Microparticulate tissue scaffold compositions, devices, methods of preparation and uses of said compositions - Patents.com

Abstract

Embodiments of the present disclosure relate to particles, compositions, tissue scaffolds, devices, and uses and methods thereof for body contouring, tissue engineering, regenerative medicine, aesthetic dermatology, and reconstructive procedures or surgery.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority under 35 U.S.C. §119(e) to U.S. Provisional Application No. 63 / 212,993, filed June 21, 2021, the entire contents of which are incorporated herein by reference.

[0002] FIELD OF THEINVENTION Embodiments of the present disclosure relate to injectable microparticulate scaffold compositions for reconstructive uses. [Background technology]

[0003] Soft tissue damage and loss caused by acute tissue injury, disease, or elective surgery, such as lumpectomy or tumor removal, occurs at a high incidence in patients. The results of these types of injury and therapeutic procedures and surgeries are mostly aesthetically undesirable, leading to scarring and tissue deformations that often require successive surgeries for reconstruction. Further defects can result from the loss of skin protein, flexibility, and smoothness as a result of the aging process.

[0004] Currently available treatment options rely on degradable fillers or fat grafting, which are surgical procedures with low success rates and associated with patient morbidity. Most biodegradable dermal fillers have drawbacks, either being temporary in effect or associated with unnatural outcomes. Permanent fillers, such as silicone or polymethyl methacrylate microspheres (PMMA), are now rarely used and are considered unsafe, as they are known to lead to serious adverse clinical outcomes, including recurrent hematomas, edema, hypertrophic scarring, nodule formation, and cancer in some cases.

[0005] Thus, there is a need for suitable agents that can assist in the reconstruction, rejuvenation of lost or reduced soft tissue volume, or the replacement of insufficient or missing tissue.

[0006] Gelatin provides an attractive implantable biomaterial for tissue engineering and regenerative medicine. Upon application of UV light to commercially available gelatin modified with pendant methacrylate groups, such as gelatin methacrylate (GelMA), to obtain crosslinked structures, crosslinked hydrogels are assembled using radical polymerization. In this example, crosslinking leaves behind toxic free radicals and biocompatibility is suboptimal.

[0007] Thus, there is a need for safe, inexpensive, and cost-effective implantable tissue supports for reconstructive uses, such as body contouring and biostimulation. Furthermore, the tissue supports should not induce adverse immune system responses (i.e., lack of immunogenicity) and should be degradable within a short time frame so as not to pose a risk of granulomas.

[0008] Yet another need is to enhance the seeding and survival of cell therapies injected into tissues, as these cells are not usually well-retained and need to be assisted by providing a supportive biocompatible scaffold in the treated tissue that serves as an initial and intermediate bed for the attachment of the cells. Summary of the Invention

[0009] One object of the present disclosure is to provide improved microparticulate porous scaffold compositions that are optionally enzymatically crosslinked and optionally injectable into the body or through a needle-containing injector.

[0010] Another object may be directed to a plurality of microparticles, the plurality of microparticles comprising: a cross-linked protein comprising at least one RGD (Arg-Gly-Asp) motif; the plurality of microparticles being essentially or substantially free of cross-linking agents; and the plurality of microparticles being water insoluble. In one embodiment of the plurality of microparticles, the cross-linked protein may be selected from the group consisting of: gelatin, collagen, casein, elastin, tropoelastin, albumin, engineered proteins thereof, and the like, or any combination thereof; or in another embodiment, the cross-linked protein is selected from: non-recombinant gelatin, recombinant gelatin, non-recombinant collagen, recombinant collagen, engineered or synthetic proteins thereof, any engineered polymer comprising an RGD motif linked thereto, and the like, or any combination thereof. Some embodiments provide such a plurality of particles of the present disclosure that are comprised of: lyophilized foam particles; a particle size (e.g., dry or wet particles) selected from 0.1 μm to 2000 μm (e.g., 40 μm to 100 μm; 60 μm to 90 μm); at least two different particle sizes selected from 0.1 μm to 2000 μm (e.g., 40 μm to 100 μm; 60 μm to 90 μm); an average particle size selected from 0.1 μm to 2000 μm (e.g., 30 μm to 500 μm; 40 μm to 100 μm; 60 μm to 90 μm), or a combination thereof.

[0011] In another object of the present disclosure, a method of preparing a plurality of microparticles as described herein comprises: (a) mixing a crosslinkable protein solution and a crosslinker solution, where the crosslinkable protein solution comprises dissolving a crosslinkable protein or an engineered polymer comprising or linked to at least one RGD (Arginine-Gly-Aspartic Acid (Arg-Gly-Asp)) motif in a liquid; the crosslinker solution comprises dissolving a crosslinker in a liquid; (b) forming a crosslinked foam comprising the mixed crosslinkable protein solution and crosslinker solution of (a); (c) removing the crosslinker from the crosslinked foam of (b) to form a crosslinker-free foam; and (d) reducing the size of the formed crosslinked foam of (b), the crosslinker-free foam of (c), or a combination of the formed crosslinked foam of (b) and the crosslinker-free foam of (c) to form a plurality of microparticles comprising reduced-sized crosslinked foam of (b) and / or reduced-sized crosslinker-free foam of (c). Another embodiment of the method provides for mixing (a) comprising: (a1) preparing a cross-linkable protein solution by adding a cross-linkable protein to a liquid (e.g., water, saline, PBS) at 50° C. with stirring or continuous stirring; dissolving the cross-linkable protein to form a cross-linkable protein solution; and (a2) preparing a cross-linker solution by adding a cross-linker to a liquid (e.g., water, saline, PBS) at 25° C. with stirring or continuous stirring; dissolving the cross-linker to form a cross-linker solution. In some embodiments of the methods disclosed herein, the cross-linked foam of (b) is enzymatically cross-linked, and the cross-linker is a transglutaminase (e.g., microbial transglutaminase). In further embodiments of the disclosed method of preparing a plurality of microparticles, forming the crosslinked foam in (b) comprises: whipping or agitating with aeration, or agitating with or without the addition of gas or air (e.g., argon, carbon dioxide, helium, hydrogen, krypton, methane, neon, nitrogen, oxygen, ozone, water vapor, xenon).In some embodiments, the method includes agitating the crosslinker solution of (a) without the addition of gas or air to form a confluent crosslinked protein (b) that is not a foam, which may be further treated to reduce size in the same manner as is done for the foams of (c)-(g). The crosslinker solution of (a) is added to the crosslinkable protein solution of (a) at 37° C. to form a crosslinked foam of (b). Whipping allows for aeration in the formation of the crosslinked foam. In some embodiments, the formation of the crosslinked foam of (b) may occur by agitation or mixing without gas or air. In yet another aspect of the method, the reduction in (d) is from 0.2 mm to 20 mm (e.g., 1 mm to 19 mm; 2 mm to 18 mm; 3 mm to 17 mm; 4 mm to 16 mm; 5 mm to 15 mm; 6 mm to 14 mm; 7 mm to 13 mm; 8 mm to 12 mm; 9 mm to 11 mm); 0.5 mm or more (e.g., 0.6 mm; 0.7 mm; 0.8 mm; 0.9 mm; 1 mm; 2 mm; 3 mm; 4 mm; 5 mm; 6 mm; 7 mm; 8 mm; 9 mm; 10 mm; 11 mm; 12 mm; 13 mm; 14 mm; 15 mm; 16 mm; 17 mm); and cutting (e.g., dicing, chopping, meshing) the formed crosslinked foam of (b) into large pieces of foam having sizes of 19.5 mm; 18 mm; 19 mm; 20 mm; 20 mm or less (e.g., 19.5 mm; 18.5 mm; 17.5 mm; 16.5 mm; 15.5 mm; 14.5 mm; 13.5 mm; 12.5 mm; 11.5 mm; 10.5 mm; 9.5 mm; 8.5 mm; 7.5 mm; 6.5 mm; 5.5 mm; 4.5 mm; 3.5 mm; 2.5 mm; 1.5 mm).Some embodiments of the method further directed to the removal of (c) may include: removing the cross-linking agent or cross-linking enzyme, for example, by washing the cross-linked foam of (b), where the cross-linked foam of (b) is reduced in size by cutting (e.g., dicing, chopping, meshing) into pieces, and the washing occurs by agitating the cross-linked foam pieces at 45° C. to 55° C. (e.g., 50° C.) to form washed foam pieces; and sieving the washed foam pieces of (c1) over a mesh sieve (e.g., one or more mesh sieves; 35 US mesh number to 5000 US mesh number; 2.5 mm to 500 mm; 0.5 mm), thereby forming cross-linker-free foam pieces (e.g., 0.2 mm to 20 mm).

[0012] Another object of the method may be directed to further comprising: (e) freezing the crosslinker-free foam of (c) or the plurality of particles of (d); (f) drying (e.g., lyophilizing, freeze-drying, oven-drying, room temperature drying, ambient temperature drying) the frozen crosslinker-free foam of (e); and (g) reducing the size of the lyophilized crosslinker-free foam of (f) to form a plurality of crosslinked foam particles. The plurality of crosslinked foam particles of the method may comprise a particle size (e.g., dry particles or wet particles) of 0.1 μm to 2000 μm (e.g., 5 μm to 150 μm; 40 μm to 100 μm; 60 μm to 90 μm). In one embodiment of the method, the crosslinkable protein may be selected from: gelatin, collagen, casein, elastin, tropoelastin, albumin, engineered proteins thereof, etc., or any combination thereof, and the crosslinkable protein may further be selected from the group consisting of: non-recombinant gelatin, recombinant gelatin, non-recombinant collagen, recombinant collagen, etc., or engineered polymers comprising or linked to at least one RGD motif, etc., or any combination thereof. Some embodiments may be directed to a crosslinking agent, which is an enzyme, such as, but not limited to, transglutaminase or oxidase. Non-limiting examples of such cross-linking agents are: native transglutaminase, modified transglutaminase, recombinant transglutaminase, microbial transglutaminase (mTG), tissue transglutaminase (tTG), keratinocyte transglutaminase, epithelial transglutaminase, prostate transglutaminase, neuronal transglutaminase, human transglutaminase, factor XIII, native oxidase, modified oxidase, lysyl oxidase, tyrosinase, laccase, peroxidase, etc., or any combination thereof. Furthermore, in another embodiment of the method of the present disclosure, the freezing in (e) can occur at -18°C to 25°C for a minimum of 2 hours (e.g., 3 hours, 4 hours, 5 to 25 hours); the lyophilization in (f) can occur at -50°C ± 10°C, 0.01 mbar to 0.1 mbar (e.g., 0.04 mbar to 0.05 mbar), and 24 hours to 96 hours (e.g., 48 hours).

[0013] Yet another embodiment may further process and reduce the size in the same manner as for the foam by agitating the protein solution of (a) in the absence of gas or air to form a confluent crosslinked protein solution (b) that is not a foam, (c) removing the crosslinker from the crosslinked foam or block of (b) to form a crosslinker-free foam or block; reducing the size of the formed crosslinked foam or block of (b); (e) freezing the crosslinker-free foam or block of (c) or the plurality of particles of (d); (f) lyophilizing the frozen crosslinker-free foam or block of (e); and (g) reducing the size of the lyophilized crosslinker-free foam of (f) to form a plurality of crosslinked foam particles.

[0014] Further aspects of the disclosed method are directed to a size reducing step (g) comprising: (e) milling the lyophilized crosslinker-free foam to form a plurality of crosslinker-free foam particles; and (g) separating the plurality of crosslinker-free foam particles by size. The size reduction can result in a plurality of crosslinker-free foam particles having a particle size (dry or wet particle size) of 0.1 μm to 2000 μm. Another aspect of the disclosed method provides for separating the milled lyophilized crosslinker-free foam (e) by size by sieving the plurality of crosslinker-free foam particles to produce a plurality of crosslinked foam particles having one or more, or at least two different particle size ranges.

[0015] A further object of the present disclosure may be directed to a composition comprising: (a) a plurality of microparticles of the present disclosure; and (b) a carrier, wherein the cross-linked protein is selected from the group consisting of: gelatin, collagen, casein, elastin, tropoelastin, albumin, engineered proteins thereof, and the like, or any combination thereof; or wherein the cross-linked protein is selected from: non-recombinant gelatin, recombinant gelatin, non-recombinant collagen, recombinant collagen, and the like, or an engineered polymer comprising at least one RGD motif, or any combination thereof. A further embodiment may provide a carrier for the composition which is a hydrogel, the carrier may be selected from: gelatin; collagen; alginate; hyaluronic acid; carboxymethylcellulose; poly(ethylene oxide) (PEO); poly(vinyl alcohol) (PVA); poly(propylene fumarate) (PPF); polyethylene glycol (PEG), and the like, or any combination thereof; or the carrier may be selected from: gelatin (e.g., non-crosslinked, crosslinked, in situ crosslinked); collagen (e.g., non-crosslinked, crosslinked); alginate (e.g., non-crosslinked, crosslinked); hyaluronic acid (e.g., non-crosslinked, crosslinked); PEG; carboxymethylcellulose, and the like, or any combination thereof.Embodiments of these compositions also include: 1 mg / ml or more (e.g., 10 mg / ml; 20 mg / ml; 30 mg / ml; 40 mg / ml; 50 mg / ml; 60 mg / ml; 70 mg / ml; 80 mg / ml; 90 mg / ml; 100 mg / ml; 110 mg / ml; 120 mg / ml; 130 mg / ml; 140 mg / ml; 150 mg / ml; 200 mg / ml; 300 mg / ml); 300 mg / ml or less (e.g., 290 mg / ml; 280 mg / ml; 270 mg / ml; l;260mg / ml;250mg / ml;240mg / ml;230mg / ml;220mg / ml;210mg / ml;200mg / ml;190mg / ml;180mg / ml;170mg / ml;160mg / ml;155m g / ml;145mg / ml;135mg / ml;125mg / ml;115mg / ml;105mg / ml;95mg / ml;85mg / ml;75mg / ml;65mg / ml;55mg / ml;45mg / ml;35mg / ml; 25mg / ml; 15mg / ml; 5mg / ml); or 1mg / ml~300mg / ml (e.g. 2mg / ml~295mg / ml; 4mg / ml~285mg / ml; 6mg / ml~275mg / ml; 8mg / ml~265m g / ml;12mg / ml~255mg / ml;14mg / ml~245mg / ml;16mg / ml~235mg / ml;18mg / ml~225mg / ml;22mg / ml~215mg / ml;24mg / ml~205mg / ml Also provided are concentrations of a plurality of microparticles in the carrier: 26mg / ml-195mg / ml; 28mg / ml-185mg / ml; 32mg / ml-175mg / ml; 34mg / ml-165mg / ml; 36mg / ml-153mg / ml; 38mg / ml-143mg / ml; 42mg / ml-133mg / ml; 52mg / ml-123mg / ml; 62mg / ml-113mg / ml; 72mg / ml-103mg / ml; 82mg / ml-93mg / ml).

[0016] Another object of the present disclosure is to provide a tissue scaffold comprising a plurality of microparticles of the present disclosure, and in some embodiments, further comprising a hydrogel carrier, wherein the hydrogel carrier is selected from: gelatin, collagen, alginate, hyaluronic acid, carboxymethylcellulose, and the like, or any combination thereof. In some other embodiments, the tissue scaffold is configured as a foam, and the crosslinked protein microparticles comprise at least one different particle size, and the particle size may be between 0.1 μm and 2000 μm.

[0017] A still further object of the present disclosure may be directed to a device comprising the composition of the present disclosure, comprising a plurality of microparticles and a carrier, which in some embodiments is a syringe, cartridge, or vial. Another embodiment provides a syringe comprising a needle or cannula selected from 14 gauge to 39 gauge (e.g., 25 gauge to 30 gauge, 27 gauge to 30 gauge). In one embodiment of the device, the device is sterilizable or configured for sterilization.

[0018] In another aspect of the present disclosure, the composition comprising a plurality of microparticles and a carrier, and / or the use of a plurality of microparticles of the present disclosure may be for body contouring in a subject (either human or animal). An embodiment of the use provides body contouring selected from: soft tissue reconstruction, volume restoration, breast augmentation, biostimulation (of cells, e.g., skin), etc., or combinations thereof. In some embodiments, the biostimulation may be selected from: fibroblast stimulation, collagen production stimulation, neo-collagenesis, tissue re-growth, wound closure, etc., or combinations thereof. Another embodiment of the use is directed to a composition of the present disclosure and / or a plurality of microparticles of the present disclosure, the composition and / or the plurality of microparticles being configured in a device described herein, which may be, for example, a syringe, cartridge, or vial.

[0019] One object of the present disclosure also provides a method of treating a subject in need of body contouring, comprising administering a composition of the present disclosure to a site of the subject in need of body contouring, the administration being, in one embodiment, by injection. Another embodiment provides for administration of a composition of the present disclosure to: (a) stimulate fibroblasts; (b) stimulate collagen production; (c) induce new collagen production; (d) induce tissue regrowth; (e) provide tissue scaffolding, etc., or (f) any combination thereof. [Brief description of the drawings]

[0020] [Figure 1] Figure 1 shows a graphical presentation of the average activity of the cross-linking agent, mTG, in various microparticle batches, where the amount of mTG used for gelatin cross-linking ranged from 3 g to 10 g. P1: assay positive control; mTG (1:100): positive control; Y-axis: mTG average activity; X-axis: microparticle batches and positive control.

