aspirated lipoprotein or aspirated bone marrow.

[0078] The cells can be obtained from a donor (allogeneic) or a recipient (autologous). The cells can also be established cell culture lines or even genetically engineered cells. In addition, the cells can be harvested from a number of hosts, including but not limited to human autologous tissues, transgenic mammals, or bacterial cultures (potentially used for probiotic treatments). In certain embodiments, the injectable composition and / or silk fibroin matrix can include human stem cells, such as mesenchymal stem cells, induced pluripotent stem cells (iPSCs), synovium-derived stem cells, embryonic stem cells, adult stem cells, umbilical cord blood cells, umbilical cord Wharton's jelly cells, bone cells, fibroblasts, neural cells, lipocytes, adipocytes, bone marrow cells, sorted immune cells, adipose tissue-derived precursor cells, bone marrow-derived progenitor cells, peripheral blood progenitor cells, stem cells isolated from adult tissues and genetically transformed cells or combinations of the above cells; or differentiated cells, such as muscle cells, adipocytes.

[0079] Stem cells can be obtained from bone marrow, adipose tissue or other sources in the body with minimally invasive procedures, are highly expandable in culture, and can be easily induced to differentiate into adipose tissue forming cells after exposure to well-established adipogenesis inducing supplements. The cells can be added to the injectable compositions and / or silk fibroin matrices described herein and cultured in vitro for a period of time before administration to an area of ​​the body, or added to the injectable compositions and / or silk fibroin matrices described herein and administered to an area of ​​the body. The cells can be seeded onto the silk fibroin matrix for a short period of time (less than one day) immediately prior to administration, or can be cultured for a longer period (more than one day) to allow the cells to proliferate and synthesize extracellular matrix within the seeded matrix prior to administration.

[0080] When utilized as a source of stem cells, adipose tissue can be obtained by any method known to those skilled in the art. For example, adipose tissue can be removed from an individual by suction-assisted lipoplasty, ultrasound-assisted lipoplasty, and excisional lipectomy. In addition, these procedures can include combinations of such procedures. Suction-assisted lipoplasty can be desirable for removing adipose tissue from an individual because it provides a minimally invasive method of tissue retrieval and reduces the potential for stem cell damage that can be associated with other techniques, such as ultrasound-assisted lipoplasty. Adipose tissue should be retrieved in a manner that ensures the viability of cellular components and minimizes the potential for contamination of the tissue with potential infectious organisms, such as bacteria and / or viruses.

[0081] In some embodiments, preparation of cell populations may require depletion of the adipocyte component containing mature fat of the adipose tissue. This is typically accomplished by a series of washing and disaggregation steps, first rinsing the tissue to reduce the presence of free fat (released from ruptured adipocytes) and peripheral blood elements (released from cut blood vessels during tissue collection), and then disaggregating it to separate intact adipocytes and other cell populations from the connective tissue matrix. Disaggregation may be accomplished using any conventional technique or method, including mechanical force (mincing or shearing force), enzymatic digestion with single or combined proteolytic enzymes, such as collagenase, trypsin, lipase, liberase HI and pepsin, or a combination of mechanical and enzymatic methods. For example, the cellular components of intact tissue fragments may be disaggregated by methods using collagenase-mediated disaggregation of adipose tissue, as well as methods for recovering microvascular endothelial cells in adipose tissue, as known to those skilled in the art. Additional methods using collagenase that may be used are also known to those skilled in the art. Additionally, the methods may use a combination of enzymes, such as a combination of collagenase and trypsin, or a combination of an enzyme, such as trypsin, and mechanical dissociation.

[0082] A cell population (processed lipoaspirate) can then be obtained from the disaggregated tissue fragments by reducing the presence of mature adipocytes. Cell separation can be achieved by buoyant density sedimentation, centrifugation, elutriation, differential adhesion to and elution from solid phase moieties, antibody-mediated selection, charge differences; immunomagnetic beads, fluorescence activated cell sorting (FACS) or other means.

[0083] After disaggregation, the active cell population can be washed / rinsed to remove additives and / or by-products of the disaggregation process (e.g., collagenase and newly released free fat).The active cell population can then be concentrated by centrifugation.In one embodiment, cells are concentrated and collagenase is removed by passing the cell population through a continuous flow rotating membrane system or the like, such as the system disclosed in U.S. Patent Nos. 5,034,135; and 5,234,608, which are incorporated herein by reference.

[0084] In addition to the above, there are many post-wash methods that can be applied to further purify the cell population. These include both positive selection (selection of target cells) or negative selection (selective removal of unwanted cells) or a combination thereof. In another embodiment, the cell pellet can be resuspended, layered over (or under) a fluid material forming a continuous or discontinuous density gradient, and centrifuged for separation of cell populations based on cell density. In a similar embodiment, continuous flow approaches, such as apheresis and elutriation (with or without counterflow) can be used. Adhesion to plastic followed by short-term cell expansion is also applied to bone marrow-derived adult stem cell populations. This approach uses culture conditions that preferentially expand one population while maintaining (reduced by dilution with the selected cells growing thereby) or disappearing due to the absence of the necessary growth conditions. The enriched, cultured and / or expanded cells can be incorporated into the silk fibroin matrices and / or injectable compositions described herein.

[0085] In one embodiment, stem cells are harvested, the harvested cells are contacted with adipogenic medium for a sufficient time to induce differentiation into adipocytes, and the adipocytes are loaded into a biocompatible matrix to be implanted. In a further embodiment, at least a portion of the stem cells can be differentiated into adipocytes, such that initially there is a mixture of both cell types, which over time changes to essentially only adipocytes, with undetectable amounts of stem cells. Adipose tissue is produced in vivo by stem cells or prepared ex vivo by stem cells.

[0086] Depending on the type of tissue to be repaired or augmented, many different cell types or combinations thereof can be used in the injectable composition. These cell types include, but are not limited to, smooth muscle cells, skeletal muscle cells, cardiac muscle cells, epidermal cells, endothelial cells, urinary tract epithelial cells, fibroblasts, myoblasts, chondrocytes, chondroblasts, osteoblasts, osteoclasts, keratinocytes, hepatocytes, bile duct cells, pancreatic islet cells, thyroid, parathyroid, adrenal, hypothalamus, pituitary, ovary, testis, salivary gland cells, adipocytes and progenitor cells. By way of example only, smooth muscle cells and endothelial cells can be used when the injectable composition is used to repair or augment muscle and / or vascular tissue, such as vascular, esophageal, intestinal, rectal or urinary tissue; chondrocytes can be included in the injectable composition for cartilage tissue; fibroblasts can be included in the injectable composition intended to replace and / or reinforce any wide variety of tissue types (e.g., skin) that contain extracellular matrix, such as collagen; adipocytes can be included in the injectable composition intended to repair or augment any wide variety of adipose tissue. In general, any cell found in a natural tissue can be included in the injectable composition used for the corresponding tissue. In addition, precursor cells, such as myoblasts or stem cells, can be included to generate their corresponding differentiated cell types.

[0087] In some embodiments, the injectable composition may further comprise a pharma- ceutically acceptable carrier. As used herein, the term "pharma- ceutically acceptable carrier" refers to a pharma- ceutically acceptable material, composition, or vehicle for administration of the silk fibroin matrix and, optionally, an active agent. A pharma- ceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, and isotonic and absorption delaying agents that are compatible with the activity of the silk fibroin matrix and, if present, the active agent, and are physiologically acceptable to the subject. Pharmaceutical preparations suitable for injection include sterile aqueous solutions or dispersions. The carrier may be, for example, a solvent or dispersion medium, including water, cell culture medium, a buffer (e.g., phosphate buffered saline), a polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), or a suitable mixture thereof. In some embodiments, the pharmaceutical carrier may be a buffer solution (e.g., PBS).

[0088] In addition, various additives that enhance the stability, sterility and isotonicity of the injectable composition may be added, including antimicrobial preservatives, antioxidants, chelating agents and buffers. Suppression of microbial activity may be brought about by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. In many cases, it may be desirable to include isotonic agents, such as sugars, sodium chloride, and the like. Injectable compositions may also contain auxiliary substances, such as wetting or emulsifying agents, pH buffers, gelling or viscosity enhancing additives, preservatives, dyes, and the like, depending on the desired preparation. Basic texts, such as "REMINGTON'S PHARMACEUTICAL SCIENCE", 17th Edition, 1985, which is incorporated herein by reference, may be of assistance in preparing suitable preparations without undue experimentation.

[0089] The viscosity of the injectable composition can be maintained at a selected level by using a pharma- ceutically acceptable thickening agent. In one embodiment, methylcellulose is used because it is easily and inexpensively available and easy to handle. Other suitable thickening agents include, for example, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, carbomer, etc. The preferred concentration of the thickening agent can depend on the selected drug and the desired injection viscosity. What is important is the use of an amount that can achieve the selected viscosity, for example, the addition of such a thickening agent to some embodiments of the injectable composition.

[0090] Typically, any additive (in addition to the silk fibroin matrix and / or additional active agent described herein) may be present in an amount of 0.001 to 50 wt% by dry weight or in a buffer solution. In some embodiments, the active agent may be present in the order of micrograms to milligrams to grams, e.g., about 0.0001 to about 5 wt%, about 0.0001 to about 1 wt%, about 0.0001 to about 0.05 wt%, or about 0.0001 to about 20 wt%, about 0.01 to about 10 wt%, and about 0.05 to about 5 wt%. When administering any pharmaceutical composition to a subject in need thereof, it is preferable to determine toxicity, e.g., by determining the lethal dose (LD) and LD50 in an appropriate animal model, e.g., rodents, e.g., mice or rats; as well as the dose of the composition, the concentration of its components, and the timing of administration of the composition that induces an appropriate response. Such determinations do not require undue experimentation within the knowledge of one of ordinary skill in the art.

[0091] Activator In some embodiments, the injectable composition and / or silk fibroin matrix described herein may further comprise at least one active agent. The active agent may be mixed, dispersed or suspended in the injectable composition and / or it may be distributed or embedded in the silk fibroin matrix. In some embodiments, the active agent may be distributed, embedded or encapsulated in the silk fibroin matrix. In some embodiments, the active agent may be coated on the surface of the silk fibroin matrix. In some embodiments, the active agent may be mixed with the silk fibroin matrix to form an injectable composition. The term "active agent" may also include a combination or mixture of two or more active agents as described below. Examples of active agents include, but are not limited to, biologically active agents (e.g., therapeutic agents), cosmetically active agents (e.g., anti-aging agents), cell adhesives (e.g., integrin binding molecules), and any combination thereof.

[0092] The term "biologically active agent" as used herein refers to any molecule that exerts at least one biological effect in vivo. For example, a biologically active agent can be a therapeutic agent for treating or preventing a disease state or condition of a subject. Examples of biologically active agents include, but are not limited to, peptides, peptidomimetics, aptamers, antibodies or portions thereof, antibody-like molecules, nucleic acids (DNA, RNA, siRNA, shRNA), polysaccharides, enzymes, receptor antagonists or agonists, hormones, growth factors, autologous bone marrow, antibiotics, antimicrobial agents, small molecules, and therapeutic agents. Biologically active agents can also include, but are not limited to, anti-inflammatory agents, anesthetic agents, active agents that stimulate tissue formation and / or healing and regrowth of natural tissue, and any combination thereof.