[0021] [Diagram 2] Figures 2A-2D show representative histopathological evaluation of the subcutaneous area implanted with the composition of the present disclosure 30 days after injection using Masson's Trichrome (MT) staining. Scales for each are as follows: 1000 μm (Figure 2A); 200 μm (Figure 2B); and 50 μm (Figures 2C-2D). Black arrows indicate the implanted composition of the present disclosure. Grey arrows indicate new collagen production. White arrows indicate interactions between infiltrating fibroblasts and the scaffold.

[0022] [Diagram 3] FIG. 3 shows the linear correlation between injection force and particle size using a 1 ml syringe containing a 27 gauge (G) needle.

[0023] [Figure 4] FIG. 4 shows an exemplary scanning electron microscope (SEM) image of particles larger than 0.1 μm (i.e., 100 nm) (e.g., 104 nm; 105 nm; 112 nm; 145 nm; 150 nm; 275 nm).

[0024] [Diagram 5] FIG. 5 shows an exemplary SEM image of microparticles having a particle size range of 60 μm to 100 μm (e.g., 75.69 μm; 88.38 μm; 91.56 μm; 99.68 μm).

[0025] [Figure 6] Figure 6A shows an exemplary optical microscope image of particles up to 2000 μm. The particles were hydrated before imaging (scale 500 μm). Figure 6B shows dry particle sizes of: 558 μm, 862 μm, and 986 μm (scale 200 μm). Figure 6C shows wet particles with a particle size of 1808 μm (scale 500 μm).

[0026] [Figure 7] Figure 7A shows dry gelatin microparticles (scale 100 μm) and Figure 7B shows hydrated or wet gelatin microparticles (scale 100 μm).

[0027] [Figure 8] Figure 8 shows a graphical representation of the size distribution of dry and hydrated microparticles. Dry microparticles (left column): particle size: 70 μm - 170 μm. Hydrated microparticles (right column): 90 μm - 310 μm.

[0028] [Figure 9] FIG. 9 shows a representative frequency sweep graph showing storage modulus G'(Pa)Δ, loss modulus G″(Pa)□, and complex viscosity η*(Pa.s)O on the Y-axis versus frequency f(Hz) on the X-axis for 0.75% gelatin carrier at 6° C.

[0029] [Figure 10] Figure 10 shows representative size distributions of exemplary foam microparticles for samples (8gr mTG), with the 96% ethanol sample (circles) peaking at 80 μm in size at 14% by volume, the DDW instant sample (diamonds) peaking at 120 μm in size at 10% by volume, and the DDW at 24 hr sample (squares) peaking at 140 μm in size at 11% by volume. See Table 6.

[0030] [Figure 11] FIG. 11 shows the injectability (N) of an exemplary formulation of crosslinked gelatin foam microparticles in different saline volumes (1.5 ml; 2 ml; 3 ml; 4 ml).

[0031] [Figure 12] FIG. 12 shows representative histological photographs of implants stained with H&E (hematoxylin and eosin, which stains cell nuclei purplish blue and extracellular matrix and cytoplasm pink) and MT, Masson's trichrome (resulting in keratin red, muscle fibers and implants, collagen and bone blue, light red or pink cytoplasm, and dark brown to black cell nuclei) in pig and rat skin 7, 30, and 180 days after implantation (H&E-pig days 7 and 30, rat days 7 and 30, and MT-pig day 180), with arrows indicating the site of the implanted composition of the present disclosure.

[0032] [Figure 13] FIG. 13 shows representative histological photographs of implants stained with H&E (hematoxylin and eosin, which stains cell nuclei purplish blue and extracellular matrix and cytoplasm pink) and Masson's trichrome (resulting in keratin red, muscle fibers and implants, collagen and bone blue, light red or pink cytoplasm, and dark brown to black cell nuclei) 7, 30, and 180 days after implantation (H&E-pig day 7, and Masson's trichrome-pig days 30 and 180, rat days 7 and 30), showing the implanted blend of new collagen fibers (white arrows) stained blue (black arrow).

[0033] [Figure 14]Figure 14 shows SDS-PAGE of 1 mg / ml FP prepared in water. Collagenase was added to the suspension to degrade the FP. Molecular weight marker (M); microbial transglutaminase (7 μg protein in 20 μl) (1); gelatin (10 μg protein in 20 μl) (2); collagenase (1.7 U in 20 μl) (3); foam particles (FP) (digested with collagenase, 10 μg protein in 20 μl) (4).

[0034] [Figure 15] Figure 15 shows the calibration curve for arginine. The R value of 0.999 indicates high linearity. Arginine concentration (μg / ml) (X-axis) versus emission intensity (Y-axis).

[0035] [Figure 16] FIG. 16 shows the fluorescence emission spectra of free arginine, raw material, and crosslinked gelatin particles.

[0036] [Figure 17] FIG. 17 shows RGD quantification in raw non-crosslinked gelatin and crosslinked gelatin particles (ie, FP and confluent particles).

[0037] [Figure 18] FIG. 18 shows the amount of RGD sequence or motif (μg / mg) (Y-axis) on FP with different crosslinked gelatin particle size ranges (X-axis): less than 63 μm; 63 μm to 99 μm; and greater than 99 μm.

[0038] [Figure 19] FIG. 19 shows the amount of RGD (μg / mg) (Y-axis) associated with various weight ratios of gelatin:mTG, gelatin, and microbial transglutaminase (mTG).

[0039] [Figure 20]Figures 20A and 20B show optical microscope images of human induced pluripotent stem (iPS) cells grown on foam particle microcarriers of the present disclosure that have differentiated into cardiomyocytes. Figure 20A has a scale of 50 μm and Figure 20B has a scale of 200 μm.

[0040] [Figure 21] Figures 20A and 20B show optical microscope images of foam particles (FP) made from cross-linked gelatin fibers. Scale 100 μm. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0041] Detailed embodiments of the present disclosure are described herein; however, the disclosed embodiments are merely exemplary of the invention, which can be embodied in various forms. Moreover, each of the examples provided in connection with the various embodiments of the invention are intended to be illustrative, and not limiting.

[0042] Porous and biodegradable polymer scaffolds can be utilized as structural support matrices or as cell adhesive substrates. The objective of the present disclosure is to provide safe, non-toxic, inexpensive or low-cost implantable tissue supports that do not induce immune responses or are not immunogenic. In one example, the implantable tissue supports of the present disclosure are synthetic and / or devoid or essentially free of non-human components. The use of materials that contain innate cell-binding elements can improve implant performance by allowing direct cell attachment and local remodeling. Tripeptide motifs (e.g., RGD (arginine (Arg)-glycine (Gly)-aspartic acid (Asp))) are found in extracellular matrix proteins, such as, but not limited to, bone sialoprotein, collagen, fibrinogen, fibronectin, gelatin, laminin, osteopontin, and vitronectin, and promote cell adhesion, cell membrane binding, and cell attachment. The RGD motif is an integrin-binding domain in ECM proteins. For example, gelatin, which is derived from collagen, contains the RGD motif, which is useful for cell attachment.

[0043] particle

[0044] In various embodiments of the present disclosure, provided herein is a microparticle or a plurality of microparticles; methods for preparing the same; compositions comprising a plurality of microparticles; devices, such as syringes or vials, comprising a composition of the present disclosure; scaffolds or tissue scaffolds comprising a plurality of microparticles or compositions of the present disclosure; uses of the disclosed microparticles or compositions of the present disclosure; and methods of treating a subject by administering a plurality of microparticles or compositions of the present disclosure.

[0045] As used herein, the term "subject" refers to any organism to which a composition according to the present disclosure may be administered, for example, for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Exemplary subjects include any animal (e.g., mammals, such as mice, rats, rabbits, dogs, cats, non-human primates, and humans, etc.). A subject in need is typically a subject for whom it is desirable to treat a disease, disorder, or condition as described herein. For example, a subject in need may be a subject who may be seeking or in need of treatment, who may need treatment, who may be undergoing treatment, or who may be undergoing treatment in the future, or a human or animal who is receiving care for a particular disease, disorder, or condition by a trained professional. In some embodiments, the subject is in need of body contouring, including, but not limited to: soft tissue reconstruction, volume restoration, breast augmentation, biostimulation (of cells, e.g., of the skin), etc., or a combination thereof. In some embodiments, the biostimulation may be selected from: fibroblast stimulation, collagen production stimulation, new collagen production, tissue re-growth, wound closure, etc., or a combination thereof.

[0046] One embodiment of the present disclosure is directed to a microparticle or microparticles having cross-linked proteins, the cross-linked proteins comprising at least one RGD (Arg-Gly-Asp) motif, inter alia, that conveys cell adhesion properties. In some embodiments, the cross-linked proteins having at least one RGD motif, but not several RGD motifs, can be fully and / or better exposed by decreasing the particle size and increasing the surface area of ​​the microparticle. The microparticle or microparticles described herein may have a concentration of 0.1 μg / mg to 50 μg / mg (e.g., 0.2 μg / mg to 45 μg / mg; 0.3 μg / mg to 40 μg / mg; 0.4 μg / mg to 35 μg / mg; 0.5 μg / mg to 30 μg / mg; 0.6 μg / mg to 25 μg / mg; 0.7 μg / mg to 20 μg / mg; 0.8 μg / mg to 15 μg / mg; 0.9 μg / mg to 10 μg / mg; 1 μg / mg to 5 μg / mg); 0.1 μg / mg or more (e.g., 2 μg; 4 μg; 6 μg; 8 μg; 10 μg; 12 μg; 14 μg; 16 μg; 18 μg; 20 μg; 22 μg; 24 μg; 26 μg; 28 μg; 30 μg; 32 μg; 34 μg; 36 μg; 38 μg; 40 μg; 42 μg; 44 μg; 46 μg; 48 μg; 50 μg); or 50 μg / mg or less (e.g., 49 μg; 47 μg; 45 μg; 43 μg; 41 μg; 39 μg; 37 μg; 35 μg; 33 μg; 31 μg; 29 μg; 27 μg; 25 μg; 23 μg; 21 μg; 19 μg; 17 μg; 15 μg; 13 μg; 11 μg; 9 μg; 7 μg; 5 μg; 3 μg; 1 μg; 0.9 μg; 0.7 μg; 0.5 μg; 0.3 μg; 0.1 μg).

[0047] The microparticle or microparticles comprising crosslinked proteins are free of or essentially free of crosslinkers, and "free of crosslinkers" as used herein means that there is no crosslinker present or that, although present, there is a small amount of crosslinker that does not affect the function or use of the microparticles or crosslinked proteins. The crosslinked proteins may be stabilized, for example, into foams, into confluent hydrogels, or into fibers, where crosslinking occurs by enzymatic crosslinking. In some embodiments, the enzymatic crosslinking is performed using an enzyme, which is subsequently removed, for example, by washing the enzyme from the particles or by inactivating the crosslinker or crosslinking enzyme. One embodiment includes the use of a transglutaminase enzyme to crosslink the proteins of the crosslinked protein, and upon completion of crosslinking, the enzyme is washed away from the crosslinked protein(s). In a further embodiment, the transglutaminase enzyme is or includes a microbial transglutaminase enzyme.

[0048] Thus, the final microparticle or microparticles comprise cross-linked proteins that are free of cross-linking agents. In some embodiments, the proteins of the cross-linked proteins of the final microparticle or microparticles comprise previously cross-linked or pre-cross-linked proteins, and the cross-linked proteins are washed to remove any cross-linking agents, such that the final microparticle or microparticles comprising cross-linked proteins are free of cross-linking agents or are essentially or substantially free of cross-linking agents. The proteins of the cross-linked proteins may be selected from the group consisting of, but are not limited to, gelatin, collagen, casein, elastin, tropoelastin, albumin, engineered proteins thereof, and the like, or any combination thereof. Another aspect of the embodiment may be directed to the proteins of the cross-linked proteins comprising: non-recombinant gelatin, recombinant gelatin, non-recombinant collagen, recombinant collagen, engineered proteins thereof, engineered polymers comprising or linked to at least one RGD motif, and the like, or any combination thereof. Further, the microparticle or microparticles of the present disclosure comprise at least one or more cross-linking proteins, where the at least one or more cross-linking proteins comprise at least one RGD (Arg-Gly-Asp) motif; the microparticle or microparticles are cross-linker-free (i.e., the cross-linker(s) are absent or essentially absent); the microparticle or microparticles are water-insoluble or essentially water-insoluble. Some embodiments of the present disclosure are directed to microparticles that are pre-cross-linked, water-insoluble, cross-linker-free, and not water-soluble.

[0049] Another embodiment is directed to a plurality of microparticles of the present disclosure, the microparticles comprising particles of foam or particles having foam-like properties, the plurality of microparticles or foam particles comprising cross-linked proteins without cross-linking agents. In some aspects of the embodiments of the present disclosure, the cross-linked proteins are stabilized into foam, into confluent hydrogels, or into fibers (as in electrospinning), and the cross-linking occurs by enzymatic cross-linking. As used herein, "foam" refers to a dispersion of gas bubbles in a liquid, solid, or semi-solid (e.g., gel). In other examples disclosed herein, the foam comprises particles or is configured as particles. These foam particles retain the properties of foam or are derived from foam, thereby having "foam-like" properties. Additionally, the plurality of microparticles or foam particles may be comprised of lyophilized particles, including lyophilized foam particles comprising cross-linked proteins without cross-linking agents. "Foam particles" (FP) as used herein means that they are derived from a stable protein foam and are not necessarily foam in their structure after milling. This may depend on the size of the gas bubbles in the initial crosslinker-free foam of (c) and the size of the resulting freeze-dried, size-reduced particles of (g). If the gas bubbles are smaller than the particle size, they may contain closed cells of the foam; however, if the particle is smaller than the gas bubbles, the bubbles or full bubbles may not remain encapsulated in the particle. In either case, the performance and intent of the embodiments described herein are not hindered, which are not limited to foam morphology.

[0050] One embodiment is directed to foam or foam particles, including microparticles, that are reduced in size by using cutting (e.g. chopping, dicing); compressing, delumping, crushing, milling (e.g. impact mill, flour mill, full screen hammer mill, mega hammer mill, air classifier mill, jet mill, ball mill, pebble mill, rod mill); grinder (fine grinder, blade grinder), etc., or combinations thereof, and include particles or are set as particles. By reducing the size, the crosslinked foam to form particles, e.g. microparticles, allows some RGD motifs to be exposed to a large surface area, which allows cell adhesion and biostimulation. The embodiment with at least two different particle sizes also benefits from the increased surface area. Particle size may be analyzed or measured by any technique commonly known and / or used by those of ordinary skill in the art. Non-limiting examples of such methods, techniques, or tools for measuring particle size include: particle size analyzers (PSA); high resolution image processing; image particle analysis (IPA) (e.g., optical microscopy, scanning electron microscope (SEM), transmission electron microscope (TEM)); dynamic image analysis (DIA); static laser light scattering (SLS, also known as laser diffraction); dynamic light scattering (DLS); acoustic spectroscopy; sieve analysis (e.g., dry sieving, wet sieving), and the like, or any combination thereof.