[0093] Anti-inflammatory agents may include, but are not limited to, naproxen, sulindac, tolmetin, ketorolac, celecoxib, ibuprofen, diclofenac, acetylsalicylic acid, nabumetone, etodolac, indomethacin, piroxicam, cox-2 inhibitors, ketoprofen, antiplatelet agents, salsalate, valdecoxib, oxaprozin, diflunisal, flurbiprofen, corticosteroids, MMP inhibitors, and leukotriene modifiers or combinations thereof.

[0094] Agents that enhance the formation of new tissue at the injection site and / or stimulate healing or regrowth of native tissue may include, but are not limited to, fibroblast growth factors (FGFs), transforming growth factor beta (TGF-β), platelet-derived growth factor (PDGF), epidermal growth factor (EGF), connective tissue activating peptide (CTAP), osteogenic factors including bone morphogenetic proteins, heparin, angiotensin II (A-II) and fragments thereof, insulin-like growth factors, tumor necrosis factors, interleukins, colony stimulating factors, erythropoietin, nerve growth factor, interferons, biologically active analogs, fragments and derivatives of such growth factors, and any combination thereof.

[0095] Anesthetic agents may include, but are not limited to, those used in coccygeal, epidural, inhalation, injection, retrobulbar and spinal applications, such as bupivacaine, lidocaine, benzocaine, cetacaine, ropivacaine and tetracaine, or combinations thereof.

[0096] In some embodiments, active agent can be cosmetic active agent.The term "cosmetic active agent" refers to compounds that have cosmetic or therapeutic effects on skin, hair or nail, such as anti-aging agents, anti-free radical agents, lightening agents, whitening agents, bleaching agents, darkening agents, such as self-tanning agents, coloring agents, anti-acne agents, shine control agents, antibacterial agents, anti-inflammatory agents, antifungal agents, antiparasitic agents, external analgesics, sunscreen agents, photoprotective agents, antioxidants, keratolytic agents, surface / surface active agents, moisturizing agents, nutrients, vitamins, energy supplements, antiperspirants, astringents, deodorants, depilatories, stabilizers, anti-callous agents, muscle relaxants, hair, nail and / or skin conditioning agents, and any combination thereof.

[0097] In one embodiment, the cosmetically active agent may be selected from the group consisting of, but is not limited to, hydroxy acids, benzoyl peroxide, sulfur resorcinol, ascorbic acid, D-panthenol, hydroquinone, octyl methoxycinnamate, titanium dioxide, octyl salicylate, homosalate, avobenzone, polyphenols, carotenoids, free radical scavengers, ceramides, polyunsaturated fatty acids, essential fatty acids, enzymes, enzyme inhibitors, minerals, hormones such as estrogen, steroids such as hydrocortisone, 2-dimethylaminoethanol, copper salts such as copper chloride, coenzyme Q10, lipoic acid, amino acids such as proline and tyrosine, vitamins, lactobionic acid, acetyl coenzyme A, niacin, riboflavin, thiamine, ribose, electron carriers such as NADH and FADH2, and other plant extracts such as aloe vera, feverfew and soybean, and derivatives and mixtures thereof. Examples of vitamins include, but are not limited to, vitamin A, vitamin B (eg, vitamin B3, vitamin B5, and vitamin B12), vitamin C, vitamin K, and vitamin E, and derivatives thereof.

[0098] In one embodiment, the cosmetic active agent can be an antioxidant.Examples of antioxidants include, but are not limited to, water-soluble antioxidants, such as sulfhydryl compounds and their derivatives (e.g., sodium metabisulfite and N-acetyl-cysteine), lipoic acid and dihydrolipoic acid, resveratrol, lactoferrin, ascorbic acid and ascorbic acid derivatives (e.g., ascorbyl palmitate and ascorbyl polypeptides).Oil-soluble antioxidants suitable for use in the compositions described herein can include, but are not limited to, butylated hydroxytoluene, tocopherols (e.g., tocopheryl acetate), tocotrienols and ubiquinones.Natural extracts containing antioxidants suitable for use in the injectable compositions described herein can include, but are not limited to, extracts containing flavonoids and isoflavonoids and their derivatives (e.g., genistein and diadzein), and extracts containing resveratrol.Examples of such natural extracts include grape seed, green tea, pine bark and propolis. Other examples of antioxidants can be found in the ICI Handbook, pages 1612-13.

[0099] In some embodiments, the active agent may be a cell adhesive. Examples of cell adhesives include, but are not limited to, hyaluronic acid, collagen, cross-linked hyaluronic acid / collagen, integrin binding molecules, chitosan, elastin, fibronectin, vitronectin, laminin, proteoglycan, any derivative thereof, any peptide or oligosaccharide variant thereof, and any combination thereof. As used herein, the term "oligosaccharide" refers to a compound that contains at least two or more sugars selected from the group consisting of glucose, fructose, galactose, xylose, and any combination thereof. In one embodiment, the oligosaccharide may be selected from the group consisting of fructooligosaccharide, galactooligosaccharide, lactosucrose, isomaltulose, glycosylsucrose, isomaltooligosaccharide, gentiooligosaccharide, xylooligosaccharide, and any combination thereof. As used herein, the term "oligosaccharide" includes disaccharides.

[0100] In some embodiments, the injectable composition and / or silk fibroin matrix may further comprise at least one additional material for soft tissue augmentation, such as, but not limited to, dermal filler materials including poly(methyl methacrylate) microspheres, hydroxylapatite, poly(L-lactic acid), collagen, gelatin, elastin and glycosaminoglycans, hyaluronic acid, commercially available dermal filler products such as BOTOX® (Allergan), DYSPORT®, COSMODERM®, EVOLENCE®, RADIESSE®, RESTYLANE®, JUVEDERM® (Allergan), SCULPTRA®, PERLANE® and CAPTIQUE®, and any combination thereof.

[0101] In some embodiments, the injectable composition and / or silk fibroin matrix may include metal nanoparticles (e.g., but not limited to, gold nanoparticles), optical molecules (e.g., but not limited to, fluorescent molecules and / or quantum dots), and any other imaging agents known in the art, e.g., for biomedical imaging.

[0102] In various embodiments, the injectable composition may be stored or transported in a dry or hydrated state.

[0103] When an active agent is embedded in a silk fibroin matrix, the bioactivity of the active agent (e.g., at least about 30% of the bioactivity of the active agent) may be stabilized for a period of time (e.g., days, weeks, or months) under certain conditions. Such conditions may include, but are not limited to, cycling through changing conditions (e.g., freeze-thaw cycles), temperature (e.g., above 0°C), air pressure, humidity, and light exposure. See U.S. Patent Application No. 61 / 477,737. Some embodiments of the injectable composition may be stored or shipped at between about 0°C and about 60°C, about 10°C and about 60°C, or about 15°C and about 60°C. In these embodiments, the injectable composition may be stored or shipped at room temperature during which the bioactivity of the active agent (e.g., at least about 30% of the bioactivity of the active agent) may be stabilized for a period of time, e.g., at least about 3 weeks or more.

[0104] Application of the injectable compositions and silk fibroin matrices described herein The injectable compositions described herein may be used in a variety of medical applications, including, but not limited to, tissue space fillers, tissue reconstruction or regeneration templates, cell scaffolds in tissue engineering applications, or drug delivery vehicles / carriers. The silk fibroin matrices described herein injected into tissues that one wishes to repair or augment may act as scaffolds to mimic the extracellular matrix (ECM) in the body and / or promote tissue regeneration. The scaffolds may function as both physical supports and / or adhesive templates for cells to grow within. In some embodiments, the silk fibroin matrices may be cell-free. Additionally, the silk fibroin matrices may be coated with cell adhesives, such as collagen, and / or chemoattractants, such as growth factors, that may attract host cells to the silk fibroin matrix and support cell growth. In some embodiments, the silk fibroin matrices may be seeded with cells prior to administration to the target tissues one wishes to repair or augment.

[0105] In some embodiments, the present invention provides an injectable composition that can be used to fill, increase volume, and / or regenerate tissues that require it.The injectable composition can generally be used for filling or increasing volume of tissues, augmenting, replacing, cosmetically enhancing, and / or repairing soft tissues in subjects.In addition, the injectable composition can be used to fill any tissue void or depression, either naturally formed (e.g., aging) or formed by surgical procedures for removing tissues (e.g., skin cysts or solid tumors), corticosteroid treatment, immunological reactions that cause lipodystrophy, traumatic injury, or therapeutic treatments (e.g., radiation therapy or chemotherapy).The injectable composition can also be used to raise depressed scars.

[0106] In certain embodiments, the injectable composition may be used for augmentation of soft tissue. As used herein, the term "augment" or "augmentation" refers to the increase, filling, restoration, strengthening or replacement of tissue. In some embodiments, the tissue may lose its elasticity, hardness, shape and / or volume. In some embodiments, the tissue may be partially or completely lost (e.g., tissue removal) or damaged. In these embodiments, the term "augment" or "augmentation" may also refer to the relief, reduction or alleviation of at least one symptom or defect in the tissue (e.g., without limitation, loss of elasticity, hardness, shape and / or volume in the tissue; presence of voids or indentations in the tissue; loss of function in the tissue) by injecting at least one injectable composition described herein into the tissue. In such embodiments, at least one symptom or defect in the tissue may be relieved, reduced or alleviated by at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80% or more compared to no treatment. In some embodiments, at least one symptom or defect in tissue can be reduced, decreased or alleviated by at least about 90%, at least about 95%, at least about 97% or more compared to no treatment. In some embodiments, at least one symptom or defect in tissue can be reduced, decreased or alleviated by 100% compared to no treatment (no defect or defect becomes undetectable to those skilled in the art). In other embodiments, the tissue can be used to prevent or delay the onset of symptoms of defects in tissue, such as loss of elasticity, firmness, shape and / or volume of tissue or signs of wrinkles. As used herein, the phrase "soft tissue augmentation" is generally used to refer to the alteration of the structure of soft tissue, including but not limited to augmentation, filling, restoration, strengthening or replacement of tissue, for example, to improve the cosmetic or aesthetic appearance of soft tissue.For example, breast augmentation (also known as breast enlargement, mammoplasty enlargement, or augmentation mammoplasty) alters the size and shape of a woman's breasts to improve her cosmetic or aesthetic appearance. Examples of soft tissue augmentation include, but are not limited to, dermal tissue augmentation, especially of the face and neck, filling of lines, folds, wrinkles, small facial hollows and cleft lip; correction of small deformities due to aging or disease, including those of the hands and feet, fingers and toes; vocal cord or glottal augmentation for vocal rehabilitation; dermal filling of sleep lines and expression lines; replacement of dermal and subcutaneous tissue loss due to aging; lip augmentation; filling of the bovine foot and orbital sulcus around the eyes; breast augmentation; chin augmentation; cheek and / or nose augmentation; periurethral support, bulking agents to fill soft tissue, dermal or subcutaneous hollows, such as those caused by excessive fat absorption or other trauma; filling of acne or traumatic scars; filling of nasolabial folds, nasoglabellar lines and intraoral lines. In some embodiments, the injectable compositions and / or silk fibroin matrices described herein may be used to treat facial lipoatrophy. In some embodiments, the injectable compositions may be used for breast augmentation and / or reconstruction.