[0051] In some embodiments, including but not limited to those derived from crosslinked proteins, the plurality of microparticles may be sized to have a diameter between 0.1 μm and 2000 μm (e.g., 0.2 μm to 1499 μm; 0.4 μm to 1450 μm; 0.5 μm to 1425 μm; 0.6 μm to 1400 μm; 0.7 μm to 1350 μm; 0.8 μm to 1300 μm; 0.9 μm to 1250 μm; 1 μm to 1200 μm; 2 μm to 1 150μm;3μm~1100μm;4μm~1050μm;5μm~1000μm;6μm~950μm;7μm~900μm;8μm~850μm;9μm~800μm;10μm~750μm; 11μm~700μm;12μm~650μm;13μm~600μm;14μm~550μm;15μm~500μm;16μm~450μm;17μm~400μm;18μm~350μm;19μ m~300μm;20μm~250μm;25μm~200μm;30μm~150μm;40μm~100μm;60μm~90μm);0.1μm or more (e.g. 0.5μm;1μm;5μm;15 μm;25μm;35μm;45μm;55μm;65μm;75μm;85μm;95μm;100μm;105μm;115μm;125μm;135μm;145μm;150μm;200μm; 250 μm; 500 μm; 1000 μm; 2000 μm); or particle sizes below 2000 μm (e.g. 1250 μm; 1000 μm; 750 μm; 500 μm; 250 μm; 200 μm; 150 μm; 140 μm; 130 μm; 120 μm; 110 μm; 90 μm; 80 μm; 70 μm; 60 μm; 50 μm; 40 μm; 30 μm; 20 μm; 10 μm; 5 μm; 4 μm; 3 μm; 2 μm).Other embodiments directed to such a plurality of microparticles include: 0.1 μm to 2000 μm (e.g., 0.2 μm to 1499 μm; 0.4 μm to 1450 μm; 0.5 μm to 1425 μm; 0.6 μm to 1400 μm; 0.7 μm to 1350 μm; 0.8 μm to 1300 μm; 0.9 μm to 1250 μm; 1 μm to 1200 μm; 2 μm to 1150 μm; 3 μm to 1100 μm; 4 μm to 1 050μm;5μm~1000μm;6μm~950μm;7μm~900μm;8μm~850μm;9μm~800μm;10μm~750μm;11μm~700μm;12μm~6 50μm;13μm~600μm;14μm~550μm;15μm~500μm;16μm~450μm;17μm~400μm;18μm~350μm;19μm~300μm;20μm ~250μm;25μm~200μm;30μm~150μm;40μm~100μm;60μm~90μm;0.1μm or more (e.g. 5μm;15μm;25μm;35μm;45μm;55μm;65μm;75μm;85μm;95μm;100μm;105μm;115μm;125μm;135μm;145μm;150μm;200μm;250μm;500μm; 1000 μm; 2000 μm); or 2000 μm or less (e.g., 1250 μm; 1000 μm; 750 μm; 500 μm; 250 μm; 200 μm; 150 μm; 140 μm; 130 μm; 120 μm; 110 μm; 90 μm; 80 μm; 70 μm; 60 μm; 50 μm; 40 μm; 30 μm; 20 μm; 10 μm; 5 μm; 4 μm; 3 μm; 2 μm). In embodiments of the present disclosure, "average particle size" as used herein refers to the average particle size of a plurality of microparticles. In some embodiments, "particle size" refers to dry particle size. In some embodiments, "particle size" refers to wet particle size. In some embodiments, the wet or hydrated particles have a larger particle size than the dry particles of the same dry size, for example by a factor of 1.4 to 2.8, with an average factor of 1.67 (1.65 to 1.67). See, for example, Table 4.

[0052] Further embodiments of the present disclosure are directed to a plurality of microparticles as described herein, wherein the plurality of microparticles may comprise at least two different particle sizes. Particle sizes may include, but are not limited to: 0.1 μm to 2000 μm (e.g., 0.2 μm to 1900 μm; 0.3 μm to 1800 μm; 0.4 μm to 1700 μm; 0.5 μm to 1600 μm; 0.6 μm to 1500 μm; 0.7 μm to 1400 μm; 0.8 μm to 1300 μm; 0.9 μm to 1250 μm; 1 μm to 1200 μm; 2 μm to 1150 μm; 3 μm to 1100 μm; 4 μm to 1000 μm; 5 μm to 1200 μm; 6 μm to 1400 μm; 7 μm to 1600 μm; 8 μm to 1800 μm; 9 μm to 1000 μm; 10 μm to 1200 μm; 11 μm to 1300 μm; 14 μm to 1500 μm; 15 μm to 1600 μm; 16 μm to 1700 μm; 17 μm to 1800 μm; 18 μm to 1900 μm; 19 μm to 2000 μm; 20 μm to 2100 μm; 21 μm to 2200 μm; 22 μm to 2300 μm; 23 μm to 2400 μm; 24 μm to 2500 μm; 25 μm to 3000 μm; 26 μm to 31 50μm;5μm~1000μm;6μm~950μm;7μm~900μm;8μm~850μm;9μm~800μm;10μm~750μm;11μm~700μm;12μm~650μm;1 3μm~600μm;14μm~550μm;15μm~500μm;16μm~450μm;17μm~400μm;18μm~350μm;19μm~300μm;20μm~250μm;25μ m~200μm;30μm~150μm;40μm~100μm;60μm~90μm) ;65μm;75μm;85μm;95μm;100μm;105μm;115μm;125μm;135μm;145μm;150μm;200μm;250μm;500μm;1000μm;20 00 μm); or 2000 μm or less (e.g., 1250 μm; 1000 μm; 750 μm; 500 μm; 250 μm; 200 μm; 150 μm; 140 μm; 130 μm; 120 μm; 110 μm; 90 μm; 80 μm; 70 μm; 60 μm; 50 μm; 40 μm; 30 μm; 20 μm; 10 μm; 5 μm; 4 μm; 3 μm; 2 μm).Other embodiments directed to a plurality of microparticles comprising at least two different particle sizes include, but are not limited to: 0.1 μm to 2000 μm (e.g., 0.2 μm to 1499 μm; 0.4 μm to 1450 μm; 0.5 μm to 1425 μm; 0.6 μm to 1400 μm; 0.7 μm to 1350 μm; 0.8 μm to 1300 μm; 0.9 μm to 1250 μm; 1 μm to 1200 μm; 2 μm to 1150 μm). ;3μm~1100μm;4μm~1050μm;5μm~1000μm;6μm~950μm;7μm~900μm;8μm~850μm;9μm~800μm;10μm~750μm;11μm~7 00μm;12μm~650μm;13μm~600μm;14μm~550μm;15μm~500μm;16μm~450μm;17μm~400μm;18μm~350μm;19μm~300μ m;20μm~250μm;25μm~200μm;30μm~150μm;40μm~100μm;60μm~90μm);0.1μm or more (e.g. 0.5μm;1μm;5μm;15μm;25μm ;35μm;45μm;55μm;65μm;75μm;85μm;95μm;100μm;105μm;115μm;125μm;135μm;145μm;150μm;200μm;250μm;5 00 μm; 1000 μm; 2000 μm); or 2000 μm or less (e.g. 1250 μm; 1000 μm; 750 μm; 500 μm; 250 μm; 200 μm; 150 μm; 140 μm; 130 μm; 120 μm; 110 μm; 90 μm; 80 μm; 70 μm; 60 μm; 50 μm; 40 μm; 30 μm; 20 μm; 10 μm; 5 μm; 4 μm; 3 μm; 2 μm).

[0053] Methods for preparing particles of the present disclosure

[0054] Gelatin microparticles have been previously produced using a variety of methods and techniques, including water-in-oil emulsion, electrospray, spray drying, and microfluidic emulsification, to name just a few. Using these methods, gelatin is crosslinked by several types of chemical crosslinkers, such as 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxysuccinimide (NHS), glycidoxyproyltrimethoxysilane (GPTMS), glutaraldehyde, and genipin. The water-in-oil method is a commonly used laboratory technique, but it has many drawbacks, including difficulty in scaling up to industrial scale, toxicity issues due to the use of oil and chemical crosslinkers, and the need for thorough removal of residual oil and chemical crosslinkers.

[0055] One embodiment of the present disclosure includes: (a) mixing a crosslinkable protein solution and a crosslinker solution, wherein the crosslinkable protein solution is a crosslinkable protein (e.g., gelatin (e.g., recombinant gelatin, non-recombinant gelatin, in or any combination thereof; or non-recombinant gelatin, recombinant gelatin, non-recombinant collagen, recombinant collagen, any engineered protein thereof, engineered polymers comprising or linked to RGD, or any combination thereof) in a liquid (e.g., water, saline, PBS); the crosslinker solution comprises dissolving a crosslinker or enzymatic crosslinker (e.g., transglutaminase (e.g., native transglutaminase, modified transglutaminase, recombinant transglutaminase, microbial transglutaminase (mTG), tissue transglutaminase (tTG), keratinocyte transglutaminase, epithelial transglutaminase, endo ... The present disclosure is directed to a method for preparing a plurality of microparticles comprising dissolving an oxidase (e.g., taminase, prostatic transglutaminase, neuronal transglutaminase, human transglutaminase, factor XIII, etc., or any combination thereof), an oxidase (e.g., native oxidase, modified oxidase, lysyl oxidase, tyrosinase, laccase, peroxidase, etc., or any combination thereof) in a liquid (e.g., water, saline, PBS), wherein the crosslinking agent is in an amount sufficient to crosslink the crosslinkable protein to form a crosslinked foam / block, a non-foam crosslinked hydrogel, or a fiber (e.g., as in electrospinning). Another embodiment is directed to a crosslinking agent in an amount sufficient to convert the crosslinkable protein from soluble to insoluble at a temperature range of 10° C. to 40° C.A method of preparing a plurality of microparticles of the present disclosure includes: (b) forming a crosslinked foam / block, non-foam crosslinked hydrogel, or fiber (e.g., as in electrospinning) comprising the mixed crosslinkable protein and crosslinker of (a); (c) comminuting the non-foam crosslinked hydrogel, fiber, or crosslinked foam of (b); (d) removing the crosslinker from the crosslinked formulation or product of (c) to form a crosslinker-free foam or hydrogel or fiber (e.g., essentially or substantially free of crosslinker); and (e) reducing the size of the formed crosslinked product of (d), the crosslinker-free product of (d), or a combination of the formed crosslinked foam of (d) and the crosslinker-free foam or hydrogel of (d) to form a plurality of particles and / or microparticles comprising reduced-sized crosslinked foam or hydrogel of (b) and / or reduced-sized crosslinker-free foam or hydrogel of (d). In some embodiments, the plurality of particles and / or microparticles comprising (b) reduced size crosslinked foam or hydrogel and / or (d) reduced size crosslinker-free foam or hydrogel may be sterilized by any suitable method that does not substantially alter the functionality, physicochemical properties, stability, toxicity, or biological effects, including, but not limited to: filtration, autoclaving (e.g., 110° C.-134° C.; 15 minutes-40 minutes; 5 psi-20 psi), irradiation (e.g., ultraviolet light (UV); gamma radiation; electron beam (e-beam); x-ray). Some embodiments of sterilization include UV treatment for 5 minutes-720 minutes (e.g., 100 minutes, 150 minutes, 200 minutes, 250 minutes) and exposure to UV wavelengths of 10 nm-400 nm (e.g., 200 nm-270 nm). Further embodiments include gamma irradiation of 10 kGy to 50 kGy (e.g., 15 kGy, 20 kGy, 25 kGy, 30 kGy, 35 kGy, 40 kGy, 45 kGy). Vetten et al. disclose a variety of useful sterilization techniques and parameters that are applicable herein and are incorporated by reference in their entirety (see Nanomedicine. 10(7):1391-1399, 2014).

[0056] Some embodiments relate to methods of preparing a plurality of microparticles as described herein, where cross-linking occurs in vitro as a manufacturing control step, as opposed to other formulations that are mixed and injected at the point of care, thereby allowing cross-linking to occur in situ. Multiple repeated and extensive washes are performed after the cross-linking reaction has occurred to remove the transglutaminase cross-linker enzyme as described in the disclosed methods.

[0057] In another embodiment, the disclosed method of preparing a plurality of microparticles is directed to a crosslinkable protein solution comprising: (i) adding a crosslinkable protein to a liquid (e.g., water, saline, PBS) at a temperature sufficient for the crosslinkable protein to dissolve, e.g., a temperature above 25° C. (e.g., 30° C., 37° C., 40° C., 45° C., 50° C.), where the crosslinkable protein is selected from, but is not limited to, a protein comprising at least one RGD (Arg-Gly-Asp) motif (e.g., gelatin (e.g., non-recombinant gelatin, recombinant gelatin), collagen (e.g., non-recombinant collagen, recombinant collagen), casein, albumin, and any combination thereof), at a temperature sufficient for the crosslinkable protein to essentially dissolve or completely dissolve, e.g., but is not limited to, between 40° C. and 60° C., e.g., 50° C., with continuous stirring; and (ii) dissolving, essentially dissolving, or completely dissolving the crosslinkable protein in the liquid to form a crosslinkable protein solution.

[0058] Some embodiments are directed to producing foamed cross-linked gelatin microparticles (MPs) by cross-linking the reaction with a transglutaminase enzyme (e.g., microbial transglutaminase (mTG); recombinant transglutaminase; bacterial transglutaminase). Briefly, a transglutaminase (e.g., mTG) solution may be added to gelatin in a liquid state in a whipping machine or may be mixed or agitated by any other means with or without the addition of gas or air (e.g., argon, carbon dioxide, helium, hydrogen, krypton, methane, neon, nitrogen, oxygen, ozone, water vapor, xenon, or any combination thereof). In some embodiments, the method comprises forming a cross-linked foam by whipping a cross-linkable protein solution of (a) while adding a cross-linker solution of (a) at 37° C. to form a cross-linked foam of (b). Other embodiments relate to methods of the present disclosure that include stirring or mixing the crosslinkable protein solution of (a) while adding a crosslinker solution of (a) at 37° C. without the addition of gas or air to form a non-bubbly crosslinked block of (b).

[0059] While mixing and foaming, the gelatin is cross-linked until a three-dimensional (3D) foam structure is stabilized. The compounded foam is then incubated at 45°C and then chopped into giant or large slices or pieces (e.g., 0.05 cm to 2 cm; 0.5 mm to 20 mm). The chopped slices are washed several times at 50°C to remove excess cross-linking agent or transglutaminase (e.g., mTG). After washing, the foamed gelatin is freeze-dried, for example, using a lyophilizer. For the production of MPs, the dried cross-linked foamed gelatin is milled and sieved into microparticles of several size ranges (e.g., 0.1 μm to 10 mm). The MPs may be sterilized by any means, including those described herein, that do not negatively affect the structure, function, or performance of the microparticles, including but not limited to irradiation.

[0060] In another embodiment of the present disclosure, crosslinked gelatin microparticles (MPs) may be produced by crosslinking the reaction with transglutaminase enzyme (e.g., microbial transglutaminase (mTG); recombinant transglutaminase; bacterial transglutaminase). Briefly, a transglutaminase (e.g., mTG) solution is added to liquid state gelatin. The gelatin is stirred (without foaming) until crosslinking results in a stable three-dimensional (3D) structure and forms a crosslinked gelatin structure. The compounded structure is then incubated at 45°C and then chopped into giant or large slices or pieces (e.g., 0.05 cm to 2 cm; 0.5 mm to 20 mm). The chopped slices are washed several times at 50°C to remove excess crosslinker or transglutaminase (e.g., mTG). After washing, the crosslinked gelatin is then freeze-dried, for example, using a freeze-drying device. To produce MPs, the dried cross-linked gelatin is milled and sieved into microparticles of several size ranges (e.g., 0.1 μm to 10 mm). The MPs may be sterilized by any means, including those described herein, that do not negatively affect the structure, function, or performance of the microparticles, including but not limited to, irradiation.

[0061] Further embodiments provide a method of preparing a plurality of microparticles that are directed to a crosslinker solution, comprising: (i) adding the crosslinker to a liquid (e.g., water, saline, PBS) at a temperature sufficient to dissolve, essentially dissolve, or completely dissolve the crosslinker, such as, but not limited to, room temperature, 15°C to 27°C, e.g., 25°C, with continuous stirring; and (ii) dissolving, essentially dissolve, or completely dissolve the crosslinker in the liquid to form a crosslinker solution. Other embodiments may be directed to a method of preparing a plurality of microparticles, wherein a crosslinkable protein is crosslinked in the presence of or when mixed with a crosslinker of the present disclosure. In further embodiments, the crosslinker is an enzyme (e.g., a transglutaminase, e.g., a microbial transglutaminase), which when mixed with the crosslinkable protein forms an enzymatically crosslinked protein, an enzymatically crosslinked foam, or an enzymatically crosslinked particle, or an enzymatically crosslinked fiber. The (b) crosslinked foam in the method of preparing a plurality of microparticles can be formed by: (b1) whipping the (a) crosslinkable protein solution or (b2) adding the (a) crosslinker solution at a temperature sufficient for whipping, stirring, while mixing or stirring with or without the addition of a gas or air (e.g., argon, carbon dioxide, helium, hydrogen, krypton, methane, neon, nitrogen, oxygen, ozone, water vapor, xenon, or combinations thereof) to form the (b) crosslinked foam or (b) crosslinked block, respectively, wherein, for example, the whipping, mixing or stirring occurs at a temperature of 30°C to 40°C (e.g., 37°C).

[0062] Removal of cross-linking agents from the cross-linked proteins, cross-linked foams, and / or microparticles, or compositions comprising the same, is beneficial in one embodiment from the standpoint of safety and control, as well as cost. Accordingly, some embodiments may be directed to a method of the present disclosure, wherein removing (c) comprises: (b) washing the crosslinked foam or block, where the size of the crosslinked foam or block of (b) has been reduced as described herein, and where washing occurs by agitating the crosslinked foam pieces in a liquid (e.g., water, saline, PBS) at a temperature (e.g., 40° C.-60° C.; 45° C.-55° C., e.g., 50° C.) and for a time (e.g., 5 minutes-1 hour; 10 minutes-45 minutes; 15 minutes-30 minutes) sufficient to remove or essentially remove the crosslinker from the crosslinked foam; and sieving the washed foam pieces to a desired size, e.g., using a suitable mesh sieve, such as, but not limited to, a sieve having a mesh number between 35 and 5000 (500 μm-2.5 μm), e.g., a sieve having a mesh number of 0.5 mm or equivalent to 35, thereby forming a crosslinker-free foam piece of the present disclosure comprising pieces of the desired size.