[0107] In some embodiments, the injectable composition may be used for soft tissue repair. The term "repair" or "repairing" as used herein with respect to tissue refers to any correction, reinforcement, repair, treatment, regeneration, filling of tissue that restores the volume, shape and / or function of the tissue. In some embodiments, "repair" includes complete repair and partial repair. For example, the volume, shape and / or function of the tissue to be repaired may be restored at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80% or more compared to the case without treatment. In some embodiments, the volume, shape and / or function of the tissue to be repaired may be restored at least about 90%, at least about 95%, at least about 97% or more compared to the case without treatment. In some embodiments, the volume, shape and / or function of the tissue to be repaired may be restored 100% compared to the case without treatment (no defect or defect becomes undetectable to the skilled artisan). In various embodiments, the injectable composition may be used to repair any soft tissue as described above, such as breast, skin and any soft tissue suitable for soft tissue augmentation. In some embodiments, the terms "repair" or "repairing" are used interchangeably with the terms "regeneration" or "regenerating," which are used herein to refer to the treatment of tissue.

[0108] In some embodiments, the injectable composition may be used for soft tissue reconstruction. As used herein, the phrase "soft tissue reconstruction" refers to reconstructing soft tissue structures that have been severely damaged or lost, for example, by major accidents or surgical removal. For example, breast reconstruction is the reconstruction of the breast, usually in women. The conventional method of constructing a natural-looking breast generally uses autologous tissue or prostheses. In some embodiments, such breast reconstruction may include the re-construction of a natural-looking areola and nipple, and such procedures may use implants or transfer flaps of the patient's own tissue. In some embodiments, administration of the injectable composition and / or silk fibroin matrix to the soft tissue area to be reconstructed may maintain the shape and / or size of the reconstructed soft tissue structure for a period of time, for example, at least 6 weeks, at least about 2 months, at least about 3 months or more.

[0109] Without wishing to be limited, some embodiments of the injectable composition may be used for hard tissue (eg, musculoskeletal) augmentation or repair, such as augmentation or repair of bone, cartilage and ligaments.

[0110] The injectable composition and silk fibroin matrix described herein can also be used to fill tissue at or near a prosthetic implant, such as, but not limited to, a conventional breast implant or knee replacement implant. In some embodiments, the injectable composition and silk fibroin matrix can be used to create an interface between the prosthetic implant and tissue, such as to fill any gaps between the prosthetic implant and tissue and / or to prevent tissue from directly contacting the prosthetic implant. By way of example only, the injectable composition described herein can be introduced into or near a prosthetic implant (e.g., breast implant) after placement in a subject to fill any gaps between the implant and tissue (e.g., breast tissue) and / or to "sculpt" the tissue to a more natural appearance.

[0111] In any of the uses described herein, the silk fibroin matrix may be combined with cells for the purpose of biologically enhancing repair. The cells may be harvested from a number of hosts, including but not limited to human autologous tissue or transgenic animals. More specifically, the human cells used may include cells selected from stem cells (e.g., adipose-derived stem cells), bone cells, fibroblasts, adipocytes, sorted immune cells, cells from lipoaspirate, or any combination thereof. In some embodiments, the cells may be added after rinsing the silk fibroin matrix itself. They may be blended into the silk fibroin matrix, carrier solution, or a mixture of silk fibroin matrix and carrier solution prior to injection.

[0112] In some embodiments, administration of cells (e.g., stem cells or lipoaspirate) together with the silk fibroin matrix or injectable composition described herein may enhance or accelerate host integration and / or tissue formation over time. The cells may be mixed with the silk fibroin matrix or injectable composition described herein, or they may be administered before, at the same time, or after the silk fibroin matrix or injectable composition is introduced to the target site. Without wishing to be bound by theory, the cells may secrete pro-angiogenic and / or growth factors at the target site. As tissue regenerates or remodels to fill voids or repair defects, the silk fibroin matrix may degrade accordingly. In some embodiments, the silk fibroin matrix may be integrated into the regenerated host tissue.

[0113] In addition, active agents, such as therapeutic agents, medicines, or specific growth factors, added to the silk fibroin matrix for the purpose of improving the results can be introduced at any of a variety of points in the silk fibroin matrix manufacturing process or a combination thereof. In some embodiments, these elements can be added to the silk fibroin solution or accelerant phase before drying and solidification, they can be immersed in the silk fibroin matrix during the accelerant rinsing process, or they can be coated on the bulk silk fibroin after rinsing. In some embodiments, small silk fibroin matrices used for tissue repair or augmentation can be cut out from the large silk fibroin matrix, and then the active agent can be introduced into the small silk fibroin matrix. For example, the active agent can be immersed in the silk fibroin matrix, coated on the silk fibroin matrix, or introduced into the carrier liquid before or after blending with the silk fibroin matrix.

[0114] In some aspects, the injectable composition and silk fibroin matrix described herein can be used as tissue void filler.In one embodiment, tissue void filler is dermal filler.Dermal filler can be used to improve the appearance or condition of skin, including but not limited to rehydrate skin, provide high elasticity to skin, reduce roughness of skin, make skin firmer, reduce or eliminate stretch lines or stretch marks, give skin better tone, luster, brightness and / or radiance, reduce or eliminate wrinkles of skin, provide wrinkle resistance to skin and replace loss of soft tissue.

[0115] Dermal fillers containing silk fibroin matrices can be tailored for their hardness and opacity through alteration of silk fibroin concentration and formulation method. In one embodiment, the dermal filler can be manufactured by forming silk fibroin matrices (or foams) from, for example, about 1% (w / v) to about 6% (w / v) silk fibroin solutions so that they can be compressed and injected into tissues through needles or cannulae. The needles or cannulae can have an outer diameter of 4 mm or less, 3 mm or less, 2 mm or less, 1 mm or less, 0.8 mm or less, 0.6 mm or less, 0.4 mm or less, 0.2 mm or less, or 0.1 mm or less. In some embodiments, the gauge of the needle or cannulae can range from 10-34, 11-34, 12-32, or 13-30. In some embodiments, the size of the needle or cannulae can be determined to achieve a suitable extrusion force of at least 40 N. Depending on the size of the silk fibroin matrix to be injected, in some embodiments, the size of the needle or cannula can be determined to achieve an appropriate extrusion force of less than 40 N (the nominal deliverable injection force by the human hand).

[0116] Therefore, another aspect provided herein relates to a method for improving the condition and / or appearance of the skin of a subject in need thereof. Non-limiting examples of the condition and / or appearance of the skin include dehydration, lack of skin elasticity, roughness, lack of skin firmness, stretch lines and / or marks, pale skin, and wrinkled skin. The method includes injecting the injectable composition described herein into the dermis area of ​​the subject, where the injection improves the condition and / or appearance of the skin. For example, improving the appearance of the skin includes, but is not limited to, rehydrating the skin, providing high elasticity to the skin, reducing roughness of the skin, making the skin firmer, reducing or eliminating stretch lines or marks, providing better tone, luster, brightness, and / or radiance to the skin to reduce pale skin, reducing or eliminating wrinkles in the skin, and providing wrinkle-resistant properties to the skin.

[0117] As used herein, the term "dermal region" refers to the region of skin that includes the epidermal-dermal junction and dermis, including the superficial dermis (papillary region) and deep dermis (reticular region). Skin is composed of three main layers: the epidermis, which provides waterproofing and acts as a barrier against infection; the dermis, which serves as a site for skin appendages; and the subcutaneous tissue (subcutaneous fat layer). The epidermis does not contain blood vessels and is nourished by diffusion from the dermis. The major cell types that form the epidermis include, but are not limited to, keratinocytes, melanocytes, Langerhans cells, and Merkel cells.

[0118] The dermis is the subepidermal skin layer composed of connective tissue that cushions the body against stress and strain. The dermis is tightly bound to the epidermis by the basement membrane. It also possesses many mechanoreceptors / nerve endings that provide the senses of touch and heat. It contains hair follicles, sweat glands, sebaceous glands, apocrine glands, lymphatic vessels and blood vessels. The blood vessels of the dermis provide nutrition and waste removal to its own cells and the basal layer of the epidermis. The dermis is structurally divided into two regions: a superficial region adjacent to the epidermis called the papillary region and a deeper, thicker region known as the reticular region.

[0119] The papillary region is composed of loose connective tissue. Its name comes from its finger-like projections called papillae that extend towards the epidermis. The papillae provide the dermis with a "rough" surface that interlocks with the epidermis, strengthening the bond between the two skin layers. The reticular region is located deep to the papillary region and is usually much thicker. It is composed of dense irregular connective tissue and gets its name from the high concentration of collagenous, elastic and reticular fibers that weave it together. These protein fibers give the dermis its strength, extensibility and elastic properties. The reticular region is also home to hair roots, sebaceous glands, sweat glands, receptors, nails and blood vessels. Stretch marks caused by pregnancy are also located in the dermis.

[0120] The hypodermis is not part of the skin, but lies below the dermis. Its purpose is to attach the skin to the underlying bones and muscles, as well as to supply it with blood vessels and nerves. It is made up of loose connective tissue and elastin. The main cell types are fibroblasts, macrophages, and adipocytes (the hypodermis contains 50% of body fat). Fat acts as padding and insulation for the body.

[0121] In one embodiment, provided herein is a method of treating skin dehydration, comprising injecting an injectable composition described herein into an area of ​​the dermis affected by skin dehydration, e.g., the composition comprises a silk fibroin matrix and optionally a carrier and / or an active agent, wherein injection of the composition rehydrates the skin, thereby treating skin dehydration.

[0122] In another embodiment, a method for treating a lack of skin elasticity comprises injecting an injectable composition described herein into an area of ​​the dermis affected by the lack of skin elasticity, e.g., the composition comprises a plurality of silk fibroin matrices and optionally a carrier and / or an active agent, where injection of the composition increases skin elasticity, thereby treating the lack of skin elasticity.

[0123] In yet another embodiment, a method of treating rough skin comprises injecting an injectable composition described herein into an area of ​​the dermis affected by rough skin, e.g., the composition comprises a silk fibroin matrix and optionally a carrier and / or an active agent, where injection of the composition reduces the roughness of the skin, thereby treating the rough skin.

[0124] In yet another embodiment, a method of treating lack of skin firmness comprises injecting an injectable composition described herein into an area of ​​the dermis affected by lack of skin firmness, e.g., the composition comprises a silk fibroin matrix described herein and optionally a carrier and / or an active agent, where injection of the composition causes the skin to become more firm, thereby treating the lack of skin firmness.

[0125] In a further embodiment, a method of treating stretch lines or stretch marks on the skin comprises injecting an injectable composition described herein into an area of ​​the dermis affected by stretch lines or stretch marks on the skin, e.g., the composition comprises a silk fibroin matrix described herein and optionally a carrier and / or an active agent, wherein injection of the composition reduces or eliminates the stretch lines or stretch marks on the skin, thereby treating the stretch lines or stretch marks on the skin.

[0126] In another embodiment, a method of treating skin wrinkles comprises injecting an injectable composition described herein into an area of ​​the dermis affected by skin wrinkles, e.g., the composition comprises a silk fibroin matrix and optionally a carrier and / or an active agent, where injection of the composition reduces or eliminates skin wrinkles, thereby treating skin wrinkles.

[0127] In yet another embodiment, a method of treating, preventing or delaying the formation of skin wrinkles comprises injecting an injectable composition described herein into an area of ​​the dermis that is prone to wrinkles or shows signs of wrinkles, e.g., the composition comprises a silk fibroin matrix and optionally a carrier and / or an active agent, where injection of the composition renders the skin resistant to skin wrinkles, thereby treating, preventing or delaying the formation of skin wrinkles.

[0128] The effective amount / size and administration schedule of the silk fibroin matrix to be injected into the dermal region can be determined by those skilled in the art, taking into consideration various factors, including, but not limited to, the type of skin condition, the location of the skin condition, the cause of the skin condition, the severity of the skin condition, the degree of relief desired, the duration of relief desired, the particular silk fibroin matrix formulation used, the degradation or volume retention rate of the particular silk fibroin matrix formulation used, the pharmacodynamics of the particular silk fibroin matrix formulation used, the nature of other compounds contained in the particular silk fibroin matrix formulation used, the individual characteristics, medical history and risk factors of the individual, such as age, weight, general health and any combination thereof. In some embodiments, the silk fibroin matrix can be injected into the dermal region every 3 months, every 6 months, every 9 months, every year, every 2 years or more.

[0129] In another aspect, the injectable composition may be used as a dermal filler for dermal augmentation to reconstruct or augment soft tissue body parts, such as lips, breasts, breast parts such as nipples, muscle or any other soft body parts where fat and / or connective tissue is used to provide shape, shielding or other biological functions. In the fillers used in these applications, the concentration of silk fibroin and / or the amount of carrier (e.g., saline) added to the silk fibroin matrix mixture may be adjusted to take into account the relevant constraints of the biological environment. For example, silk fibroin matrices for breast augmentation may be tailored in terms of matrix hardness and volume retention through the alteration of silk fibroin concentration and processing method. For example, a silk fibroin concentration of about 1% (w / v) to about 10% (w / v) may be used to fabricate silk fibroin matrices, optionally including an active agent, such as adipocytes, e.g., adipose-derived stem cells or cells derived from liposuction. The carrier content may be on the order of 0% to 25% (v / v) in the case of saline. Other factors such as the type of defect, the size of the defect and the required injection depth of the particular filler material should also be considered.

[0130] Although not wishing to be limited, although injection is minimally invasive, other administration methods, such as implants, may also be used when required, for example to repair or augment large defect areas. For example, for injection into the dermis or lip augmentation, a syringe needle of size 26g-30g may be used. In applications using large amounts of filler, such as breast reconstruction or augmentation, larger matrix sizes and larger caliber needles or smaller needle gauges, such as 23g-27g, may be used to administer the filler. In some embodiments, surgery, such as implants, may also be used to administer large amounts of filler and / or to reach certain tissue depths.

[0131] Thus, in a further aspect, the present disclosure relates to a method for reconstructing, repairing or enhancing soft tissue, comprising administering an injectable composition as described herein to a soft tissue region of an individual in need thereof; the composition comprises a silk fibroin matrix as described herein and optionally an active agent and / or a carrier. The method of administration of the injectable composition as described herein can be determined by those skilled in the art. In some embodiments, the method of administration can be injection. In some embodiments, the method of administration can be surgery, such as implantation.

[0132] While the injectable compositions and / or silk fibroin matrices described herein can be applied directly to the target area (e.g., by injection or surgery), in some embodiments, the injectable compositions and / or silk fibroin matrices disclosed herein can also be used to fill an expandable implantable medical device, such as an expandable breast implant shell, that is placed in the defect area. In such embodiments, provided herein is a method of soft tissue reconstruction, repair, or augmentation, comprising placing an implantable medical device at a desired location in an area of ​​soft tissue of an individual; and expanding the device by filling the device with the silk fibroin matrix and / or injectable composition described herein, wherein the expansion of the medical device by filling the silk fibroin matrix and / or injectable composition described herein can reconstruct or augment the soft tissue.

[0133] The silk fibroin matrices or injectable compositions disclosed herein may also be used in conjunction with interventional radiology embolization procedures to block abnormal blood vessels (arteries) (e.g., to stop bleeding) or organs (e.g., embolization of the spleen in cases of hypersplenism to stop excessive function), including uterine artery embolization for percutaneous treatment of uterine fibroids. Tuning of the stiffness and volume retention of the silk fibroin matrix may be achieved through alteration of silk fibroin concentration and processing methods, as described above.

[0134] The silk fibroin matrix or injectable composition disclosed herein may be used to repair spinal voids, for example, formed by spinal disc nucleotomy surgery, to help maintain the normal distance between adjacent vertebral bodies. In some embodiments, a disc filler comprising a plurality of silk fibroin matrices may be used to repair voids present in the spine, for example, between vertebral bodies, and / or in a disrupted spinal disc. In such embodiments, a silk fibroin concentration of about 1% (w / v) to about 10% (w / v) may be used to fabricate the silk fibroin matrix described herein. Accelerators and / or activators may also be mixed into the silk fibroin matrix and / or injectable composition before, during, or after injection into the site of interest.

[0135] The silk fibroin matrix or injectable composition disclosed herein can be used to fill the vitreous cavity to support the structure of the eye and maintain the position of the retina. The viscosity of the injectable composition described herein can be adjusted by one skilled in the art to the viscosity of the vitreous humor in the eye.

[0136] In some embodiments, the silk fibroin matrix and / or injectable composition can be used as a template for tissue reconstruction or augmentation, such as soft tissue reconstruction or augmentation (e.g., breast augmentation), and even for ossicular or cartilage defects, such as fractures. Administration of the silk fibroin matrix or injectable composition described herein can be used to promote ingrowth and proliferation of chondrocytes and support collagen matrix accumulation to improve cartilage / bone repair. In another aspect, donor chondrocytes are seeded or mixed into the silk fibroin matrix and / or injectable composition described herein prior to administration to expand the cell population and promote cartilage tissue development. In some embodiments, specific growth factors that support cartilage or bone tissue formation, such as TGF-β or bone morphogenetic protein (BMP), respectively, can be added to the silk fibroin matrix.

[0137] In another embodiment, the silk fibroin matrix and / or injectable composition described herein can be used for facial plastic surgery, such as nose reconstruction. The above-mentioned reconstructive strategies for repairing cartilage / bone defects can also be applicable to facial plastic surgery.

[0138] In some embodiments, the silk fibroin matrix and / or injectable composition described herein can be used as a scaffold to support cell growth in tissue engineering.For example, the silk fibroin matrix and / or injectable composition described herein can be administered to an incision or wound site to promote wound healing or wound revealing.The method generally includes administering the injectable composition or silk fibroin matrix described herein to the wound or incision site, and allowing the wound or incision to heal while the silk fibroin matrix erodes or is absorbed in the body and replaces it with the individual's own living tissue.The method may further include seeding the silk fibroin matrix or mixing it with the injectable composition with live cell material, either from the individual or from a donor, before or during administration.

[0139] In another aspect, the injectable composition comprising the silk fibroin matrix can be used directly or indirectly in a method for repairing, augmenting or reconstructing a tissue of a subject, for example, augmenting or reconstructing a human breast. In some embodiments, the injectable composition or silk fibroin matrix can be placed directly into the tissue (e.g., breast tissue) that is desired to be repaired or augmented, for example, by injection. The injectable composition or silk fibroin matrix can be injected into the tissue (e.g., breast tissue) every six months, every year, every two years, every three years, or at longer intervals. In other embodiments, the injectable composition or silk fibroin matrix can be used to enhance the support of a conventional tissue implant, for example, by enhancing the support of the lower strut position of the breast implant. In alternative embodiments, the method can include administering the injectable composition and / or silk fibroin matrix generally near or proximal to the tissue implant, for example, a conventional breast implant, and seeding the injectable composition and / or silk fibroin matrix with live cell material before or during administration. In yet another embodiment, the injectable composition and / or silk fibroin matrix can be used to partially or completely cover a tissue implant (e.g., a breast implant) to provide a superior interface with the host's cells and to reduce the possibility of malposition or capsular contracture.

[0140] In some embodiments, the silk fibroin matrix and / or injectable composition described herein can be used as a filler to promote or support adipogenesis, for example to treat lipodystrophy of the face.In such embodiments, the injectable composition and / or silk fibroin matrix can be seeded or mixed with adipose-related cells, such as adipose-derived stem cells, or lipoaspirate, before or at the same time as injection into the lipodystrophy-affected target area of ​​the subject's face.In some embodiments, the silk fibroin matrix can be injected every 3 months, every 6 months, every 9 months, every year, or every 2 years, or longer, to maintain treatment.

[0141] In yet another embodiment, the silk fibroin matrix and / or injectable composition described herein may be used as a scaffold for cells useful for peripheral nerve repair. The silk fibroin matrix may be delivered (e.g., through injection) to the nerve defect site with or without additional devices to aid in connection with the nerve endings. For such purposes, certain growth factors that support nerve regeneration, such as nerve growth factor (NGF), may be added to the injectable composition and / or mixed into the silk fibroin matrix prior to or during administration. In such embodiments, soft silk fibroin matrices may be used, for example, using silk fibroin concentrations of about 0.5 (w / v) to about 3% (w / v). Depending on the brain microenvironment, stiffer silk fibroin matrices may also be used. The silk fibroin matrix and / or injectable composition may be injected with or loaded with appropriate therapeutic factors according to the methods described above.

[0142] Any of the cells described herein can be seeded on the surface of the silk fibroin matrix described herein. For example, the silk fibroin matrix can be immersed in a growth medium suitable for the cells of interest and then directly exposed to the cells. The cells grow on the surface and interstices of the silk fibroin matrix. The silk fibroin matrix is ​​then removed from the growth medium, washed if necessary, and administered. Alternatively, the cells can be placed in a suitable buffer or liquid growth medium and wicked through the silk fibroin matrix by using vacuum filtration. The cells can also be mixed with the silk fibroin solution prior to forming the silk fibroin matrix, and at least some of the cells can be trapped within the silk fibroin matrix. In another embodiment, the cells of interest can be dispersed in a suitable solution (e.g., growth medium or buffer) and then sprayed onto the silk fibroin matrix. For example, electrospraying involves subjecting a cell-containing solution having a suitable viscosity and concentration to an electric field sufficient to obtain a spray of small charged droplets of the cell-containing solution.