[0063] In one embodiment, such a method may provide a size reduction step (d) that includes cutting (e.g., dicing, chopping, meshing) the formed crosslinked foam or block of (b), the crosslinker-free foam or block of (c), or a combination of the crosslinked foam or block of (b) and the crosslinker-free foam or block of (c). Other non-limiting examples of techniques, methods, or tools for reducing the size of the crosslinked foam or block of (b) and / or the crosslinker-free foam or block of (c) include: cutting (e.g., dicing, chopping, meshing, sieving) using compression, lump breakers, crushers, mills (e.g., impact mills, flour mills, full screen hammer mills, mega hammer mills, air classifier mills, jet mills, ball mills, pebble mills, rod mills); grinders (fine grinders, blade grinders), and the like, or combinations thereof. The size reduction of these methods ranges from 0.1 μm to 10 mm (e.g., 0.2 μm to 9 mm; 0.3 μm to 8 mm; 0.4 μm to 7 mm; 0.5 μm to 7 mm; 1 μm to 6 mm; 5 μm to 5 mm; 10 μm to 4 mm; 20 μm to 1 mm; 40 μm to 500 μm; 60 μm to 200 μm; 90 μm to 150 μm; 95 μm to 100 μm); and more than 1 μm (e.g., 2 μm, 4 μm, 6 μm, 8 μm, 12 μm, 15 μm, 25 μm, 35 μm, 45 μm, 55 μm, 65 μm, 75 μm, 85 μm, 95 μm, 105 μm, 115 μm, 125 μm, 135 μm). , 145 μm, 150 μm, 200 μm, 300 μm, 400 μm, 500 μm, 1 mm, 3 mm, 5 mm, 7 mm, 9 mm); 10 mm or less (e.g., 8 mm, 6 mm, 4 mm, 2 mm, 900 μm, 800 μm, 700 μm, 600 μm, 550 μm, 450 μm, 350 μm, 250 μm, 175 μm, 165 μm, 155 μm, 140 μm, 130 μm, 120 μm, 100 μm, 90 μm, 80 μm, 70 μm, 60 μm, 50 μm, 40 μm, 30 μm, 20 μm, 10 μm, 5 μm, 3 μm, 1 μm).Further embodiments provide methods, wherein the reducing step of (d) results in the formed crosslinked foam or crosslinked foam pieces of (b) having a size of 0.5 mm to 10 mm (e.g., 1 mm to 8 mm; 2 mm to 7 mm; 3 mm to 6 mm; 4 mm to 5 mm); 0.5 mm or more (e.g., 1.5 mm, 2.5 mm, 3.5 mm, 4.5 mm, 5.5 mm, 6.5 mm, 7.5 mm, 8.5 mm, 9.5 mm); 10 mm or less (e.g., 9 mm, 8 mm, 7 mm, 6 mm, 5 mm, 4 mm, 3 mm, 2 mm, 1 mm).

[0064] In yet another embodiment, a method of preparing a plurality of microparticles includes: (e) freezing the crosslinker-free foam or block of (c) or the plurality of particles of (d); lyophilizing the frozen crosslinker-free foam or block of (e); and (f) reducing the size of the lyophilized crosslinker-free foam or block of (f) to form a plurality of crosslinked foam or block particles. Another embodiment of a method of preparing a plurality of microparticles includes: drying the crosslinker-free foam or block of (c) or the plurality of particles of (d); and reducing the size of the dried crosslinker-free foam or block of (c) to form a plurality of dried crosslinked foam or block particles. The crosslinked foam particles may be, for example, 0.1 μm to 2000 μm (e.g., 0.2 μm to 1499 μm; 0.4 μm to 1450 μm; 0.5 μm to 1425 μm; 0.6 μm to 1400 μm; 0.7 μm to 1350 μm; 0.8 μm to 1300 μm; 0.9 μm to 1250 μm; 1 μm to 1200 μm; 2 μm to 1150 μm; 3 μm to 1100 μm; 4 μm to 1050 μm; 5 μm to 1000 μm; 6 μm to 950 μm; 7 μm to 900 μm). ;8μm~850μm;9μm~800μm;10μm~750μm;11μm~700μm;12μm~650μm;13μm~600μm;14μm~550μm;15μm~500μm;16μm~450μm;17μm~400μm;18μm~350μm;19μm~300μm;20μm~250μm;25μm~200μm;30μm~150μm;40μm~100μm;60μm~90μm). Such methods include a crosslinkable protein selected from the group consisting of: gelatin, collagen, casein, albumin, tropoelastin, elastin and any combination thereof; or non-recombinant gelatin, recombinant gelatin, non-recombinant collagen, recombinant collagen, any engineered protein thereof, an engineered polymer containing or linked to at least one RGD motif, etc., or any combination thereof. Further embodiments of such methods also include an enzymatic crosslinker, which may be selected from transglutaminase or oxidase.Other embodiments of the present disclosure provide an enzymatic crosslinker selected from the group consisting of: native transglutaminase, modified transglutaminase, recombinant transglutaminase, microbial transglutaminase (mTG), tissue transglutaminase (tTG), keratinocyte transglutaminase, epithelial transglutaminase, prostate transglutaminase, neuronal transglutaminase, human transglutaminase, factor XIII, etc., or any combination thereof. Some other embodiments may provide an enzymatic crosslinker selected from the group consisting of: native oxidase, modified oxidase, lysyl oxidase, tyrosinase, laccase, peroxidase, etc., or any combination thereof.

[0065] Further embodiments include those in which the freezing in (e) occurs at a temperature sufficient to prepare a lyophilized product, and the temperature is between -18°C and 25°C (e.g., -15°C to 23°C; -10°C to 20°C; -5°C to 15°C; 0°C to 10°C); -18°C or higher (e.g., -16°C; -14°C; -12°C; -8°C; -6°C; -4°C; -2°C; 2°C; 4°C; 6°C; 8°C; 10°C; 12°C; 14°C; 16°C; 18°C; 20°C; 22°C; 24°C); or 25°C or lower (e.g., 23°C; 21°C; 19°C; 17°C; 15°C; 13°C; 11°C; 9°C; 7°C; 5°C; 3°C; 1°C); for a time sufficient to prepare a lyophilized product. ;-1°C;-3°C;-5°C;-7°C;-9°C;-11°C;-13°C;-15°C;-17°C), and for a period of time including: 2 hours to 25 hours (e.g., 7 hours to 24 hours; 9 hours to 22 hours; 11 hours to 20 hours; 13 hours to 18 hours; 15 hours to 16 hours); 5 hours or more (e.g., 6 hours; 8 hours; 10 hours; 12 hours; 14 hours; 16 hours; 18 hours; 20 hours; 22 hours; 24 hours); or 25 hours or less (e.g., 23 hours; 21 hours; 19 hours; 17 hours; 15 hours; 13 hours; 11 hours; 9 hours; 7 hours; 5 hours).

[0066] Still other embodiments include a method for freeze-drying (f) at a temperature selected from: -50°C±10°C (e.g., -60°C to -40°C; -55°C to -35°C; -50°C to -30°C; -45°C to -25°C; -40°C to -20°C; -35°C to -30°C); -60°C or higher (e.g., -58°C; -56°C; -54°C; -52°C; -50°C; -48°C; -46°C; -44°C; -42°C; -40°C); -40°C or lower (e.g., -41°C; -43°C; -45°C; -47°C; -49°C; -51°C; -53°C; -55°C; -57°C; -59°C), at a temperature between 0.01 mbar and 0.1 mbar (e.g., 0.0 in an atmosphere of 2mbar to 0.08mbar; 0.04mbar to 0.06mbar; 0.01mbar or more (e.g. 0.03mbar; 0.05mbar; 0.07mbar; 0.09mbar); or 0.1mbar or less (0.08mbar; 0.06mbar; 0.04mbar; 0.02mbar; 0.01mbar); for 24 hours to 96 hours (e.g. 48 hours to 95 hours; 50 hours to 94 hours; 52 hours to 92 hours; 54 hours to 90 hours; 56 hours to 88 hours; 58 hours to 86 hours; 60 hours to 84 hours; 62 hours to 82 hours; 64 hours to 80 hours) between 66 hours and 78 hours; between 68 hours and 76 hours; between 70 hours and 74 hours; more than 48 hours (e.g. 49 hours; 51 hours; 53 hours; 55 hours; 57 hours; 59 hours; 61 hours; 63 hours; 65 hours; 67 hours; 69 hours; 71 hours; 73 hours; 75 hours; 77 hours; 79 hours; 81 hours; 83 hours; 85 hours; 87 hours; 89 hours); or less than 96 hours (e.g. 94 hours; 92 hours; 90 hours; 88 hours; 86 hours; 84 hours; 82 hours; 80 hours; 78 hours; 76 hours; 74 hours; 72 hours; 70 hours; 68 hours; 66 hours; 64 hours; 62 hours; 60 hours 58 hours; 56 hours; 54 hours; 52 hours; 50 hours; 48 hours; 36 hours); the temperature, pressure, and time are sufficient to produce a (c) lyophilized frozen crosslinker-free foam or (d) lyophilized plurality of particles, and a "lyophilized" product, such as, but not limited to, as used herein, a (c) lyophilized crosslinker-free foam or (d) lyophilized plurality of particles, is 4% or less (e.g., 3.8%; 3.6%; 3.4%; 3.2%; 3%; 2.8%; 2.6%; 2.4%; 2.2%; 2%; 1.8%;1.6%;1.4%;1.2%;1%;0.8%;0.6%;0.4%;0.2%;0.08%;0.06%;0.04%;0.02%;0%);0% or more (e.g. 0.01%;0.03%;0.05%;0.07%;0.09%;1.1%;1.3%;1.5%;1.7%;1.9%;2. The present invention may be directed to a method of producing a product having a moisture content of 1%; 2.3%; 2.5%; 2.7%; 2.9%; 3.1%; 3.3%; 3.5%; 3.7%; 3.9%; or 0%-4% (0.5%-3.5%; 0.7%-3.3%; 1.1%-2.9%; 1.3%-2.7%; 1.5%-2.5%). The temperature, pressure, and time required to adequately freeze-dry the crosslinker-free foam or plurality of particles are understood by one of ordinary skill in the art and do not require undue experimentation to optimize these parameters.

[0067] In yet another embodiment, such method of preparing a plurality of microparticles comprises: (e) freezing the crosslinker-free foam or block of (c) or the plurality of microparticles of (d); and / or (f) drying the crosslinker-free foam or hydrogel block of (c) or (e) or the plurality of microparticles of (d). Some embodiments are directed to drying, including but not limited to lyophilization or freeze drying, oven drying, and room or ambient temperature drying. The method of preparing a plurality of microparticles further comprises: (g) reducing the size of the dried crosslinker-free foam or hydrogel block of (e) and / or (f) to form a plurality of crosslinked foam particles or non-foam crosslinked hydrogel particles. The multiple crosslinked microparticles may be, for example, 0.1 μm to 2000 μm (e.g., 0.2 μm to 1499 μm; 0.4 μm to 1450 μm; 0.5 μm to 1425 μm; 0.6 μm to 1400 μm; 0.7 μm to 1350 μm; 0.8 μm to 1300 μm; 0.9 μm to 1250 μm; 1 μm to 1200 μm; 2 μm to 1150 μm; 3 μm to 1100 μm; 4 μm to 1050 μm; 5 μm to 1000 μm; 6 μm to 950 μm; 7 μm to 900 μm). m; 8μm~850μm; 9μm~800μm; 10μm~750μm; 11μm~700μm; 12μm~650μm; 13μm~600μm; 14μm~550μm; 15μm~500μm; 16μm~450μm; 17μm~400μm; 18μm~350μm; 19μm~300μm; 20μm~250μm; 25μm~200μm; 30μm~150μm; 40μm~100μm; 60μm~90μm).

[0068] In another embodiment, the size reduction in (g) comprises: grinding the dried (e.g., lyophilized) crosslinker-free foam or hydrogel block of (e) to form a plurality of crosslinker-free foam particles; and separating by size the plurality of crosslinker-free foam or hydrogel particles of the present disclosure. Such methods, where the plurality of crosslinker-free foam or hydrogel particles comprises a particle size, e.g., from 0.1 μm to 2000 μm, may comprise reducing the size or separating by size the plurality of crosslinker-free foam particles by sieving the plurality of crosslinker-free foam particles sufficiently to produce a plurality of crosslinked foam particles having different particle size ranges selected from a particle size or average particle size of 0.1 μm to 2000 μm, the different particle size ranges comprising at least two different particle size ranges.

[0069] composition

[0070] In some embodiments, the present disclosure may relate to a composition comprising (a) a plurality of microparticles as described herein, with or without (b) a carrier. The composition of the present disclosure comprises (a) a plurality of microparticles, the plurality of microparticles comprising a crosslinked protein, the crosslinked protein comprising at least one RGD (Arg-Gly-Asp) motif; the plurality of microparticles not comprising a crosslinker; the plurality of microparticles being all or independently water-insoluble; and (b) with or without a carrier. Furthermore, the composition of the present disclosure is injectable. Some embodiments are directed to a composition comprising a plurality of microparticles comprising at least two different particle sizes ranging from 0.1 μm to 2000 μm (e.g., 5 μm to 150 μm); or a combination thereof.

[0071] In another embodiment, a composition of the present disclosure comprises: (a) a plurality of microparticles as described herein, wherein the microparticle or plurality of microparticles comprises a crosslinked protein, wherein the protein of the crosslinked protein comprises at least one RGD (Arg-Gly-Asp) motif, wherein the plurality of microparticles is essentially or substantially free of crosslinkers, and wherein the plurality of microparticles is water-insoluble; and optionally (b) a carrier.Such compositions comprising a plurality of microparticles of the present disclosure may have a particle size ranging from 0.1 μm to 2000 μm (e.g., 0.2 μm to 1499 μm; 0.4 μm to 1450 μm; 0.5 μm to 1425 μm; 0.6 μm to 1400 μm; 0.7 μm to 1350 μm; 0.8 μm to 1300 μm; 0.9 μm to 1250 μm; 1 μm to 1200 μm; 2 μm to 1150 μm; 3 μm to 1100 μm; 4 μm to 1050 μm; 5 μm to 1000 μm; 6 μm to 950 μm; 7 μm to 900 μm). At least two different particle sizes in the ranges of;8μm-850μm;9μm-800μm;10μm-750μm;11μm-700μm;12μm-650μm;13μm-600μm;14μm-550μm;15μm-500μm;16μm-450μm;17μm-400μm;18μm-350μm;19μm-300μm;20μm-250μm;25μm-200μm;30μm-150μm;40μm-100μm;60μm-90μm or at least two different particle sizes are between 0.1 μm and 2000 μm (e.g. 0.2 μm to 1499 μm; 0.4 μm to 1450 μm; 0.5 μm to 1425 μm; 0.6 μm to 1400 μm; 0.7 μm to 1350 μm; 0.8 μm to 1300 μm; 0.9 μm to 1250 μm; 1 μm to 1200 μm; 2 μm to 1150 μm; 3 μm to 1100 μm; 4 μm to 1050 μm; 5 μm to 1000 μm; 6 μm to 950 μm; 7 μm to 9 00μm; 8μm to 850μm; 9μm to 800μm; 10μm to 750μm; 11μm to 700μm; 12μm to 650μm; 13μm to 600μm; 14μm to 550μm; 15μm to 500μm; 16μm to 450μm; 17μm to 400μm; 18μm to 350μm; 19μm to 300μm; 20μm to 250μm; 30μm to 150μm; 40μm to 100μm; 60μm to 90μm); and (b) a carrier.

[0072] Yet another embodiment of the present disclosure provides a composition as disclosed herein, wherein the cross-linked protein is selected from the group consisting of: gelatin, collagen, elastin, tropoelastin, casein, albumin, any engineered protein thereof, analogous proteins thereof, etc., or combinations thereof. In a further embodiment, the cross-linked protein may be selected from the group consisting of: non-recombinant gelatin, recombinant gelatin, non-recombinant collagen, recombinant collagen, engineered proteins thereof, any engineered polymer containing or linked to an RGD motif, etc., or combinations thereof. Other embodiments may be directed to such compositions as described herein comprising a plurality of microparticles and such a plurality of microparticles, wherein the cross-linked protein comprises gelatin or collagen. In a further embodiment, the compositions as described herein comprising a plurality of microparticles and such a plurality of microparticles are directed to a cross-linked protein comprised of gelatin.