[0143] In some embodiments, silk fibroin matrices or injectable compositions containing at least one active agent can be used as a platform for drug delivery. For example, silk fibroin matrices can be formed with agents either incorporated in the matrix or bound to the matrix, and then administered (e.g., injected, implanted, or even orally) into the body. In some embodiments, active agents can be mixed with silk fibroin matrices and / or injectable compositions, and then administered (e.g., injected, implanted, or even orally) into the body. For extended or sustained release, silk fibroin matrices can be engineered to adjust their beta-sheet content, for example, for their volume retention and / or degradation rate. To further control the drug release profile, silk fibroin matrices containing pharma- ceutically active drugs can be mixed with additional silk fibroin gel phases that act as carriers, either with or without viscosity-inducing components, surfactants, and / or included lubricant fluids such as saline. Silk fibroin matrices with therapeutic agents bound to them can also be crosslinked to further enhance their stability and extend the release period. In an alternative approach, the silk fibroin matrix can be mixed with other polymers, such as hyaluronic acid, to prolong the release of certain growth factors or cytokines and stabilize their function. Furthermore, the silk fibroin matrix and / or the injectable composition can also be used to coat a coaxial drug delivery system, for example by spraying.

[0144] As used herein, the term "sustained release" refers to the release of a pharma- ceutically active drug over a period of about 7 days or more. In aspects of this embodiment, the drug delivery platform comprising the silk fibroin matrix and / or the injectable composition releases the pharma- ceutically active drug over a period of, for example, at least about 7 days after administration, at least about 15 days after administration, at least about 30 days after administration, at least about 45 days after administration, at least about 60 days after administration, at least about 75 days after administration, or at least about 90 days after administration.

[0145] As used herein, the term "extended release" refers to the release of a pharma- ceutically active drug for a period of less than about 7 days.In such an embodiment, the drug delivery platform comprising the silk fibroin matrix and / or injectable composition described herein can release a pharma-ceutically active drug for a period of, for example, about 1 day after administration, about 2 days after administration, about 3 days after administration, about 4 days after administration, about 5 days after administration, or about 6 days after administration.

[0146] Depending on the silk fibroin matrix formulation and processing method and the associated application, the injectable composition or silk fibroin matrix can be administered (e.g., by injection) periodically, for example, every 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, yearly, every 2 years or more. In some embodiments, the injectable composition or silk fibroin matrix can be administered once to the tissue to be repaired or augmented, and the tissue can regenerate over time to replace the silk fibroin matrix.

[0147] In some embodiments of any of the applications described herein, the injectable composition or silk fibroin matrix may be at least partially dry when administered to the tissue to be repaired or augmented, in some embodiments, the injectable composition or silk fibroin matrix may be dry (e.g., in the absence of a carrier) when administered to the tissue to be repaired or augmented.

[0148] In some embodiments of any application described herein, the injectable composition or silk fibroin matrix may be in an at least partially hydrated state when administered to the tissue to be repaired or augmented. In some embodiments, the injectable composition or silk fibroin matrix may be in a hydrated state (e.g., in the presence of a carrier, such as a buffer solution and / or lipoaspirate) when administered to the tissue to be repaired or augmented.

[0149] In some embodiments of any of the applications described herein, the injectable composition or silk fibroin matrix may be injected subcutaneously, submuscularly, or intramuscularly.

[0150] In some embodiments, the methods and / or compositions described herein may be used in the dermis region. In some embodiments, the methods and / or compositions described herein may be used in the epidermis layer, the dermis layer, the subcutaneous tissue layer, or any combination thereof.

[0151] DELIVERY DEVICES AND KITS COMPRISING SILK FIBROIN MATRIX - Patent application Also provided herein is a delivery device comprising the injectable composition or silk fibroin matrix described herein.The delivery device can be any conventional delivery device used for injection purposes, such as a syringe, or a custom-made delivery device, such as an injection gun.Therefore, a further aspect provided herein is an injection device comprising the injectable composition or silk fibroin matrix.

[0152] In some embodiments, the delivery device may further comprise a tubular structure for introducing the silk fibroin matrix into the tissue to be repaired or augmented. In some embodiments, the tubular structure may be tapered. For example, the tapered tubular structure may comprise a cone-shaped internal space. Examples of tubular structures may include, but are not limited to, needles, cannulas, catheters, any injection applicators known in the art, and any combination thereof.

[0153] In some embodiments, the delivery device may further comprise a mechanical element (eg, an elongated structure, such as a ramrod) that facilitates ejection through the tubular structure of the compressed silk fibroin matrix.

[0154] In various embodiments, the delivery device may include a carrier for injection, such as a buffer solution.

[0155] In various embodiments, the delivery device (eg, a syringe) can contain an anesthetic agent.

[0156] Further provided herein is a kit including one embodiment of the injectable composition or silk fibroin matrix packaged in a delivery applicator, such as a catheter, needle, or cannula. In some embodiments, a local anesthetic may be blended with the injectable composition or silk fibroin matrix. In alternative embodiments, the local anesthetic may be packaged in a separate container. For example, it may be desirable to apply a local anesthetic to the target tissue to be treated prior to further treatment. Exemplary anesthetics include, but are not limited to, lidocaine. Depending on the application, the kit may include a syringe with a size of 0.5 mL to 60 mL, with applications requiring larger volumes (e.g., bone filler, disc filler) being provided with a larger size syringe. Additionally, the gauge of the needle may be adjusted according to the injection site, with a needle of 10 g to 30 g being the acceptable range. For example, a needle of 10 g to 20 g may be used for intradermal injection.

[0157] In some embodiments, the kit may further comprise a plurality of delivery devices preloaded with the injectable compositions or silk fibroin matrices described herein, each of which may be individually packaged.

[0158] In some embodiments, the kit may further comprise a container containing a buffer solution or a carrier for injection.

[0159] In some embodiments, the kit may further include at least one additional empty syringe. In some embodiments, the kit may further include at least one additional needle. In some embodiments, the kit may further include at least one catheter or cannula.

[0160] Embodiments of the various aspects described herein can be illustrated by the following numbered items: 1. A method for repairing or augmenting tissue in a subject, comprising placing a composition comprising a compressed silk fibroin matrix into the tissue to be repaired or augmented, wherein the compressed silk fibroin matrix expands upon placement in the tissue and retains at least about 1% of its original expanded volume within the tissue for at least about two weeks. 2. The method of claim 1, wherein the compressed silk fibroin matrix expands in volume by at least about two times the volume of the compressed silk fibroin matrix. 3. The method of item 1 or 2, wherein the silk fibroin matrix retains at least about 50% of its initial expanded volume within the tissue for at least about 2 weeks, at least about 6 weeks, or at least about 3 months. 4. The method according to any of items 1 to 3, wherein the silk fibroin matrix retains at least about 50% of its initial expanded volume within the tissue for at least about 6 months. 5. The method according to any of items 1 to 4, wherein the silk fibroin matrix retains at least about 60% of its initial expanded volume within the tissue for at least about 6 weeks. 6. The method of item 5, wherein the silk fibroin matrix retains at least about 70% of its initial expanded volume within the tissue for at least about 6 weeks. 7. The method of item 6, wherein the silk fibroin matrix retains at least about 80% of its initial expanded volume within the tissue for at least about 6 weeks. 8. The method according to any of items 1 to 7, wherein the silk fibroin matrix retains at least about 70% of its initial expanded volume within the tissue for at least 3 months. 9. The method of any of items 1 to 8, wherein the silk fibroin matrix is ​​adapted to degrade to no more than 50% of its initial swollen volume in at least about 6 weeks. 10. The method of item 9, wherein the silk fibroin matrix is ​​adapted to degrade to no more than 50% of its initial expanded volume in at least about 3 months. 11. The method of any of items 1 to 10, wherein the silk fibroin matrix is ​​adapted to degrade to no more than 30% of its initial swollen volume in at least about 6 weeks. 12. The method of item 11, wherein the silk fibroin matrix is ​​adapted to degrade to no more than 10% of its initial expanded volume in at least about 6 weeks. 13. The method of any of items 1 to 12, wherein the silk fibroin matrix is ​​adapted to degrade to no more than 30% of its initial expanded volume in at least about 3 months. 14. The method according to any one of items 1 to 13, wherein the silk fibroin matrix is ​​porous. 15. The method of item 14, wherein the porous silk fibroin matrix has a porosity of at least about 1%, at least about 5%, at least about 10%, at least about 15%, or at least about 30%. 16. The method of item 15, wherein the porous silk fibroin matrix has a porosity of at least about 50%. 17. The method of item 16, wherein the porous silk fibroin matrix has a porosity of at least about 70%. 18. The method according to any one of items 14 to 17, wherein the pores have a size of about 1 μm to about 1500 μm. 19. The method of item 18, wherein the pores have a size of about 50 μm to about 650 μm. 20. The method according to any one of items 1 to 19, wherein the silk fibroin matrix is ​​formed from about 0.1% w / v to about 30% w / v silk fibroin solution. 21. The method of item 20, wherein the silk fibroin solution is about 0.5% w / v to about 10% w / v. 22. The method of item 21, wherein the silk fibroin solution is about 1% w / v to about 6% w / v. 23. The method according to any one of items 1 to 22, wherein the silk fibroin matrix is ​​freezer-treated. 24. The method according to any one of items 1 to 23, wherein the silk fibroin matrix is ​​a silk fibroin foam. 25. The method according to any of items 1 to 24, wherein the composition or silk fibroin matrix further comprises at least one active agent. 26. The method of item 25, wherein the at least one active agent is a biologically active agent, a cosmetically active agent, a cell adhesive agent, or any combination thereof. 27. The method of item 26, wherein the biologically active agent is selected from the group consisting of therapeutic agents, anesthetic agents, cell growth factors, peptides, peptidomimetics, antibodies or portions thereof, antibody-like molecules, nucleic acids, polysaccharides, and any combination thereof. 28. The method of item 26, wherein the cell adhesive is selected from the group consisting of hyaluronic acid, collagen, cross-linked hyaluronic acid / collagen, integrin binding molecules, chitosan, elastin, fibronectin, vitronectin, laminin, proteoglycans, any derivative thereof, any peptide or oligosaccharide variant thereof, and any combination thereof. 29. The method according to item 26, wherein the cosmetically active agent is selected from the group consisting of anti-aging agents, anti-free radical agents, antioxidants, hydrating agents, whitening agents, coloring agents, bleaching agents, sunscreens, muscle relaxants, and any combination thereof. 30. The method of any one of items 1 to 29, wherein the composition further comprises cells. 31. The method of item 30, wherein the cell is a stem cell. 32. The method of any one of items 1 to 31, wherein the composition further comprises a biological fluid or concentrate. 33. The method of item 32, wherein the biological fluid or concentrate is lipoaspirate, bone marrow aspirate, or any combination thereof. 34. The method of any one of items 1 to 33, wherein the composition or silk fibroin matrix further comprises a hydrogel. 35. The method of any of items 1 to 34, wherein the composition or silk fibroin matrix further comprises a dermal filling material. 36. The method of item 35, wherein the dermal filling material is selected from the group consisting of poly(methyl methacrylate) microspheres, hydroxylapatite, poly(L-lactic acid), hyaluronic acid, collagen, gelatin, and any combination thereof. 37. The method of any one of items 1 to 36, wherein the composition further comprises a carrier. 38. The method according to any one of items 1 to 37, wherein the silk fibroin matrix does not contain amphiphilic peptides. 39. The method of claim 38, wherein the amphipathic peptide comprises an RGD motif. 40. The method according to any one of items 1 to 39, wherein the placement of the compressed silk fibroin matrix is ​​performed by injection. 41. The method according to item 40, wherein the injection is performed subcutaneously, submuscularly or intramuscularly. 42. The method according to any one of items 1 to 41, wherein the tissue is a soft tissue. 43. The method of item 42, wherein the soft tissue is selected from the group consisting of tendons, ligaments, skin, breast tissue, fibrous tissue, connective tissue, muscle, and any combination thereof. 44. The method according to item 43, wherein the soft tissue is skin. 45. The method of item 44, wherein the soft tissue is breast tissue. 46. ​​The method according to any one of items 1 to 45, wherein the subject is a mammalian subject. 47. The method of item 46, wherein the mammalian subject is a human. 48. The method of any of items 1 to 47, wherein the silk fibroin matrix is ​​compressed by loading the silk fibroin matrix into an interior space of a delivery applicator, wherein the interior space has a volume smaller than the volume of the silk fibroin matrix in an uncompressed state. 49. The method of item 48, wherein the delivery applicator comprises a needle, a cannula, a catheter, or any combination thereof. 50. An injectable composition for use in repairing or augmenting tissue in a subject, comprising a compressed silk fibroin matrix, wherein the compressed silk fibroin matrix expands upon injection into the tissue and retains at least about 1% of its initial expanded volume within the tissue for at least about two weeks. 51. The composition described in item 50, wherein the compressed silk fibroin matrix expands in volume by at least about two times the volume of the compressed silk fibroin matrix. 52. The composition according to item 50 or 51, wherein the compressed silk fibroin matrix does not contain an amphiphilic peptide. 53. The composition described in item 52, wherein the amphipathic peptide contains an RGD motif. 54. The composition according to any of items 50 to 53, wherein the silk fibroin matrix retains at least about 50% of its initial expanded volume within the tissue for at least about 2 weeks, at least about 6 weeks, or at least about 3 months. 55. The composition according to any of items 50 to 54, wherein the silk fibroin matrix retains at least about 50% of its initial expanded volume within the tissue for at least about 6 months. 56. The composition according to any of items 50 to 55, wherein the silk fibroin matrix retains at least about 60% of its initial expanded volume within the tissue for at least about 6 weeks. 57. The composition described in item 56, wherein the silk fibroin matrix retains at least about 70% of its initial expanded volume within the tissue for at least about 6 weeks. 58. The composition described in item 57, wherein the silk fibroin matrix retains at least about 80% of its initial expanded volume within the tissue for at least about 6 weeks. 59. The composition according to any of items 50 to 58, wherein the silk fibroin matrix retains at least about 70% of its initial expanded volume within the tissue for at least about 3 months. 60. The composition according to any of items 50 to 59, wherein the silk fibroin matrix is ​​adapted to degrade to no more than 50% of its initial expanded volume in at least about 6 weeks. 61. The composition according to item 60, wherein the silk fibroin matrix is ​​adapted to degrade to no more than 50% of its initial expanded volume in at least about 3 months. 62. The composition according to any of items 50 to 62, wherein the silk fibroin matrix is ​​adapted to degrade to no more than 30% of its initial expanded volume in at least about 6 weeks. 63. The composition of item 62, wherein the silk fibroin matrix is ​​adapted to degrade to no more than 10% of its initial expanded volume in at least about 6 weeks. 64. The composition according to any of items 50 to 63, wherein the silk fibroin matrix is ​​adapted to degrade to no more than 30% of its initial expanded volume in at least about 3 months. 65. The composition according to any of items 50 to 64, wherein the silk fibroin matrix is ​​porous. 66. The composition described in item 65, wherein the porous silk fibroin matrix has a porosity of at least about 1%, at least about 5%, at least about 10%, at least about 15%, or at least about 30%. 67. The composition described in item 66, wherein the porous silk fibroin matrix has a porosity of at least about 50%. 68. The composition described in item 67, wherein the porous silk fibroin matrix has a porosity of at least about 70%. 69. The composition according to any of items 65 to 68, wherein the pores have a size of about 1 μm to about 1500 μm. 70. The composition according to item 69, wherein the pores have a size of about 50 μm to about 650 μm. 71. The composition according to any of items 50 to 70, wherein the silk fibroin matrix is ​​formed from about 0.1% w / v to about 30% w / v silk fibroin solution. 72. The composition according to item 71, wherein the silk fibroin solution is about 0.5% w / v to about 10% w / v. 73. The composition according to item 72, wherein the silk fibroin solution is about 1% w / v to about 6% w / v. 74. The composition according to any of items 50 to 73, wherein the silk fibroin matrix is ​​freezer-treated. 75. The composition according to any of items 50 to 74, wherein the silk fibroin matrix is ​​a silk fibroin foam. 76. The composition according to any of items 50 to 75, wherein the injectable composition or the silk fibroin matrix further comprises at least one active agent. 77. The composition according to item 76, wherein at least one active agent is a biologically active agent, a cosmetically active agent, a cell adhesive agent, or any combination thereof. 78. The composition according to item 77, wherein the biologically active agent is selected from the group consisting of therapeutic agents, anesthetic agents, cell growth factors, peptides, peptidomimetics, antibodies or portions thereof, antibody-like molecules, nucleic acids, polysaccharides, and any combination thereof. 79. The composition according to item 77, wherein the cell adhesive is selected from the group consisting of hyaluronic acid, collagen, cross-linked hyaluronic acid / collagen, integrin binding molecules, chitosan, elastin, fibronectin, vitronectin, laminin, proteoglycan, any derivative thereof, any peptide or oligosaccharide variant thereof, and any combination thereof. 80. The composition according to item 77, wherein the cosmetically active agent is selected from the group consisting of anti-aging agents, anti-free radical agents, antioxidants, hydrating agents, whitening agents, colorants, bleaching agents, sunscreens, muscle relaxants, and any combination thereof. 81. The composition according to any one of items 50 to 80, further comprising cells. 82. The composition of item 81, wherein the cell is a stem cell. 83. The composition according to any of items 50 to 82, further comprising a biological fluid or concentrate. 84. The composition according to item 83, wherein the biological fluid or concentrate is lipoaspirate, bone marrow aspiration, or any combination thereof. 85. The composition according to any of items 50 to 84, wherein the injectable composition or silk fibroin matrix further comprises a hydrogel. 86. The composition according to any of items 50 to 85, wherein the injectable composition or silk fibroin matrix further comprises a dermal filling material. 87. The composition according to item 86, wherein the dermal filling material is selected from the group consisting of poly(methyl methacrylate) microspheres, hydroxylapatite, poly(L-lactic acid), hyaluronic acid, collagen, gelatin, and any combination thereof. 88. The composition according to any one of items 50 to 87, further comprising a carrier. 89. The composition according to any of items 50 to 88, wherein the compressed silk fibroin matrix has a volume of about 10% to about 90% of its original volume before compression. 90. The composition according to any of items 50 to 88, wherein the compressed silk fibroin matrix has a volume of 70% or less of its original volume before compression. 91. A delivery device comprising an injectable composition according to any one of items 50 to 90. 92. The delivery device according to item 91, further comprising a tubular structure for introducing the injectable composition into the tissue to be repaired or augmented. 93. The delivery device according to item 92, wherein the tubular structure is tapered. 94. The delivery device according to item 93, wherein the tapered tubular structure comprises a conical interior space. 95. A delivery device described in any of items 91 to 94, wherein the tubular structure is a needle, a cannula, a catheter, or any combination thereof. 96. A delivery device described in any of items 91 to 95, further comprising a mechanical element that facilitates ejection of the compressed silk fibroin matrix through the tubular structure. 97. A delivery device according to any one of items 91 to 96, further comprising an injectable carrier.

[0161] Definitions of Some Selected Terms As used herein, "subject" means a human or an animal. Usually, an animal is a vertebrate, such as a primate, a rodent, a livestock animal, or a game animal. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, such as rhesus monkeys. Rodents include mice, rats, woodchucks, ferrets, rabbits, and hamsters. Livestock and game animals include cattle, horses, pigs, deer, bison, buffalo, feline species, such as domestic cats, canine species, such as dogs, foxes, wolves, bird species, such as chickens, emus, ostriches, and fish, such as trout, catfish, and salmon. In certain embodiments of the aspects described herein, the subject is a mammal, such as a primate, such as a human. The subject can be male or female. Preferably, the subject is a mammal. The mammal can be, but is not limited to, a human, a non-human primate, a mouse, a rat, a dog, a cat, a horse, or a cow. Advantageously, non-human mammals can be used as animal model subjects for tissue repair, regeneration and / or reconstruction. In addition, the methods and compositions described herein can be used to treat domesticated animals and / or pets.

[0162] The term "statistically significant" or "significant" refers to statistical significance, generally meaning two standard deviations (2SD) above or below a reference level. The term refers to statistical evidence that a difference exists. It is defined as the probability that a decision is made to reject the null hypothesis when the null hypothesis is in fact true. This decision is often made using a p-value.

[0163] As used herein, the terms "protein" and "peptide" are used interchangeably herein to refer to a series of amino acid residues connected to another by a peptide bond between the alpha amino and carboxy groups of adjacent residues. The terms "protein" and "peptide" are used interchangeably herein to refer to a polymer of protein amino acids, including modified amino acids (e.g., phosphorylated, glycosylated, etc.) and amino acid analogs, regardless of their size or function. Although "protein" is often used to refer to relatively large polypeptides and "peptide" is often used to refer to small polypeptides, the usage of these terms in the art overlaps and varies. The term "peptide" as used herein refers to peptides, polypeptides, proteins and fragments of proteins, unless otherwise indicated. The terms "protein" and "peptide" are used interchangeably herein when referring to gene products and fragments thereof. Thus, exemplary peptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, fragments and analogs of the above.

[0164] The term "nucleic acid" as used herein refers to polynucleotides, such as deoxyribonucleic acid (DNA) and, where appropriate, ribonucleic acid (RNA), polymers thereof in either single-stranded or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, each nucleic acid sequence implicitly encompasses the sequence explicitly indicated, as well as conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer, et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka, et al., J. Biol. Chem. 260:2605-2608 (1985), and Rossolini, et al., Mol. Cell. Probes 8:91-98 (1994)). The term "nucleic acid" should also be understood to include, as equivalents, derivatives, variants and analogs of either RNA or DNA generated from nucleotide analogs, as well as single-stranded (sense or antisense) and double-stranded polynucleotides.

[0165] The term "short interfering RNA" (siRNA), also referred to herein as "small interfering RNA," is defined as an agent that functions to inhibit expression of a target gene, for example, by RNAi. siRNAs can be chemically synthesized, produced by in vitro transcription, or produced in a host cell. siRNA molecules can also be generated by cleavage of double-stranded RNA, one strand of which is identical to the message one wishes to inactivate. The term "siRNA" refers to small inhibitory RNA duplexes that induce the RNA interference (RNAi) pathway. These molecules can be of various lengths (generally 18-30 base pairs) and contain various degrees of complementarity to their target mRNA in their antisense strand. Some, but not all, siRNAs have unpaired overhanging bases at the 5' or 3' end of the sense and / or antisense strands. The term "siRNA" includes duplexes of two separate strands and single strands that can form hairpin structures containing duplex regions.