[0073] In some embodiments of the composition, the carrier may comprise a hydrogel. Some aspects of the embodiment provide a hydrogel carrier, which as used herein, in one embodiment, refers to a gel or semi-solid hydrophilic polymer of at least 10% HO. The carrier and / or lubricant may also be selected from the group consisting of, but not limited to: gelatin (e.g., cross-linked (2% w / v); non-cross-linked gelatin (0.25%-2% w / v)); or in situ cross-linked gelatin (0.1% w / v-10% w / v); collagen (e.g., cross-linked; non-cross-linked); alginate; carboxymethylcellulose (CMC) (1%-3.5% w / v); poly(ethylene oxide) (PEO); poly(vinyl alcohol) (PVA); poly(propylene fumarate) (PPF); polyethylene glycol (PEG); glycosaminoglycan polymers such as hyaluronic acid (HA) (e.g., cross-linked and non-cross-linked HA (0.01%-10% w / v)), and the like, or any combination thereof. The carrier may comprise a single carrier or a mixture of two or more carriers (e.g., a first carrier and a second carrier of the same but different weight average molecular weight). Non-limiting examples of carriers include glycosaminoglycan polymers (e.g., hyaluronic acid, cross-linked hyaluronic acid, keratan sulfate, chondroitin sulfate, and / or heparin), extracellular matrix protein polymers (e.g., gelatin, collagen, elastin, and / or fibronectin). Other embodiments are directed to a composition of the present disclosure comprising a plurality of microparticles and a carrier, wherein the carrier is selected from the group consisting of: gelatin; collagen; alginate; glycosaminoglycan (GAG); polyethylene glycol (PEG); carboxymethylcellulose; and combinations thereof.Some embodiments provide compositions of the present disclosure comprising a carrier selected from the group consisting of: non-crosslinked chondroitin sulfate polymers, non-crosslinked dermatan sulfate polymers, non-crosslinked keratan sulfate polymers, non-crosslinked heparan polymers, non-crosslinked heparan sulfate polymers, non-crosslinked hyaluronan polymers, non-crosslinked glycosaminoglycan polymers, non-crosslinked elastin and / or fibronectin, and combinations thereof.

[0074] A further embodiment provided herein is an injectable composition comprising a crosslinked hyaluronic acid carrier and a plurality of microparticles, wherein the crosslinked hyaluronic acid has a crosslink density of about 3 mol % to about 40 mol %.

[0075] In some embodiments where there are at least two carriers, a first carrier may comprise hyaluronic acid having a weight average molecular weight of about 200 kDa to about 1 MDa, and optionally a second carrier comprises hyaluronic acid having a weight average molecular weight of about 200 kDa to about 5 MDa. In some embodiments, the hyaluronic acid polymer may have a concentration of about 0.1% w / v to 10% w / v.

[0076] The average particle size of the protein microparticles in some embodiments involving the compositions described herein may be selected to suit the needs of each application. For example, a smaller average particle size may be desirable for the treatment of fine lines and wrinkles, while a larger average particle size may be more suitable for vocal fold augmentation or even large volume reconstruction (e.g., breast reconstruction).

[0077] Another embodiment relates to a composition of the present disclosure comprising: a plurality of microparticles as described herein and a carrier. Non-limiting examples of carriers useful in the embodiments of the present disclosure are selected from the group consisting of: non-crosslinked gelatin; non-crosslinked collagen; non-crosslinked alginate; non-crosslinked hyaluronic acid; and combinations thereof. An inactive crosslinker, such as a reserve agent, may be added to react with the non-crosslinked carrier in situ, thereby maintaining the particles at the injection site. Another embodiment provides, for example, non-crosslinked gelatin crosslinkable proteins and active crosslinker enzymes capable of crosslinking in situ, thereby maintaining the particles in the hydrogel for a longer period of time in situ compared to using non-active crosslinkers.

[0078] In one embodiment, a composition of the present disclosure that does not include a crosslinker but includes a plurality of microparticles comprising crosslinked proteins, and a carrier, is provided that is at a concentration of: 1 mg / ml or more (e.g., 10 mg / ml; 20 mg / ml; 30 mg / ml; 40 mg / ml; 50 mg / ml; 60 mg / ml; 70 mg / ml; 80 mg / ml; 90 mg / ml; 100 mg / ml; 110 mg / ml; 120 mg / ml; 130 mg / ml; 140 mg / ml; 150 mg / ml; 200 mg / ml; 300 mg / ml); 300 mg / ml; 400 mg / ml; 500 mg / ml; 600 mg / ml; 70 mg / ml; 80 mg / ml; 90 mg / ml; 100 mg / ml; 110 mg / ml; 120 mg / ml; 130 mg / ml; 140 mg / ml; 150 mg / ml; 200 mg / ml; 300 mg / ml); mg / ml or less (e.g. 290mg / ml; 280mg / ml; 270mg / ml; 260mg / ml; 250mg / ml; 240mg / ml; 230mg / ml; 220mg / ml; 210mg / ml; 200mg / ml; 190mg / ml; 1 80mg / ml;170mg / ml;160mg / ml;155mg / ml;145mg / ml;135mg / ml;125mg / ml;115mg / ml;105mg / ml;95mg / ml;85mg / ml;75mg / ml;65mg / ml; 55mg / ml; 45mg / ml; 35mg / ml; 25mg / ml; 15mg / ml; 5mg / ml); 5mg / ml;8mg / ml~265mg / ml;12mg / ml~255mg / ml;14mg / ml~245mg / ml;16mg / ml~235mg / ml;18mg / ml~225mg / ml;22mg / ml~215mg / ml;24m The microparticles have a concentration of 100mg / ml-205mg / ml; 26mg / ml-195mg / ml; 28mg / ml-185mg / ml; 32mg / ml-175mg / ml; 34mg / ml-165mg / ml; 36mg / ml-153mg / ml; 38mg / ml-143mg / ml; 42mg / ml-133mg / ml; 52mg / ml-123mg / ml; 62mg / ml-113mg / ml; 72mg / ml-103mg / ml; 82mg / ml-93mg / ml).

[0079] In some embodiments involving foam particles described herein, the foam particle population may have a modulus of elasticity of at least about 0.5 kPa or greater (as measured in a 0.1 Hz to 10 Hz frequency sweep).

[0080] Some embodiments provide a microparticle or microparticles described herein, wherein at least about 40% (e.g., at least about 50%, at least about 60%, at least about 70% or more) of the microparticle pores have an aspect ratio of about 1.0 to about 2.0.

[0081] In further embodiments involving the particles described herein, the particle pores have an average aspect ratio of from about 1 to about 2.5.

[0082] Further embodiments provide a microparticle or microparticles as described herein, wherein the microparticles may be hydrated in an aqueous solution, including, for example and without limitation, water, saline, a buffer solution, such as phosphate buffer solution, or a combination thereof.

[0083] Tissue scaffolds

[0084] Another embodiment provides: a tissue scaffold comprising a plurality of microparticles as described herein, wherein the plurality of microparticles comprises crosslinked protein microparticles, the plurality of microparticles comprising a protein, for example, a crosslinked protein selected from gelatin; collagen; and combinations thereof, the plurality of microparticles being water-insoluble, the plurality of microparticles having a particle size of 1 μm to 2000 μm (e.g., 5 μm to 150 μm) or an average particle size of 1 μm to 1500 μm (e.g., 5 μm to 150 μm). In some embodiments, the tissue scaffold further comprises a hydrogel carrier, the hydrogel carrier being selected from, but not limited to, gelatin; collagen; alginate; hyaluronic acid; carboxymethylcellulose; poly(ethylene oxide) (PEO); poly(vinyl alcohol) (PVA); poly(propylene fumarate) (PPF); polyethylene glycol (PEG), and the like, or any combination thereof. Other embodiments are directed to such tissue scaffolds comprising a dispersion of crosslinked protein microparticles or a dispersion of a plurality of microparticles as described herein in a hydrogel carrier. Further embodiments provide such tissue scaffolds, wherein the tissue scaffold is configured as a foam. In yet another embodiment, the tissue scaffold of the present disclosure comprises a plurality of microparticles of crosslinked protein microparticles, the plurality of microparticles being free of crosslinking agents and water insoluble, and the crosslinked protein microparticle or plurality of microparticles comprises at least two different or independent particle sizes. Still other embodiments provide such tissue scaffolds of the present disclosure comprising or configured in a three-dimensional shape. One embodiment relates to a tissue scaffold having at least two different or independent particle sizes, including particle sizes selected from 1 μm to 2000 μm (e.g., 5 μm to 120 μm; 40 μm to 100 μm; 60 μm to 90 μm).

[0085] Device

[0086] Further embodiments of the present disclosure provide a device comprising the composition described herein. In one embodiment, the device of the present disclosure comprises: a plurality of microparticles, the plurality of microparticles comprising crosslinked proteins, the protein of the crosslinked proteins comprising at least one RGD (Arg-Gly-Asp) motif, the plurality of microparticles comprising crosslinked proteins or the composition comprising the plurality of microparticles is free of crosslinking agents and insoluble in water; and a carrier, such as a hydrogel; the device is a syringe, cartridge or vial. Other embodiments provide: (a) a plurality of microparticles comprising crosslinked gelatin, the plurality of microparticles being essentially or substantially free of crosslinking agents and insoluble in water; and (b) a syringe comprising a hydrogel carrier or a composition comprising the hydrogel carrier, the syringe and / or its contents being sterile, sterilizable or configured for sterilization. Non-limiting examples of sterilization methods, techniques, or tools thereof include: steam sterilization (e.g., autoclaves); flaming; heat sterilization (e.g., hot air ovens for dry heat sterilization; glass bead sterilizers); chemical sterilization (e.g., ethylene oxide gas sterilization, nitrogen dioxide sterilization, sterilization with glutaraldehyde and formaldehyde solutions, hydrogen peroxide sterilization (e.g., liquid and evaporation), peracetic acid sterilization); irradiation sterilization (e.g., electromagnetic irradiation with ultraviolet (UV) light sterilization (e.g., UV-C or germicidal UV sterilization (e.g., far-UVC sterilization)); irradiation with gamma rays, x-rays, or electron beams); broad-spectrum UV (including but not limited to UV-A, UV-B, and UV-C wavelengths, or any combination thereof); low-temperature sterilization (e.g., evaporated hydrogen peroxide, peracetic acid soak, ozone), and the like, or any combination thereof.Further embodiments of the present disclosure provide a device, e.g., a syringe, comprising a needle selected from 14 gauge (G) to 39 gauge (e.g., 18 gauge to 30 gauge; 20 gauge to 29 gauge; 22 gauge to 27 gauge; 25 gauge to 26 gauge; 27 gauge to 30 gauge; 17G; 18G; 19G; 20G; 21G; 22G; 23G; 24G; 25G; 26G; 27G; 28G; 29G; 30G), where the lower gauge (i.e., thicker needle) allows for easier injection of the plurality of microparticles or composition of the present disclosure; whereas the higher gauge (i.e., thinner needle) causes less damage to the dermis of a subject requiring a tissue scaffold, a plurality of microparticles; or a composition comprising a plurality of microparticles. Some embodiments for dermatological applications may include, for example, a syringe device that may be attached or configured to be attached to a number of different needles, e.g., 27 gauge to 39 gauge needles. For example, a 27 gauge needle may be included, the biomaterial may be included, and the needle may be 2N to 70N (e.g., 3N to 60N; 4N to 50N; 5N to 40N; 6N to 30N; 7N to 20N); 70N or less than 70N (e.g., 65N; 55N; 45N; 35N; 25N; 15N; 5N); 2N or more than 2N (e.g., 3N; 4N; 5N; 6N; 7N; 8N; 9N; 10N; 11N A syringe capable of injecting the particles and / or compositions of the present disclosure while maintaining an injection force or load of 0.1 N; 0.1 N; 0.1 N; 0.1 N; 0.1 N; 0.1 N; 0.2 N; 0.2 N; 0.3 N; 0.4 N; 0.5 N; 0.6 N; 0.7 N) is included by some methods of the present disclosure. The tissue scaffold may be a porous gelatinous tissue scaffold in some embodiments. Other such embodiments provide a three-dimensional tissue scaffold. Further embodiments may be directed to a device, such as a syringe, that includes a plurality of microparticles, a composition comprising a plurality of microparticles of the present disclosure, or a tissue scaffold as described herein.

[0087] Further embodiments of the present disclosure provide devices, such as syringes, cartridges, vials, or additive manufacturing devices (e.g., for biofabrication), comprising the microparticles of the present disclosure, wherein the microparticles are disposed in a plate or supporting hydrogel, in tissue, or on or in the body of a subject.

[0088] In some embodiments of the present disclosure, the composition of any embodiment described herein provides an injectable composition that may be preloaded into a device or delivery device, such as a syringe. In some embodiments, the syringe is coupled to a tube through a handle, so that the composition can be injected through the tube. The tube may be further coupled to an endoscope or cystoscope during a procedure. The needle may be a hollow needle attached to the tube. The tube may be disposed within an outer barrel tube and movable within the tube. The needle may be movable between a retracted position within the barrel tube and an extended position where the tip of the needle is outside the barrel to control the injection of the composition. In some embodiments, the outer barrel tube is inserted into a channel of an endoscope with the needle and inner tube within the outer barrel tube. The delivery device may include a handle that is actuated by a user, which moves the inner tube distally relative to the outer barrel, thereby advancing the needle distally through the outer barrel tube to an extended position where the needle tip is exposed and injecting a composition or a plurality of microparticles as described herein into a tissue or region of interest.

[0089] In other embodiments for small volume bulking applications, the composition or plurality of microparticles may be injected with a 14 gauge to 39 gauge needle using an average extrusion force of about 30 N or less. Examples of small volume bulking applications include, but are not limited to, dermal fillers for skin tissue (e.g., to treat facial skin tissue having facial lines, wrinkles, or scars to be filled), urethral bulking (e.g., to treat stress urinary incontinence), cervical tissue bulking (e.g., to treat cervical insufficiency), or vocal cord bulking (e.g., to correct vocal cord paralysis or other causes of vocal cord insufficiency).

[0090] Uses and Treatments

[0091] Yet further embodiments provide for the use of a plurality of microparticles, a composition comprising a plurality of microparticles, a tissue scaffold, a device comprising a plurality of microparticles and / or a composition comprising a plurality of microparticles for any one or more of: body contouring, tissue engineering, regenerative medicine, and aesthetic dermatology, some embodiments further providing body contouring selected from the group consisting of: soft tissue reconstruction, volume restoration, breast augmentation, biostimulation, etc., or combinations thereof. Another use embodiment of the present disclosure includes biostimulation as used herein, selected from the group consisting of: fibroblast stimulation, collagen production stimulation, de novo collagen production (i.e., the process of making new collagen), tissue re-growth, angiogenesis induction, tissue scaffolding provision, etc., or any combinations thereof. Yet further use embodiments of the present disclosure relate to a composition and / or a plurality of microparticles set in a device as described herein, the device being, for example, a syringe, cartridge, or vial.

[0092] The method of the present disclosure provides a method of treating a subject (including an animal, including a human) in need of body contouring as described herein, comprising administering a composition of a plurality of microparticles and a carrier to the site of the subject in need of body contouring. Such a method includes, for example, administering by injecting a composition of a plurality of microparticles and a carrier to the site of the subject in need of body contouring. Another embodiment of the present disclosure provides such a method of treating a subject in need of body contouring, wherein the administration includes: stimulating fibroblasts; stimulating collagen production; inducing new collagen production; inducing tissue re-growth; inducing angiogenesis; providing tissue scaffolding, etc., or any combination thereof.

[0093] In some embodiments of the present disclosure, a plurality of microparticles suspended in a hydrogel carrier to form a composition may be injected through a sterile syringe containing the composition into a site of a subject where the subject is in need of therapeutic and / or aesthetic application. The compositions or formulations described herein may be injected into the subcutaneous layer (also known as subcutis, hypodermis), soft tissue, and mammary gland as needed. This technique may be utilized in combination with others, such as ultrasound and x-ray, to visualize injection placement. Furthermore, injecting a tissue scaffold into a subject using a minimally invasive method minimizes the risk of infection, costs associated with surgery, and / or potential for medical errors due to the minimal exposure of the body cavity: performing an open surgery. Additionally, the method of treating a subject described herein by injecting a plurality of microparticles suspended in a hydrogel carrier as a composition also reduces recovery time and pain compared to typical surgery that requires a large incision or opening larger than the size of the syringe and / or needle used herein.

[0094] Some embodiments are directed to the type of administration of the compositions or formulations described herein, wherein the compositions or formulations are administered into the subcutaneous layer of the skin (also known as subcutaneous tissue). The skin may include facial skin, buttock skin, or any soft tissue. The compositions and formulations described herein may also include administration into the mammary gland or into adipose tissue for breast reconstruction procedures in subjects. Preclinical data has shown gelatin microparticles of the present disclosure with a non-crosslinked gelatin carrier injected into the subcutaneous (SC) layer of the skin in rat and pig models, and into the mammary gland in pig models. See, for example, Example 2.

[0095] In some embodiments, the microparticles or compositions comprising the microparticles of the present disclosure as a scaffold can be used to provide immediate physical and mechanical stabilization of tissue damage or skin lifting / expansion through the biomechanical force of the scaffold, which may be an implant. The implants of the present disclosure can also be used as temporary scaffolds for soft tissue support to repair and reinforce defects at sites of weakness or voids that require the addition of material to achieve a desired surgical outcome. After implantation, the implant and / or the ingrowth-in native tissue resulting from the implant can maintain at least 10% volume of the implant volume at time zero (i.e., 100% volume at time zero) at 1 month, 3 months, or 6 months. The implant is not immediately degraded, but is replaced by tissue stimulated by the implant (e.g., stimulate fibroblast and / or collagen production; induce new collagen production; induce tissue repopulation; provide tissue scaffold, etc., or any combination thereof). The implant may act as a biostimulant, so that stimulated cells or tissue remain in the subject for 3 to 6 months, e.g., 10% to 100% (e.g., 20% to 50%) of the volume of the original implant. New cells or tissue may be induced by the implant and may replace the microparticle implant.