[0166] The term "shRNA" as used herein refers to short hairpin RNA, which functions as RNAi and / or siRNA species, but differs in that shRNA species is a double-stranded hairpin-like structure with enhanced stability.The term "RNAi" as used herein refers to interfering RNA, or RNA interference molecule is a nucleic acid molecule or its analog, such as an RNA-based molecule that inhibits gene expression.RNAi represents a means of selective post-transcriptional gene silencing.RNAi can destroy specific mRNA or suppress the processing or translation of RNA, such as mRNA.

[0167] The term "enzyme" as used herein refers to a protein molecule that catalyzes a chemical reaction of other substances and is not destroyed or substantially altered upon completion of the reaction. The term may include naturally occurring enzymes and bioengineered enzymes or mixtures thereof. Examples of enzyme families include kinases, dehydrogenases, oxidoreductases, GTPases, carboxyltransferases, acyltransferases, decarboxylases, transaminases, racemases, methyltransferases, formyltransferases, and α-ketodecarboxylases.

[0168] As used herein, the term "aptamer" refers to a single-stranded, partially single-stranded, partially double-stranded or double-stranded nucleotide sequence capable of specifically recognizing a selected non-oligonucleotide molecule or group of molecules. In some embodiments, an aptamer recognizes a non-oligonucleotide molecule or group of molecules by a mechanism other than Watson-Crick base pairing or triplex formation. Aptamers can include defined sequence segments and sequences, including, but not limited to, nucleotides, ribonucleotides, deoxyribonucleotides, nucleotide analogs, modified nucleotides and nucleotides containing backbone modifications, branch points and non-nucleotide residues, groups or bridges. Methods for selecting aptamers that bind to molecules are widely known in the art and readily accessible to those skilled in the art.

[0169] As used herein, the term "antibody" or "antibodies" refers to intact immunoglobulins or monoclonal or polyclonal antigen-binding fragments having an Fc (fragment crystallizable) region or an FcRn-binding fragment of the Fc region. The term "antibody" also includes "antibody-like molecules", such as fragments of antibodies, such as antigen-binding fragments. Antigen-binding fragments can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. "Antigen-binding fragments" include, among others, Fab, Fab', F(ab')2, Fv, dAb and complementarity determining region (CDR) fragments, single chain antibodies (scFv), single domain antibodies, chimeric antibodies, diabodies, and polypeptides that contain at least a portion of an immunoglobulin sufficient to confer specific antigen-binding properties to the polypeptide. For purposes described herein, linear antibodies are also included. The terms Fab, Fc, pFc', F(ab')2 and Fv are used in their standard immunological sense (Klein, Immunology (John Wiley, New York, NY, 1982); Clark, WR (1986) The Experimental Foundations of Modern Immunology (Wiley & Sons, Inc., New York); and Roitt, I. (1991) Essential Immunology, 7th Ed., (Blackwell Scientific Publications, Oxford)). Antibodies or antigen-binding fragments specific for various antigens are commercially available from sources such as R&D Systems, BD Biosciences, e-Biosciences and Miltenyi, or they can be raised against these cell surface markers by methods known to those of skill in the art.

[0170] As used herein, the term "complementarity determining region" (CDR; i.e., CDR1, CDR2 and CDR3) refers to the amino acid residues of the variable domain of an antibody that must be present for binding to an antigen. Each variable domain typically has three CDR regions identified as CDR1, CDR2 and CDR3. Each complementarity determining region may be selected from the amino acid residues in the "complementarity determining region" defined by Kabat (i.e., approximately 24-34 (L1), 50-56 (L2), and 89-97 (L3) residues in the light chain variable domain and approximately 31-35 (H1), 50-65 (H2), and 95-102 (H3) residues in the heavy chain variable domain; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)) and / or the residues in the "hypervariable loops" (i.e., approximately 26-32 (L1), 50-52 (L2), and 91-96 (L3) residues in the light chain variable domain and approximately 26-32 (H1), 53-55 (H2), and 96-101 (H3) residues in the heavy chain variable domain; Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). In some instances, the complementarity determining regions may include amino acids of both the CDR regions and the hypervariable loops as defined according to Kabat.

[0171] The term "linear antibodies" refers to antibodies as described in Zapata et al., Protein Eng., 8(10):1057-1062 (1995). Briefly, these antibodies comprise a pair of tandem Fd segments (VH-CH1-VH-CH1) that form a pair of antigen-binding regions with complementary light chain polypeptides. Linear antibodies can be bispecific or monospecific.

[0172] As used herein, the term "single-chain Fv" or "scFv" antibody fragment is intended to mean an antibody fragment comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Preferably, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding (Pluckthun, The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer- Verlag, New York, pp. 269-315 (1994)).

[0173] As used herein, the term "diabody" refers to a small antibody fragment that has two antigen-binding sites and comprises a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains in the same chain, these domains are forced to pair with complementary domains in another chain to generate two antigen-binding sites (EP 404,097; WO 93 / 11161; Hollinger et al., Proc. Natl. Acad. Sd. USA, P0:6444-6448 (1993)).

[0174] As used herein, the term "small molecule" refers to naturally occurring or synthetic molecules, including, but not limited to, peptides, peptidomimetics, amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, aptamers, nucleotides, nucleotide analogs, organic or inorganic compounds having a molecular weight of less than about 10,000 grams / mole (i.e., including heteroorganic compounds and organometallic compounds), organic or inorganic compounds having a molecular weight of less than about 5,000 grams / mole, organic or inorganic compounds having a molecular weight of less than about 1,000 grams / mole, organic or inorganic compounds having a molecular weight of less than about 500 grams / mole, as well as salts, esters and other pharma- ceutically acceptable forms of such compounds.

[0175] The term "antibiotic" is used herein to refer to a compound or composition that reduces the viability or inhibits the growth or replication of a microorganism. As used in this disclosure, antibiotics are further intended to include antibacterial, bacteriostatic or bactericidal agents. Exemplary antibiotics include, but are not limited to, penicillins, cephalosporins, penems, carbapenems, monobactams, aminoglycosides, sulfonamides, macrolides, tetracyclines, lincosides, quinolones, chloramphenicol, vancomycin, metronidazole, rifampin, isoniazid, spectinomycin, trimethoprim and sulfamethoxazole.

[0176] The term "therapeutic agent" is known in the art and refers to any chemical moiety that is a biologically, physiologically, or pharmacologically active substance that acts locally or systemically in a subject. Examples of therapeutic agents, also referred to as "drugs", are described in well-known references, such as the Merck Index, the Physicians Desk Reference, and The Pharmacological Basis of Therapeutics, and include, but are not limited to, medicines; vitamins; mineral supplements; substances used to treat, prevent, diagnose, heal, or alleviate diseases or illnesses; substances that affect the structure or function of the body; or prodrugs that become biologically active or more active after being placed in a physiological environment. Various forms of therapeutic agents that can be released from the compositions of the present invention into adjacent tissues or fluids when administered to a subject can be used. Examples include steroids and esters of steroids (e.g., estrogen, progesterone, testosterone, androsterone, cholesterol, norethindrone, digoxigenin, cholic acid, deoxycholic acid, and chenodeoxycholic acid), boron-containing compounds (e.g., carboranes), chemotherapeutic nucleotides, drugs (e.g., antibiotics, antivirals, and antifungals), enediynes (e.g., calicheamicin, esperamicin, dynemicin, neocarzinostatin chromophore, and kedarcidin chromophore), heavy metal complexes (e.g., cisplatin, These include, but are not limited to, antibodies, hormone antagonists (e.g., tamoxifen), non-specific (non-antibody) proteins (e.g., sugar oligomers), oligonucleotides (e.g., antisense oligonucleotides that bind to a target nucleic acid sequence (e.g., an mRNA sequence)), peptides, proteins, antibodies, photodynamic agents (e.g., rhodamine 123), radionuclides (e.g., I-131, Re-186, Re-188, Y-90, Bi-212, At-211, Sr-89, Ho-166, Sm-153, Cu-67, and Cu-64), toxins (e.g., ricin), and transcription-based medicines.

[0177] As used herein, the term "hormone" generally refers to naturally occurring or non-naturally occurring hormones, their analogs and mimetics. In certain embodiments, the term "hormone" refers to any hormone used in therapeutic treatment, such as growth hormone treatment. As used herein, "growth hormone" or "GH" refers to growth hormone, natural, synthetic or recombinant, from any source, in native sequence or variant form. Examples include human growth hormone (hGH) (somatotropin or somatropin), which is natural or recombinant GH with human native sequence, and recombinant growth hormone (rGH), which refers to any GH or variant produced by recombinant DNA technology, including somatrem, somatotropin and somatropin. In one embodiment, the hormone includes insulin.

[0178] As used herein, "imaging agent" can be any chemical moiety that is used to increase the degree of difference between the brightest and darkest parts of a scan or image, for example, during medical scanning or imaging, compared to the scan performed without the use of contrast agent.For example, imaging agent can include radioisotope-containing imaging agent, for example, indium or technetium; iodine, gadolinium or cyanine-containing dye; enzyme, for example, horseradish peroxidase, GFP, alkaline phosphatase or β-galactosidase; fluorescent substance, for example, europium derivative; luminescent substance, for example, N-methylacridium derivative, etc. In some embodiments, imaging agent can include gold nanoparticles and / or quantum dots.

[0179] As used herein, the term "substantially" refers to a proportion of at least about 60%, or preferably at least about 70%, or at least about 80%, or at least about 90%, at least about 95%, at least about 97%, or at least about 99% or more, or any integer between 70% and 100%. In some embodiments, the term "substantially" refers to a proportion of at least about 90%, at least about 95%, at least about 98%, at least about 99% or more, or any integer between 90% and 100%. In some embodiments, the term "substantially" can include 100%.

[0180] As used herein, the term "comprising" means that other elements may be present in addition to the defined elements presented. The use of "comprising" indicates inclusion rather than limitation.

[0181] The term "consisting of" refers to that component described herein exclusively with respect to any elements not mentioned in the description of that embodiment.

[0182] As used herein, the term "consisting essentially of" refers to those elements required for a given embodiment. The term permits the presence of elements that do not materially affect the basic and novel or functional characteristics of the embodiment of the invention.

[0183] Other than in the working examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein are to be understood as being modified in all instances by the term "about." The term "about" when used in connection with percentages can mean ±1%.

[0184] Singular terms ("a," "an," and "the") include plural references unless the context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The abbreviation "eg" is of Latin origin from exempli gratia and is used herein to indicate non-limiting examples. Thus, the abbreviation "eg" is synonymous with the term "for example."

[0185] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this disclosure belongs. Definitions of general terms relating to diseases and disorders, isolation and detection techniques can be found in The Merck Manual of Diagnosis and Therapy, 18th Edition, Merck Research Laboratories, 2006 (ISBN 0-911910-18-2); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Biology, Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

[0186] It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc. described herein, as these may vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is defined solely by the claims.

[0187] All patents and other publications identified throughout this specification are expressly incorporated herein by reference for purposes of illustration and disclosure, including for purposes of describing and disclosing methodologies in such publications that may be used in connection with the present invention. These publications are provided solely for their disclosure prior to the filing date of this application. Nothing in this connection should be treated as an admission that the inventors are not entitled to claim antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the dates or representations as to the contents of these documents are based on the information available to the applicants and do not constitute any admission as to the accuracy of those dates or the contents of these documents.