[0096] Another embodiment provides a plurality of microparticles and / or compositions of the present disclosure that act as biostimulants and tissue scaffolds. For example, upon administration of the plurality of microparticles and / or compositions, fibroblast and collagen production may be stimulated, new collagen production and / or tissue re-growth may be induced, and / or tissue scaffolds may be utilized, all within the bounds of a safe, effective, and inexpensive tissue scaffold and / or biostimulant for use in therapeutic, aesthetic dermatology, and reconstructive procedures or surgery.

[0097] As illustrated in Example 16, some embodiments of the present disclosure provide a composition comprising a plurality of microparticles or a use of a plurality of microparticles, the composition or the plurality of microparticles acting as a microcarrier for viable cells for either in vitro or in vivo applications. For example, in vitro culture of cells comprising the foam particles (FP) described herein may be used for the study of proteins, micro-organs for drug development, etc., for the manufacture of biomaterials for medical purposes, microstructures for tissue engineering, or as agents for the enhancement of cell-based therapies. In some embodiments, these cells can be grown and maintained in large numbers, e.g., in a continuous manner. However, this is difficult to achieve with standard two-dimensional cell culture methods (i.e., surface culture on plastic plates, flasks, etc.), and the microparticles (e.g., FP) of the present disclosure can act as microcarriers, for example, allowing three-dimensional suspension culture, optimally utilizing culture volume and medium, and allowing single batch as well as continuous culture processes. Furthermore, such microcarriers can provide support for cells used in cell-based therapies in vivo, e.g., cells injected into tissue, enhancing cell survival in vivo. The microparticles (eg, FPs) of the present disclosure may also be used for cell differentiation using stem cells or satellite cells at optimal conditions, such as environmental conditions, seeding concentrations and media selection, for the desired cell line differentiation.

[0098] Another embodiment provides ex vivo tissue engineering, for example to support cell growth and promote the formation of tissue-like micro-organs for transplantation, or to provide three-dimensional (3D) scaffolding for use in drug screening or for protein production.

[0099] In some embodiments, the composition or microparticles of the present disclosure can be used for in vitro tissue or cell culture, and when cells (e.g., mammalian cells) are contacted with the composition or microparticles, the cells can grow and, as such, express certain proteins, and the cells can expand and express more proteins. Some embodiments are directed to mammalian cells that require an RGD-rich scaffold for growth or expansion. Non-limiting examples of cells include: fibroblasts, epithelial, Chinese hamster ovary (CHO), NS0 and Sp2 / 0 murine myeloma cells, HEK293 cells, human diploid (HeLa) cells, baby hamster kidney (BHK21) cells, etc. that express proteins selected from the group consisting of structural extracellular matrix (ECM) components, such as collagen, elastin, gelatin, hormones, monoclonal antibodies, enzymes, FC fusion proteins, cytokines and growth factors, clotting factors, respectively. For example, a composition or a plurality of microparticles of the present disclosure may be used as a microcarrier or scaffold for cell attachment, growth, expansion, or a combination thereof, and the cells may be any commonly known and used mammalian adherent cells, such as, but not limited to: fibroblasts, epithelial, Chinese hamster ovary (CHO), NS0 and Sp2 / 0 murine myeloma cells, HEK293 cells, human diploid (HeLa) cells, baby hamster kidney (BHK21) cells, cardiomyocytes, induced pluripotent stem cells, etc. In some embodiments, fibroblasts, cardiomyocytes, and induced pluripotent stem cells are commonly used cells that are representative of other cell types used in the development and study of small organs.

[0100] Further embodiments are directed to the use of the compositions or a plurality of microparticles described herein for protein purification by in vitro tissue or cell culture. In some embodiments, protein purification may be achieved by harvesting the expressed protein and filtering the protein in the medium, where filtering or separating the water-soluble protein from the water-insoluble microparticles occurs, for example, by filtration or centrifugation for collection.

[0101] Some embodiments relate to a method of producing a protein (e.g., cell-free) comprising: growing or culturing a plurality of protein-producing or expressing cells in a cell culture medium comprising a plurality of microparticles or a composition comprising a plurality of microparticles as described herein, and the medium under conditions sufficient to culture the cells, and inducing protein expression or synthesis. In some embodiments, the cells are: Hormones: chorionic gonadotropin alpha, follitropin alpha, follitropin beta, luteinizing hormone, bone morphogenetic protein-1, thyroid stimulating hormone alpha, clotting factors, factor VIII, factor IX, insulin, somatropin, collagen, Antibodies: adalimumab, alemtuzumab, bevacizumab, brentuximab, denosumab, golimumab, ibritumomab tiuxetan, ipilimumab, obinutuzumab, omalizumab, pertuzumab, rituximab, siltuximab, tocilizumab, trastuzumab, vedolizumab, ado-trastuzumab emtansine, ustekinumab, Enzymes: agalsidase beta, alglucosidase alpha, alteplase, elosulfase, GalNAc Mammalian cells (e.g., fibroblasts, epithelial cells, Chinese hamster ovary (CHO), NS0 and Sp2 / 0 murine myeloma cells, HEK293 cells, human diploid (HeLa) cells, baby hamster kidney (BHK21) cells) that can be used to produce proteins selected from the group consisting of structural ECM components such as collagen, elastin, hormones, monoclonal antibodies, enzymes, FC fusion proteins, cytokines and growth factors, including 4-sulfatase, human DNase, hyaluronidase, imiglucerase, laronidase, tenecteplase, growth factors, and cytokines such as darbepoetin alfa, interferon beta-1a, epoetin alfa, epoetin beta, epoetin theta, etc.

[0102] Embodiments of the present disclosure are also directed to methods of culturing any of the aforementioned cells (e.g., mammalian adherent) on microcarriers, wherein the microcarriers are a plurality of microparticles as described herein having a dry particle size of 5 μm to 2000 μm (e.g., 99 μm to 700 μm). In some embodiments, the cells are adherent mammalian cells, such as human fibroblasts, epithelial cells, Chinese hamster ovary (CHO), NS0 and Sp2 / 0 murine myeloma cells, HEK293 cells, human diploid (HeLa) cells, baby hamster kidney (BHK21) cells, and any of the aforementioned cells, which are representative of cell types that are common and useful for in vitro cell culture for protein expression or purification.

[0103] Some embodiments of the present disclosure relate to a method of producing a protein, e.g., a cell-free protein, comprising: growing a plurality of protein-producing cells in a cell culture comprising a plurality of microparticles of the present disclosure and a medium, wherein the growth occurs under conditions that induce protein synthesis, thereby producing a cell-free protein. Non-limiting examples of protein-producing cells include: fibroblasts for collagen production, epithelial cells, monoclonal antibodies: adalimumab, alemtuzumab, bevacizumab, brentuximab, denosumab, golimumab, ibritumomab tiuxetan, ipilimumab, obinutuzumab, omalizumab, pertuzumab, rituximab, siltuximab, tocilizumab, trastuzumab, vedolizumab, ado-trastuzumab emtansine, ustekinumab production, or: agalsidase beta, alglucosidase alpha, alteplase, elosulfase, GalNAc Chinese hamster ovary (CHO) cells for the production of enzymes such as 4-sulfatase, human DNase, hyaluronidase, imiglucerase, laronidase, tenecteplase, or for the production of hormones such as: chorionic gonadotropin alpha, follitropin alpha, follitropin beta, luteinizing hormone, bone morphogenetic protein-1, thyroid stimulating hormone alpha, clotting factors, factor VIII, factor IX, insulin, somatropin; NS0 and Sp2 / 0 murine myeloma cells for the production of monoclonal antibodies such as belimumab, natalizumab, ofatumumab, palivizumab, ramucirumab, abciximab, basiliximab, canakinumab, cetuximab, infliximab; and HEK293 cells, human diploid (HeLa) cells, baby hamster kidney (BHK21) cells for the production of clotting factors such as factor VIIa or factor VIII. In some embodiments, the cell-free or essentially cell-free methods of producing proteins described herein produce a protein or cell-free protein selected from the group consisting of: collagen; hormones; monoclonal antibodies; enzymes; growth factors; cytokines; and combinations thereof.

[0104] In some embodiments, a method for producing a differentiated cell or a plurality of differentiated cells includes: growing a plurality of cells, including but not limited to induced pluripotent stem cells, skin stem cells, epidermal stem cells, etc. The plurality of cells is grown in a cell culture or cell medium comprising a plurality of microparticles or a composition comprising a plurality of microparticles of the present disclosure (i.e., a crosslinked protein comprising at least one RGD motif, wherein the plurality of microparticles is free, substantially free, or essentially free of crosslinking agents), and the cells are grown under conditions sufficient to induce cell differentiation, thereby producing differentiated cells. For example, the plurality of cells described above can be differentiated into functional cells, such as functional cardiomyocytes.

[0105] Further embodiments of the present disclosure relate to methods of culturing cells (e.g., mammalian adherent) on microcarriers, wherein the microcarriers comprise a plurality of microparticles described herein having a dry particle size of 5 μm to 2000 μm. In some embodiments, the cells are adherent mammalian cells suitable for differentiation, such as induced pluripotent stem cells (iPS), embryonic stem cells, hematopoietic stem cells, mesenchymal stem cells, satellite cells, and any of the cells described above.

[0106] All terms used herein are intended to have their usual meaning in the art unless otherwise provided. All concentrations refer to the weight percent of the specified component relative to the total weight of the topical composition unless otherwise defined.

[0107] As used herein, "a" or "an" shall mean one or more. As used herein, the word "a" or "an" when used in conjunction with the word "comprising" shall mean one or more than one. As used herein, "another" means at least a second or more.

[0108] As used herein, all ranges of numerical values ​​include the endpoints and all possible values ​​disclosed between the disclosed values. All half-integer numerical exact values ​​are also specifically disclosed and are intended as limitations on all subsets of the disclosed ranges. For example, the range of 0.1% to 3% specifically discloses the percentages 0.1%, 1%, 1.5%, 2.0%, 2.5%, and 3%. Furthermore, the range of 0.1% to 3% includes subsets of the original range including 0.5% to 2.5%, 1% to 3%, 0.1% to 2.5%, etc. It will be understood that the sum of all weight percentages of the individual components does not exceed 100%.

[0109] By "consisting essentially of," it is meant that the composition includes only the recited components, along with normal impurities present in commercially available materials, and any other additives present at levels that do not affect the operation of the invention as described in the disclosed embodiments, such as less than 5% by weight, or less than 1%, or even 0.5% by weight. EXAMPLES

[0110] The following examples illustrate certain aspects of the present description. The examples should not be construed as limiting, but merely to provide a particular understanding and implementation of the embodiments and their various aspects.

[0111] For example, the Examples herein describe the preparation of enzymatically (mTG) crosslinked gelatin foam microparticles that essentially form tissue scaffolds, describe the rheological properties of the disclosed compositions in the context of injectability, demonstrate the safety and efficacy of the disclosed microparticles and compositions as injectable dermal fillers, and show low inflammation and significant new collagen production in animal model studies.

[0112] Example 1: Preparation of crosslinked gelatin foam microparticles.

[0113] Crosslinked gelatin foam microparticles were prepared as follows:

[0114] 1) Gelatin powder was gradually added with continuous stirring to 50°C water until completely dissolved.

[0115] 2) Separately, microbial transglutaminase (mTG) was gradually added with continuous stirring to water at 25° C. until completely dissolved.

[0116] 3) The dissolved gelatin solution is whipped into a foam at ~37°C using a whipping machine, aerated, or stirred or agitated, for example by adding gas or air (e.g., argon, carbon dioxide, helium, hydrogen, krypton, methane, neon, nitrogen, oxygen, ozone, water vapor, xenon).

[0117] 4) The dissolved mTG solution was gradually added into the gelatin solution with stirring, e.g., without the addition of gas or air, until a cross-linked gelatin confluent hydrogel block was formed.

[0118] 5) Dice or cut the foam or hydrogel block into pieces (e.g., 5 mm to 20 mm (i.e., 2 cm)).

[0119] 6) The diced hydrogel block or foam was washed twice with agitation in water at 50°C until the mTG enzyme was removed or washed away or until most of the enzyme was removed to form a diced hydrogel or foam without crosslinker.

[0120] 7) The washed, diced crosslinked gelatin foam or hydrogel block was frozen on a tray at -18°C overnight (e.g., 2 h to 25 h) and then lyophilized at 0.04 mbar to 0.05 mbar for 48 h.

[0121] 8) The lyophilized foam or hydrogel block was ground using either a jet mill or a grade grinder and separated into particle size groups (e.g., 0.1 μm to 2000 μm) by passing the powder through a sieve (e.g., 35 US mesh number to 5000 US mesh number; 2.5 μm to 2000 μm).

[0122] Gelatin was cross-linked with different microbial transglutaminase concentrations to generate stable structures. The stable structures were chopped, milled, sieved into several size ranges to form microparticles, and washed to remove transglutaminase as mentioned in the manufacturing process. Prior to injection, the microparticles were diluted to different concentrations in carrier or lubricant. The microparticles and carrier were injected as a confluent homogenous gel-like liquid in the absence of air (composed mainly of nitrogen and oxygen, and may also contain small amounts of, e.g., carbon dioxide, hydrogen, helium, argon, neon, etc.). Cross-linked gelatin foam microparticles of the desired size range (e.g., 0.1 μm to 2000 μm) were dispersed in the selected liquid carrier (e.g., gelatin; hyaluronic acid; carboxymethylcellulose; water) (see, e.g., Tables 1-3) or mixed with dry powders of the carrier / lubricant components, filled into syringes, and sterilized by autoclaving or irradiation.

[0123] The amount of mTG crosslinker in the microparticles was measured using mTG activity assay and SDS PAGE. The concentrations tested showed lower mTG activity and mTG protein values ​​than those of the positive control, proving that the microparticles are essentially or substantially free of crosslinker, as that term is used herein. See Figure 1.

[0124] Example 2: Histopathological evaluation of cross-linked gelatin foam microparticle formulations.

[0125] Acute and subchronic responses to subcutaneous injection formulations of cross-linked gelatin foam microparticles in a rat skin model were performed to evaluate safety, tolerability, and performance with respect to tissue augmentation and skin remodeling. Parameters evaluated were external skin response and cellular and tissue response to the implanted cross-linked gelatin foam microparticle formulations.

[0126] For this experiment, a formulation of the present disclosure was tested that consisted of 125 mg of freeze-dried gelatin foam microparticles, sterilized by irradiation (10 kilograys), and suspended in 1.2 ml of sterile saline. The dried gelatin foam microparticles were mixed with saline 3 hours prior to injection. The preparation of the microparticles is described in more detail in Example 1.

[0127] The gelatin foam microparticle formulation was implanted by injection into the subcutaneous tissue of three rats. One to two sites on each rat were implanted with 0.3 ml of the formulation at each site. One site was implanted with 0.3 ml of a competing product, Radiesse TM (Merz Aesthetics; collagen stimulating factor composed of calcium hydroxyapatite microspheres in an aqueous gel carrier) was injected and used as a positive control. Implantation sites were collected for histopathological evaluation by hematoxylin and eosin (HE) and Masson's trichrome (MT) staining on days 7 and 30.

[0128] Histopathological evaluation was based on a semiquantitative scoring method and was assessed in a "blind" fashion by an independent pathologist. The assessment of implant acceptance and performance consisted of parameters of local tissue reaction at the implant site, presence of necrosis, cavity formation, type of cellular infiltration, presence of foreign body reaction, amount of new collagen fibers (new collagen production), and material resorption. Each parameter was scored on a scale of 0-4 (each number on the scale corresponded to the following: 0-no change, 1-minimal, 2-mild, 3-moderate, 4-severe). See Figures 12-13.

[0129] A histopathological evaluation of the injectability, safety, tolerability and performance of the gelatin foam microparticle formulations of the present disclosure for tissue augmentation and skin remodeling was performed.

[0130] The following formulations were prepared with various amounts of crosslinked gelatin foam microparticles and various carrier hydrogels per milliliter of product.

[0131] [Table 1]

[0132] All formulations in Table 1 (1-8) were successfully injected into each injection site: porcine abdomen, 1 ml per 3 cm x 3 cm square; carboxymethylcellulose (CMC); hyaluronic acid (HA);

[0133] The pig skin was examined for macroscopic adverse events for one week and no adverse reactions were observed at the injection site.

[0134] result:

[0135] On day 7, implants of the disclosed gelatin foam microparticle formulation harvested from two sites showed a grade 1 foreign body reaction and grades 1-2 new collagen production. Implants from three sites were harvested on day 30 and showed a grade 1-2 foreign body reaction with grade 2 new collagen production, with some resorption of the implant. Also observed was the abundance of fibroblasts associated with and infiltrating the implants or compositions comprising a plurality of microparticles described herein. No necrosis, cavity formation, or edema was present at any site or time point, providing excellent tissue tolerance (Figures 2A-2D).