[0188] Certain aspects described herein are further illustrated by the following non-limiting examples.

[0189] The contents of all references cited throughout this application, the examples, and the figures and tables are hereby incorporated by reference in their entireties. EXAMPLES

[0190] Example 1. Fabrication of an exemplary injectable silk fibroin-based foam Silk fibroin foam sheets can be manufactured by any method known in the art. In this example, silk foam sheets were made using freezer processing techniques, for example, by directly freezing a silk fibroin solution.

[0191] To prepare silk fibroin solution, Bombyx mori silkworm cocoons purchased from Tajimia Shoji Co. (Yokohama, Japan) or a Taiwanese supplier were cut into small pieces and boiled in 0.02 M Na2CO3 for about 10 to 60 min, preferably about 30 min. The resulting silk fibroin fibers were rinsed with distilled water and dried. The dried silk fibroin fibers were redissolved in 9.3 M LiBr at 60 °C for about 1 to 4 h until dissolved. The silk fibroin solution was dialyzed against distilled water with a molecular weight cutoff of 3500 Daltons with at least six water changes.

[0192] Silk fibroin solution (e.g., having a concentration of about 1% to about 6% w / v) obtained from Japanese or Taiwanese cocoons was poured into a container (e.g., a plastic Petri dish). The silk fibroin solution was then stored and maintained (e.g., stored in an EdgeStar Model FP430 thermoelectric cooler) at about 20°F (approximately −7°C) for about 3 days. The resulting silk fibroin material was gel-like rather than a hard solid. The gel-like silk fibroin material was then freeze-dried (e.g., using a VirTis Genesis (Model 25L Genesis SQ Super XL-70) freeze dryer) for about 3 days. After removal from the freeze dryer, the freeze-dried silk fibroin material was a foam-like material with a highly uniform interconnected pore structure. In some embodiments, the silk fibroin foam was further immersed in alcohol (e.g., 70% methanol) to induce beta-sheet formation. In such an embodiment, the volume of the silk fibroin foam sheet may shrink by about 10% after alcohol treatment. The alcohol-treated foam sheet showed excellent stiffness and robustness. A 4 mm diameter biopsy punch (FIG. 1A) was then used to cut small silk fibroin foam disks (approximately 2 mm thick) from the silk fibroin foam sheet, as shown in FIG. 1B.

[0193] Example 2. Evaluation of silk fibroin-based foam for tissue injection To evaluate the feasibility of injecting one or more embodiments of the injectable silk fibroin-based foam construct into tissue, experiments were performed in live chicken thighs. FIG. 2A shows silk fibroin-based foam loaded onto a pointed cone-shaped applicator tip, e.g., a pipette tip. The cone-shaped interior space of the pipette tip allows the foam to be ejected with less friction and force than a straight applicator tip (e.g., a straight needle). The foam was pushed into the pipette tip using a stiff wire (FIG. 2B). By way of example only, in some embodiments, silk fibroin-based foam injection into live chicken thigh tissue was performed as follows: A straight 14-gauge needle (approximately 1.6 mm inner diameter) was first used to puncture a hole in the fleshy part of the chicken thigh (FIG. 3A). A pipette tip loaded with silk fibroin-based foam was inserted into this hole (Figure 3B) and slowly withdrawn using a stiff wire while ejecting the foam (Figure 3C). Figure 3D shows the foam ejected and placed in a live chicken thigh. In this way, silk fibroin-based foam can be injected into tissue, for example to fill tissue voids or to augment tissue.

[0194] Figures 4A-4F show that the injected silk fibroin-based foam was extracted. For example, a razor blade was used to slice raw chicken meat (Figures 4A to 4D). Then, tweezers were used to extract the silk fibroin-based foam (Figures 4E and 4F). Figure 4F shows that the extracted silk fibroin-based foam was intact, indicating that silk fibroin-based foam can be maintained intact after injection into tissue.

[0195] Example 3. In vivo study of injectable silk fibroin-based foams A rat or mouse model was used to evaluate some aspects of the silk fibroin-based foam described herein. Depending on the tissue modeled for application and treatment of the injectable silk fibroin-based foam, other mammalian models (e.g., rabbit, dog, or pig models) can also be used. Prior to injection, the rat or mouse was weighed and anesthetized with isoflurane under oxygen supply. Silk fibroin-based foam with a size of about 5 mm in diameter and 2 mm in height was used for injection. Briefly, the silk fibroin foam in a dry state was immersed in saline immediately before loading into the catheter. Alternatively, the silk fibroin foam in a dry state can be immersed in aspirated fat immediately before loading into the catheter (e.g., as shown in FIG. 5). Subcutaneous injections were performed above the pectoral muscle. Intramuscular and submuscular injections were performed between the pectoralis major and minor muscles or under the pectoral muscle, respectively. A fanning subcutaneous injection method was performed in the dorsus of the rat or mouse. Injected samples were explanted after 1, 14, 40 and 60 days and evaluated for volume retention, which was performed by two methods, i.e., scale measurement and volume displacement.

[0196] Silk fibroin foam can be injected into tissue using a rod and plunger system. For example, FIG. 7A shows an exemplary Hauptner syringe that has been custom modified to inject silk fibroin foam subcutaneously in vivo, for example in a mouse or rat model. The design is based, at least in part, on a commercially available Ideal Instruments pistol-style (Hauptner) syringe. This Hauptner syringe device typically has a spring-loaded handle that urges the Injector Drawrod into the syringe body a pre-set distance (using an Injection Stroke Adjuster). To modify the Hauptner syringe device to inject foam rather than a solution or gel, a foam ramrod was fabricated that passes through the end of the syringe where the Catheter Adaptor is located, as shown in FIG. 7A. The catheter is attached to the adapter. Typically, a catheter is a tube used to remove fluids from the body. In some embodiments, a tapered catheter (i.e., one in which the barrel of the catheter is larger than the tip of the catheter) can be used to inject silk fibroin foam into tissue. This taper allows the foam to be pre-placed in the barrel before the catheter is attached to the Hauptner syringe, and allows for gradual compression of the foam during the injection process.

[0197] By way of example only, in a rat or mouse study protocol, a small hole is first made in the skin of the rat or mouse using a 14-gauge needle attached to a catheter (e.g., as shown in FIG. 7B, step 1: left panel). The outer diameter of the catheter was small enough to pass through the hole, and its inner diameter was large enough to deliver the compressed foam to the subcutaneous region of the rat or mouse. FIGS. 7A-7B show exemplary steps of injecting silk fibroin foam in an animal study. The foam ramrod is inserted into a syringe (FIG. 7A). The needle attached to the catheter is used to facilitate insertion of the catheter at the desired location (FIG. 7B, step 1). After placing the catheter at the desired location in the tissue, the needle may be removed from the needle / catheter. The silk fibroin foam is placed into the barrel of the catheter using tweezers (FIG. 7B, step 2). The silk fibroin foam-loaded catheter is then connected to the catheter adapter of an injection gun (FIG. 7B, step 3). The Injection Handle of the injection gun is then repeatedly squeezed to slowly inject the foam into the animal (FIG. 7B, 4th step).

[0198] Figures 8A-8C show images of silk fibroin-based foams injected into a rat model in vivo after removal of the rat skin. Silk fibroin-based foams made from different concentrations of silk fibroin solutions (e.g., 1%, 3%, 6% silk fibroin) and cocoon sources (Japan: JP vs. Taiwan: TW) were evaluated after 1 day (Figure 8A), 14 days (Figure 8B), and 30 days (Figure 8C) of injection. Figure 8A shows that the injected silk fibroin-based foams maintained transparency 1 day after injection unless stained by blood from puncturing a blood vessel (e.g., TW3). Figures 8B-8C show that the injected silk fibroin-based foams turned reddish hue about 14 days and about 30 days after injection, respectively. However, no significant changes were observed in terms of vascularization into the injected foams. Figure 8D shows images of the injected foams that can be seen from the outside of the rat skin. FIG. 8E is a set of images showing the gross morphology of explanted silk fibroin-based injectable foams (corresponding to those of FIGS. 8A-8C) after the indicated post-injection periods (e.g., 1 day, 14 days, and 30 days post-injection). There is no observable visual difference in gross morphology at the indicated time points. Silk fibroin foams consistently become stiffer with increasing weight percent of silk. All explants are soft to the touch and revert to their original shape after deformation. Histology of explanted tissue-adhered silk fibroin foams is performed to assess vascularization and tissue integration.

[0199] FIG. 8F shows the results of the volume retention of silk fibroin-based foams after injection into rat tissue for 1 day or 14 days. FIG. 8G shows the results of the volume retention of silk fibroin-based foams after injection into tissue for 14 days, 30 days, or 60 days. The results in FIG. 8F and 8G are expressed as a percentage of volume retention relative to their original volume (i.e., the volume of the silk fibroin-based foam before compression). FIG. 8F and 8G show that silk fibroin foams made from about 1%, 3%, or 6% silk fibroin solutions can maintain at least about 80% of their original volume (including at least about 90%, at least about 95%, at least about 100% or more of their original volume) for at least about 30 days or more, and at least about 50% or more of their original volume for at least about 60 days or more. As shown in Figures 8F-8G, the volume retained in tissue can be greater than its original volume, likely because silk fibroin-based foams can absorb water and swell. The stiffness of silk fibroin foams generally increases with the concentration of silk fibroin solution. Thus, silk fibroin foams with higher silk fibroin concentrations can generally maintain their volume for a longer period of time than those with lower silk fibroin concentrations. Furthermore, silk fibroin foams made from about 1%, 3%, or 6% silk fibroin solutions maintain a soft, spongy state for at least 60 days after injection into rats.

[0200] Presented herein are several embodiments of silk fibroin-based foams that can be ejected from needles, pipette tips, catheters, or other tubular structures (e.g., including tubular structures with tapered ends). The silk fibroin-based foams can be compressed prior to injection and then expanded, e.g., at least about 2.5 times, when released from the compressed state and / or injected into tissue. In some embodiments, the injected silk fibroin-based foams can have sufficient physical and mechanical integrity to raise the surface of raw chicken meat and provide appreciable bulk when injected subcutaneously into rats (Figure 8D). The properties of the silk fibroin-based foams can be controlled by various factors, including, but not limited to, the degumming time during silk fibroin solution preparation, the concentration of the silk fibroin solution used, and the use of methanol or other suitable treatments to control the crystalline (beta sheet) content.

[0201] In some embodiments, longer silk fibroin-based foam constructs can be used to fill larger soft tissue voids through incisions, cannulas, needles, pipette tips, catheters or other tubular structures (e.g., including tubular structures with tapered ends), thus requiring the use of smaller holes for tissue penetration compared to traditional invasive procedures.

Claims

[Claim 1] A method for repairing or augmenting tissue in a subject, comprising placing a composition comprising a compressed silk fibroin matrix into the tissue to be repaired or augmented, wherein the compressed silk fibroin matrix expands upon placement in the tissue and retains at least about 1% of its original expanded volume within the tissue for at least about two weeks.

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