[0136] In comparison, a similar amount of the positive control, Radiesse TM(Merz Aesthetics) were implanted subcutaneously and harvested on day 30 for histopathological evaluation. At the implantation site, new collagen production was graded 0-1, with major foreign body reaction graded 3. In contrast, the gelatin foam microparticle formulation of the present disclosure was graded 2 for new collagen production, with prominent foreign body reaction graded 1-2. TM The implants allowed cell migration around the particles but not infiltration into the particle mass as seen with gelatin foam microparticles.

[0137] In summary, the gelatin foam microparticle formulations of the present disclosure were found to be safe. The implants were well tolerated and had no negative effects on tissues such as muscle, blood vessels, nerves and epithelium. The implants promote skin regeneration by stimulating new collagen production, which is superior to a positive control competitive product.

[0138] Example 3: Injectability characteristics of gelatin foam microparticle formulations.

[0139] In one embodiment, for aesthetic dermatology and reconstructive surgery, gelatin foam or hydrogel microparticle formulations of the present disclosure were developed to provide optimal scaffold support for fibroblast stimulation and tissue re-growth. A product that lifts the skin in a similar manner to that achieved with dermal fillers and with long-lasting results is desirable, while solving the critical need for a safe, injectable biostimulant with immediate clinical outcomes.

[0140] Injectability is considered to be the ability of a product to be successfully administered through a syringe and a suitable needle. The injectability of the gelatin foam microparticle formulation of the present disclosure was evaluated using a Lloyd compression system (LLOYD Instruments). This method was developed for the characterization of adhesive 3D foam structures according to the ASTM F2900-11 standard guide and characterization of hydrogels used in regenerative medicine. This analysis provided mechanical data of the force required to inject material through a syringe. Needle size and syringe brand and size affect the force.

[0141] The purpose of this study was to evaluate different optimal formulations for administration of the gelatin foam microparticle formulation of the present disclosure. The challenges in developing such a product included producing a uniform, cohesive paste that would support the gelatin foam particles of the present disclosure maintaining their 3D structure in the target tissue and preventing premature clearance, for example, for 3 months to 2 years. The ideal formulation would be stable for a reasonable storage period, either refrigerated (e.g., 4°C) or at room temperature (e.g., 20°C to 25°C).

[0142] Formulations are referred to in terms of particular combinations of gelatin foam microparticle size, particle to carrier ratio, carrier type, and carrier concentration.

[0143] result

[0144] The effect of particle size on injection force was tested. Different sizes of particles were suspended in a carboxymethylcellulose (CMC) carrier. In particular, 120 mg of each particle size (e.g., 30 μm to 100 μm) was suspended in 1 ml of 1% CMC. Using a 1 ml syringe and a 27 gauge needle, the injection force was measured. A linear correlation (dotted line) was observed between particle size and measured force (solid line) (Figure 3).

[0145] compound

[0146] Dozens of different formulations were initially tested. Once the texture of the prepared formulation was identified as visually smooth and viscous, a syringe was prepared and its injectability was tested. Thus, optimization of each formulation was performed in a step-by-step manner. The research results presented herein used particle sizes of 50 μm to 100 μm (e.g., 60 μm to 90 μm), but additional formulations containing smaller particle sizes below 60 μm (e.g., 30 μm, 40 μm) were also prepared.

[0147] [Table 2]

[0148] Stability was tested after storage at 2°C to 8°C. For Formulation 1 in Table 2, three time points were tested: 1 day, 3-4 days, and 7 days. Prior to force testing, the syringes were equilibrated to room temperature. As can be seen in Table 3, there was no significant change in injection force after the 1 day, 3-4 days, and 7 days refrigeration period test time points.

[0149] [Table 3]

[0150] Gelatin foam microparticle formulations of various particle sizes and carrier hydrogels were prepared. Particles were milled to particle sizes of 30 μm, 40 μm, 60 μm, and 90 μm. Injectability was found to be affected by particle size, with a linear correlation of particle size versus required injection force. Data was obtained for two different needle sizes: 30G and 27G, which are known to be suitable for minimally invasive dermatological applications (Figure 3).

[0151] Example 4: Morphological profiles of gelatin foam microparticle formulations of different size ranges.

[0152] The morphological structure of gelatin foam microparticles (MPs) was evaluated using high-resolution scanning electron microscopy (HR-SEM) and bright-field microscopy. The morphological parameters of MPs, such as shape, size and size distribution, as well as the porosity and surface texture of MPs, were investigated.

[0153] A small amount of MP sample was placed in a 1.5 ml microcentrifuge flip-cap tube for transport. Samples were prepared for high-resolution scanning electron microscopy (HR-SEM) (Technion; "Soft Material Electron Microscopy" instrument). Specifically, a piece of double-sided adhesive carbo tape was adhered to a designated metal mold, on which the sample MP was spread and adhered evenly.

[0154] Analysis: Several measurements were performed on the images using the HR-SEM software. All other analyses were in principle qualitative, as a visual evaluation of the images and graphical presentation. See Figures 4-6, Figures 7A-7B, Figure 8.

[0155] As seen in the HR-SEM images, particles of several size ranges were prepared. Particles with particle sizes greater than (or decreasing to) 0.1 μm were imaged. See FIG. 4; 104 nm, 105 nm, 112 nm, 145 nm, 150 nm, 275 nm. MPs with particle sizes between 60 μm and 99 μm were observed. See FIG. 5; 75.69 μm; 88.38 μm; 91.56 μm; 99.68 μm. The size range was controlled and adjusted during the particle milling and sieving manufacturing process.

[0156] Using bright field microscopy, large particles up to 2000 μm in size were observed. The particles were hydrated prior to imaging. Cross-linked gelatin was milled and sieved to a size range of 99 μm to 710 μm. The resulting particles had a swelling factor (wet / dry) of 1.65. When wet, the particles swelled and the size increased by a factor of 1.65 from the initial size to a maximum of approximately 2000 μm. See Figure 6, which shows hydrated gelatin particles of 1172 μm.

[0157] Example 5: Particle size determination.

[0158] Lyophilized microparticles of cross-linked gelatin were dispersed in phosphate buffered saline (PBS) at room temperature (RT) for 24 hours. Hydrated microparticles were visualized under an optical microscope and compared to dry particles. Size distribution was assessed manually by ImageJ software. See Figure 7A, Figure 7B, Figure 8.

[0159] [Table 4]

[0160] Example 6: Mechanical Properties of Gelatin Foam Microparticle Formulations

[0161] Gelatin foam microparticle formulations were prepared, mixed, stored at room temperature (RT) and at 6°C, and then tested for mechanical properties.

[0162] Wet and dry blends of gelatin foam microparticles (MP) (i.e., 60 μm-99 μm, 120 mg / ml) in 0.5%, 0.75% or 1% non-crosslinked gelatin carrier were prepared and introduced into 1 ml syringes. The mechanical properties of the blends were measured using an AG-R2 rheometer in a frequency sweep test ranging from 0.1 Hz to 10 Hz. See FIG. 9. These different frequencies correspond to different levels of shear force being applied to the sample. Measurement of the gel stiffness and therefore its ability to resist deformation under applied pressure may provide an indication of how the blend will be extruded through an injection needle or cannula or how it will subsequently be subjected to the movements of the facial musculature and overlying skin.

[0163] The dry particle formulations produced lower coefficients than those of the wet particle formulations. This may be due to differences between the formulations: the dry particle formulations were sterilized before mixing with the liquid, while the wet particle formulations were sterilized after mixing with the liquid. Additionally, the percentage of non-crosslinked gelatin carrier was lower in the dry particle formulations.

[0164] [Table 5]

[0165] Example 7: Size characterization of foam gelatin microparticles.

[0166] Samples from different batches of foam particles made with different amounts of microbial transglutaminase (mTG) per 20 grams of gelatin were taken for measurement on a Mastersizer 3000 (ITI Faculty of Biotechnology and Food Engineering, Technion, Haifa, IL). Samples were dispersed in deuterium-depleted water (DDW) or 96% ethanol immediately prior to measurement, or dispersed in water for 24 h prior to measurement.

[0167] [Table 6]

[0168] result:

[0169] Figure 10 shows the size distribution of non-hydrated foam MP (BC 81-9) and hydrated foam MP. Foam MP was dispersed in deuterium depleted water (DDW) (immediate (DDW) or 24 hours (DDW, 24 hours)) and in 96% ethanol. Dispersion of particles in 96% ethanol was the non-hydrated state of the particles, which indicated the dry particle size after sieving.

[0170] A narrower size distribution was observed when the particles were dispersed in 96% ethanol, with a size range of 50 μm to 150 μm, and D x (50) was approximately 80 μm. The foam MP was sieved into a size range of 60 μm to 90 μm, and the 80 μm size D x (50), which was approximately in the middle of the expected sieving range.

[0171] A broader size distribution was observed when the particles were dispersed in water (DDW). This was due to the water uptake of the gelatin foam MP, which caused the particles to swell, resulting in a shift in the size distribution plot. The D of the particles immediately after hydration in water or after 24 hours of hydration x (50) No significant difference was observed, indicating that the foam MP was fully hydrated soon after being dispersed in water.

[0172] D of hydrated particles (DDW-24 hours) vs. non-hydrated particles (96% ethanol) x The resulting ratio between (50) was 1.67.

[0173] Example 8: Injectability of foam gelatin microparticles in different saline dilutions.

[0174] 220 mg of foam microparticles containing 10 mg of non-crosslinked gelatin used as a carrier were mixed with 1.5 ml, 2 ml, and 3 ml of saline in a 2.5 ml syringe in a syringe-to-syringe (STS) fashion for 30 seconds.

[0175] Injectability was measured 10 minutes after mixing using a Lloyd's mechanical testing apparatus with a 27 gauge (27G) needle.

[0176] [Table 7]

[0177] As shown in Table 7 and Figure 11, saline mixing volume affects the injectability values ​​of cross-linked gelatin foam MP formulations. Increasing the mixing volume from 1.5 ml to 4 ml decreased the injectability from 41 N to 3 N, respectively. This ability to adjust the injectability of the formulation may be advantageous when injection of the formulation in different locations and volumes is dependent on tissue tolerance.

[0178] Example 9: Sterility of Foam Gelatin Microparticle Formulations

[0179] After sterilization using E-beam irradiation at 12 kGy (Sor-van, IL), the sterility of the gelatin foam MP was evaluated using endotoxin and bioburden testing.

[0180] To assess endotoxin levels, 20 mg of foam gelatin MP was dispersed in 5 ml of endotoxin-free water containing 4 U or 8 U collagenase and incubated overnight at 37° C. under 150 rpm shaking until foam MP was completely degraded. Endotoxin levels were quantified using the EndoZyme™ II assay.

[0181] Bioburden testing was performed for the assessment and quantification of bacterial levels or microbial contamination in water, raw materials, or final products for manufacturing product safety purposes. Bioburden testing was performed in an external Miloda laboratory (SOP 200.04.01) according to ISO 11737-1. Samples (0.1 g) were placed in 1 ml Buffered Sodium Chloride-Peptone (BSCP) + 0.1% Tween. Extraction was performed by manual mixing for 60 seconds, then 1 ml of the extract was plated on a Tryptic Soy Agar (TSA) plate and incubated at 30°C-35°C. The amount of microorganisms growing on the plate was counted after 72 hours. The Petri dishes were then transferred to 25°C for another 72 hours, then the number of yeasts and molds was counted as colony forming units (CFU).

[0182] [Table 8]

[0183] As seen in Table 8, the endotoxin levels (endotoxin units (EU)) of the foam particle samples ranged between 0.0126EU / mg and 0.0466EU / mg. When calculating EU per device (220mg of foam particles (FP)), the EU value was a maximum of 10.2, which is lower than the acceptable EU value of 20EU per device, indicating sterility of the formulation and validating the sterilization method using E-beam irradiation. This was also verified by bioburden testing, which was less than or equal to 1 colony forming units (CFU) / gram (g).

[0184] Example 10: Water insolubility test of microparticles (MP)

[0185] Microparticles of the present disclosure were placed in wells (6 plate wells) with 5 ml saline and incubated at 55° C. on a shaker at 100 rpm. Water insolubility of MP was assessed at time zero (before incubation), 1 hour, and 4 days for visual assessment. No visual differentiation was observed over time.

[0186] In another study, microparticles of the present disclosure were placed into a filter that had been pre-dried at 60° C. overnight. The filter was weighed containing 50 mg to 55 mg particles and placed into a 2 ml Eppendorf tube. Water was added inside the filter to ensure that the particles were covered and that the filter mesh was in contact with water (~2.5 ml). The filter and Eppendorf tube were covered with aluminum foil, sealed with tape, and placed at 60° C. for incubation. After 1 hour or 48 hours, the samples were washed, dried, and the weight loss was measured. The samples were washed with 3 ml to 4 ml of water each (10 rounds of 300 μl to 400 μl) and placed at 60° C. to dry overnight. After the drying process, the filter containing the FP sample was weighed. The study was performed in triplicate. The weight ratio of the dried FP before incubation or immersion in water and after 1 hour and 48 hours of incubation at 60° C. was 1.006 and 1.005, respectively. Thus, the material dry mass remained unchanged upon incubation in water for 1 or 48 hours, even when the water was warm (60° C.). Thus, the FP is crosslinked and not water soluble.

[0187] Example 11: Animal Transplant Safety Study

[0188] In a porcine implant safety study, different types of formulations were injected into the subcutaneous tissue of pigs. The injection sites were analyzed up to 180 days after injection (BC010 study in a porcine model). The tested microparticle (MP) + carrier or lubricant formulations showed good acute and subchronic tolerance and were determined to be safe. Fibroblast recruitment was shown even at an early time point of day 7, which continued until the process of new collagen production was seen at day 30. Collagen stimulation was shown, leading to the generation of new, vital collagenous tissue, with support of local angiogenesis (new capillary formation).

[0189] In a rat implantation study, MP and carrier or lubricant formulations were injected into the subcutaneous tissue of rats. The study showed high safety and tolerability of the injection formulations at different dosages up to 2 ml per injection site (in a rat model with an extreme 100-fold overdose) with no adverse events, edema, or necrosis up to 30 days after injection. Collagen stimulation was demonstrated, leading to the generation of new collagenous tissue with support for local angiogenesis (new capillary formation).

[0190] In a rat implantation study, FP containing carrier dry particles was mixed with different hydration liquids: saline, phosphate buffered saline (PBS), or water for injection (WFI). For example, 110 mg FP and 5 mg non-crosslinked gelatin were mixed with 1 ml saline or WFI. The study showed high safety and tolerability of the injection formulations in the different liquids, with no adverse events, edema, or necrosis up to 30 days after injection. Collagen stimulation was demonstrated, leading to the generation of new collagenous tissue with support of local angiogenesis (new capillary formation).

[0191] In a rat implantation study, FP dry particles were mixed with different carriers: (a) 120 mg FP mixed with 0.5 ml saline and 0.5 ml cross-linked hyaluronic acid (HA; 3000KD; 10 mg / ml; 0.05 BDDE / 1 mg HA); (b) 120 mg FP mixed with 5 mg non-cross-linked gelatin hygroscopic dry powder (e.g., for particles, see U.S. Pat. Nos. 10,596,194 and 11,331,412) and 12.5 enzyme units of microbial transglutaminase (mTG) dry powder mixed with 1 ml saline. Both formulations (a) and (b) showed high safety and tolerability with no adverse events, edema, or necrosis up to 30 days after injection. Collagen stimulation was observed (only around FP, but not around hyaluronic acid), leading to the generation of new collagenous tissue, indicating that cross-linked gelatin microparticles FP are crucial for cell ingrowth and remodeling.

[0192] In a rat implantation study, dry FP was produced by cross-linking gelatin with different concentrations of mTG. 120 mg FP of the various cross-linked mTG formulations were mixed with 1 ml saline. All formulations demonstrated high safety and tolerability with no adverse events, edema, or necrosis up to 30 days after injection. Collagen stimulation was demonstrated, leading to the generation of new collagenous tissue with support of local angiogenesis (new capillary formation).

[0193] Example 12: Evaluation of in vivo implantation formulations

[0194] In the porcine implantation study (BC010), the injected MP and carrier formulation was observed at the implantation site at day 7, with remnants still present at the one-month time point. At 180 days, the MP and carrier formulation was completely degraded with no remnants observed. In the rat study (PCR007), the injected MP and carrier formulation was present at the implantation site one month after injection.

[0195] For the pig study, formulations of the present disclosure were tested consisting of 30 mg to 120 mg of lyophilized gelatin foam microparticles in different carriers, with a final volume of 1 ml, sterilized by autoclaving. The preparation of the microparticles is described in more detail in Example 1.

[0196] For rat studies, a formulation of the present disclosure was tested consisting of 220 mg of freeze-dried gelatin foam microparticles mixed with 10 mg of carrier powder, sterilized by irradiation (10 kilogray), and suspended in 2 ml of sterile saline. The dried gelatin foam microparticles were mixed with 2 ml of saline immediately prior to injection. The preparation of the microparticles was described in more detail in Example 1.

[0197] Gelatin foam microparticle formulations were implanted by injection into the subcutaneous tissue of two pigs and 18 rats. Each rat was implanted at one to four sites with 0.3ml to 2ml of the formulation at each site. Arrows indicate the implanted composition of the present disclosure. Implantation sites were collected at days 7 and 30 in the rat model and days 7, 30, and 180 in the pig model for histopathological evaluation by hematoxylin and eosin (HE) and Masson's trichrome (MT) staining. In pig and rat skin, 7, 30, and 180 days after implantation, implants were stained with H&E (hematoxylin and eosin, which stains cell nuclei purplish blue and extracellular matrix and cytoplasm pink) and MT, Masson's trichrome (resulting in keratin red, muscle fibers and implants, collagen and bone blue, light red or pink cytoplasm, and dark brown to black cell nuclei) (H&E-pig days 7 and 30, rat days 7 and 30, and MT-pig day 180). See FIG. 12.

[0198] The microparticle compositions or formulations described herein may be classified as biodegradable, which has the advantage of risk mitigation. Other commercial products, such as calcium hydroxyapatite (CaHA) or poly-L-lactic acid (PLA), have shown that their degradation rates can cause many adverse events and complications. Treatment with CaHA has the highest complication rate, with the most common adverse events being the formation of nodules and granulomas in the injected tissue. CaHA-CMC (calcium hydroxyapatite-carboxymethylcellulose) implants allow cell migration around the particles but do not allow infiltration into the particle mass, as seen with gelatin microparticles.

[0199] In rat studies, the injected microparticle formulation was present at the implant site one month after implantation.

[0200] Example 13: Evaluation of biostimulation processes

[0201] Based on the pig and rat implantation studies as described herein, a biostimulatory process of the material was observed only 7 days after implantation. This was indicated by an ongoing collagen production process (grade 2) and the formation of new collagen fibers in the implanted area. Representative histological photographs of the implants were stained with H&E (hematoxylin and eosin, which stains cell nuclei purplish blue and extracellular matrix and cytoplasm pink) and Masson's trichrome (resulting in keratin red, muscle fibers and implants, collagen and bone blue, light red or pink cytoplasm, and dark brown to black cell nuclei) 7, 30, and 180 days after implantation (H&E-pig day 7, and Masson's trichrome-pig days 30 and 180, rat days 7 and 30). The implant compound of new collagen fibers (black arrow) was stained blue (white arrow). See Figure 13.

[0202] Example 14: mTG Residues in Foam Particles (FP) Measured by SDS-PAGE:

[0203] This study analyzed the qualitative determination of microbial transglutaminase (mTG) enzyme residues in FP products by SDS-PAGE. The appearance of mTG bands in the FP suspension indicated the presence of the enzyme. This study was performed in duplicate. See Figure 14.

[0204] Test controls of mTG (1), gelatin (2), and collagenase (3) produced the expected protein patterns and sizes. See Example 14.

[0205] The FP results (4) showed no evidence or traces of mTG enzyme in the suspension, suggesting that the FP contained no or very little, if any, mTG and that the mTG enzyme was undetectable when measured by this method.

[0206] Example 15: RGD quantification

[0207] A fluorometric assay was used to quantify the amount of RGD (arginine-glycine-aspartic acid) motifs on the surface of crosslinked gelatin microparticles and raw materials through the amino groups of arginine. The reaction between the amino groups of arginine and 9,10-phenanthrenequinone produced a fluorescent compound. This reaction typically occurs at high pH after acidification, which produces a fluorescent compound or molecule.

[0208] Samples and standards were mixed separately with 9,10-phenanthrenequinone reagent in a high pH environment and incubated at 60°C and 100 rpm for 3 hours. The mixture was then mixed with HCl and incubated at room temperature (RT) for 1 hour to obtain fluorescent molecules. Fluorescence intensity was measured at an excitation wavelength of 312 nm and an emission wavelength of 395 nm. A blank control without RGD was prepared with deionized water. Samples were tested in triplicate.

[0209] result:

[0210] The fluorescence emission of arginine was measured at different concentrations and is shown in the arginine calibration curve in FIG.

[0211] The fluorescence emission spectra of the different materials were measured and are shown in Figure 16. At a wavelength of 395 nm, from top to bottom the curves are: Arg 80 μg / ml; non-crosslinked gelatin; BC-82-8; BC-81-8; BC-82-7; BC-82-5; BC-82-4; BC-82-6; mTG; blank.

[0212] The RGD motif in the tested materials was quantified and calculated according to the arginine calibration curve (Figure 15). The free arginine used in the calibration curve had two primary amino groups, and the RGD motif sequence had one amino group. The calculation included the fluorescence emission at a wavelength of 395 nm.

[0213] Figure 17 shows the amount of RGD (μg / mg) in non-crosslinked gelatin, microbial transglutaminase, foam particles (FP), and confluent particles. Non-crosslinked gelatin had more than 30 μg / mg of RGD motifs, while FP and confluent particles had about 12 μg / mg and 14 μg / mg, respectively. Meanwhile, microbial transglutaminase (mTG) had essentially zero or barely detectable amounts of RGD.

[0214] FIG. 18 shows the measured amount of RGD on crosslinked foam particles (FP) of different size ranges.

[0215] The amount of RGD (Y-axis) on FP crosslinked with different amounts of mTG (X-axis) was also measured and shown in Figure 19. The ratio of various batches of different gelatin to enzyme (mTG) weight when compared to gelatin or mTG alone is illustrated on the X-axis (BC-82-7; BC-81-8; BC-82-5; BC-82-4; BC-82-6; and BC-82-8).

[0216] Conclusion:

[0217] Increasing the amount of arginine showed a characteristic increase in emission at a wavelength of 395 nm, which was also seen in gelatin raw materials. mTG showed minimal fluorescence emission spectrum at a wavelength of 395 nm. The low signal in mTG may be due to the negligible amount of arginine by weight relative to the total enzyme weight, indicating that quantification of RGD in FP refers to cross-linked gelatin. This indicated that the RGD sequence could be quantified by using a fluorescence measurement method.

[0218] As expected, the amount of RGD in non-crosslinked gelatin is higher than that in crosslinked gelatin microparticles, which served as a positive control in this study.Gelatin is soluble, allowing a large amount of exposed RGD sites compared to insoluble crosslinked particles, where some of the RGD sites are captured or not exposed.Non-crosslinked gelatin exhibits more RGD, but non-crosslinked gelatin dissolves or degrades very quickly at 37°C, and has no biological effect, so its use is not practical for the suggested microparticles.

[0219] A positive characteristic release was also seen for crosslinked gelatin microparticles in both confluent and bubble particles, which differed in their manufacturing methods and sizes but had similar amounts of RGD on the particle surface.

[0220] FPs of different size ranges showed similar RGD amounts. No correlation was found between the amount of RGD and the amount of mTG used to crosslink the FPs. Combining the results, the amount of RGD on the crosslinked gelatin particles ranged from 11 μg / mg to 36 μg / mg.

[0221] Example 16: In vitro culture of primary fibroblasts on microparticles in suspension

[0222] Primary bovine dermal fibroblasts were incubated with the microparticles of the present disclosure and plated in non-tissue culture plates. For comparison, cells were seeded in non-tissue culture plates without microparticles and in regular tissue culture plates. Viability was measured.

[0223] Cells: Primary bovine dermal fibroblasts (BDF) were isolated from 14-month-old bulls using the explant method. In the explant method, small pieces of skin, e.g., from a cow, are plated onto tissue culture plates until substantial proliferation of cells occurs. This technique has been used historically as a model for wound healing. Cells were dispersed from adhesion to the culture plate by washing the ~80% confluent cell monolayer with PBS for 5 min, followed by enzymatic dispersion with 0.25% trypsin for 4 min. For this experiment, cells at passage 5 were used.

[0224] Microparticles: Microparticles as described in this disclosure with a dry particle size range of 100 μm to 700 μm were used. Prior to incubation with cells, the microparticles were suspended in complete medium for 48 hours for hydration.

[0225] Adhesion: ~1x10 5 of suspended cells were added to 120 mg of the microparticle suspension to a final volume of 4 ml in complete medium in a 15 ml cap tube. The cells and microparticles were pipetted to mix and placed in a cell culture incubator (37° C., 5% CO2) for 2 hours to allow for adequate cell-microparticle attachment.

[0226] Seeding: The cell microparticle suspension was gently suspended and seeded into a non-tissue culture 96-well U-bottom plate. BDF in the same ratio of cells / medium volume was seeded into the wells of the same plate as a control. As another control, BDF in the same ratio of cells / medium volume was seeded into the wells of a regular 96-well tissue culture plate.

[0227] Viability: Cell viability was measured 7 days after seeding using the Alamar Blue viability / proliferation / cytotoxicity assay (Bio-Rad). Half of the medium volume in each test well was removed and replaced with fresh medium and supplemented with 20% (v / v) Alamar Blue reagent to a final concentration of 10%. Reagent was also added to wells containing medium but no cells to serve as negative controls. After 4 hours of incubation with reagent, 60 μL of medium was extracted from each test or control well and diluted 1:10 in PBS. Absorbance was measured at 570 nm and 600 nm (Shimadzu UV-1280 spectrophotometer). Viability was calculated according to the equation: ((O2xA1)-(O1xA2)) / ((R1xN2)-(R2xN1))*100 and expressed as percent reduction in Alamar Blue, where O1=molar extinction coefficient (E) of oxidized Alamar Blue (blue) at 570 nm, O2=E of oxidized Alamar Blue at 600 nm, R1=E of reduced Alamar Blue (red) at 570 nm, R2=E of reduced Alamar Blue at 600 nm, A1=absorbance of test wells at 570 nm, A2=absorbance of test wells at 600 nm, N1=absorbance of negative control wells (containing medium plus Alamar Blue and no cells) at 570 nm, N2=absorbance of negative control wells (containing medium plus Alamar Blue and no cells) at 600 nm. Cultures were dried uniformly, fixed and analyzed by SEM for surface morphology.

[0228] Results: The viability of cells attached to the microparticles of the present disclosure was comparable to that of the same number of cells seeded on flat-bottom 96-well tissue culture plates. Cells seeded in non-tissue culture 96-well U-bottom plates without microparticles showed limited, if any, viability. SEM analysis showed elongated fibroblasts with collagen fiber deposition around the cells.

[0229] Conclusion: The microparticles of the present disclosure provided a surface for cell attachment and support for primary bovine dermal fibroblasts. The studies described herein also demonstrated the survival of fibroblasts in the presence of the microparticles of the present disclosure, which acted as microcarriers in suspension. Meanwhile, cells incubated without the microparticles of the present disclosure did not survive. See Table 9.

[0230] [Table 9]

[0231] Primary bovine skin fibroblasts were isolated from a 14-month-old bull. Cell culture was performed under standard conditions (e.g., 100% relative humidity (RH), 37°C, 5% CO2) using a growth medium containing high glucose DMEM supplemented with 10% fetal calf serum (FCS), L-glutamine, sodium pyruvate, and antibiotics and / or antimycotics. Approximately 100,000 cells were cultured by incubating the cells with the microcarriers or multiple microparticles (120 mg) of the present disclosure in a final volume of growth medium (4 ml) for a sufficient time to allow the cells to adhere to the microcarriers, which is about 2 hours, resuspending the cells and the microcarriers of the present disclosure, and seeding the suspension in a non-tissue culture plate (e.g., a 96-well U-bottom plate). The same ratio of cells to medium volume was seeded into the wells of the same plate as the control, but without microcarriers, and as another control, the same ratio of cells to medium volume was seeded into the wells of a regular 96-well tissue culture plate, but without microcarriers.

[0232] Viability was measured, for example, 3 days after plating using the alamarBlue viability / proliferation / cytotoxicity assay (Bio-Rad) according to the recommended manufacturer's instructions. The viability (day 3) of cells plated on microcarriers was similar to that of cells plated on tissue culture plates, while cells plated directly on non-tissue culture plates were not viable. See Table 9.

[0233] For example, 6x10 6 were incubated with the disclosed FP (particle size range 500 μm-2000 μm) in an incubator overnight on a shaker (80 RPM) in a non-coated 55 mm Petri dish for adhesion to the FP. The cell aggregates were then incubated at 37° C. for 8 days for proliferation and differentiation. The cell aggregates began to beat after 7 days of culture, indicating that the iPS cells were successfully differentiated into cardiomyocytes and were functional.

[0234] Example 17: In vitro culture of induced pluripotent stem cells for cell differentiation

[0235] iPS cells (6 million) were attached with 100 mg of FPs (particle size range 25 μm-2000 μm) in a non-coated 55 mm Petri dish on a shaker (80 RPM) in an incubator for overnight incubation. The resulting cell aggregates were then incubated at 37°C for 8 days for proliferation and differentiation.

[0236] Results: FP-loaded cells were observed under an optical microscope (see FIG. 20A; FIG. 20B). Beating of FP-loaded cells was observed after 8 days, indicating that iPS cells were successfully differentiated into cardiomyocytes.

[0237] Example 18: Foam gelatin particles made from crosslinked gelatin fibers:

[0238] Crosslinked gelatin was prepared from 1 mg of milled gelatin and 1 g mTG. The powders were mixed and placed in a 10 ml syringe (syringe 1). An additional syringe (syringe 2) was filled with 8 ml saline and connected to syringe 1. The saline from syringe 2 was mixed with the powder from syringe 1 for 60 seconds through a syringe-to-syringe mixing method. The produced foam was injected through needles of various sizes: 27G, 25G, and 21G needles into a cold (4°C) microbial transglutaminase (mTG) solution in a Petri dish at a concentration of 0.2% w / v and kept at room temperature (RT) for 2 hours. Half of the Petri dish was stored at 37°C for an additional hour. Finally, the produced fibrils were filtered from the mTG solution, dried overnight at RT, milled in a mortar and pestle, and their morphology was characterized by optical microscopy.

[0239] Results: Optical microscope images of the particles showed that foam particles (FP) with particle size range of 22 μm to 752 μm were successfully prepared from foam cross-linked gelatin fibers. See Figure 21A; Figure 21B.

[0240] Since various changes can be made in the subject matter described above without departing from the scope and spirit of the present disclosure, it is intended that all subject matter contained in the above description or defined in the appended claims be construed as describing and illustrating the present disclosure. Many modifications and variations of the present disclosure are possible in light of the above teachings. Accordingly, the present specification is intended to embrace all such alternatives, modifications and variations that fall within the scope of the appended claims.

[0241] All documents cited or referred to in this specification, and all documents cited or referred to in the documents cited herein, together with any manufacturer's instructions, descriptions, product specifications, and product sheets of any products referred to herein, or in any document incorporated by reference herein, are hereby incorporated by reference and may be used in the practice of this disclosure.

Claims

1. A plurality of microparticles, comprising: (a) containing a cross-linked protein, said cross-linked protein containing at least one RGD (Arg-Gly-Asp) motif; said plurality of microparticles essentially not containing a cross-linking agent; said plurality of microparticles being water-insoluble.

2. A composition comprising the plurality of microparticles of Claim 1.

3. Further: (b) containing a carrier, the composition of Claim 2.

4. said cross-linked protein being selected from the group consisting of gelatin, collagen, tropoelastin, elastin, casein, albumin, engineered proteins thereof, any engineered polymer containing an RGD motif, and any combination thereof, the composition of Claim 2.

5. said cross-linked protein containing an RGD motif in the range of 0.1 μg / mg to 50 μg / mg, the composition of Claim 2.

6. said plurality of particles containing dry foam particles or dry cross-linked gelatin block particles, the composition of Claim 2.

7. said plurality of particles containing a particle size selected from 0.1 μm to 2000 μm, the composition of Claim 2.

8. said plurality of particles containing at least two different particle sizes, the composition of Claim 2.

9. said at least two different particle sizes being selected from 0.1 μm to 2000 μm, the composition of Claim 8.

10. said particle size containing an average particle size of 30 μm to 500 μm, the composition of Claim 6.

11. said carrier being a hydrogel, the composition of Claim 3.

12. The composition of claim 3, wherein the carrier is selected from the group consisting of: gelatin; collagen; alginate; glycosaminoglycan (GAG); hyaluronic acid; carboxymethylcellulose; polyethylene glycol (PEG); poly(ethylene oxide) (PEO); poly(vinyl alcohol) (PVA); poly(propylene fumarate) (PPF); polyethylene glycol (PEG); and any combination thereof.

13. The composition of claim 3, wherein the carrier is selected from the group consisting of: uncrosslinked chondroitin sulfate polymer, uncrosslinked dermatan sulfate polymer, uncrosslinked keratan sulfate polymer, uncrosslinked heparan polymer, uncrosslinked heparan sulfate polymer, uncrosslinked hyaluronan polymer, uncrosslinked glycosaminoglycan polymer, uncrosslinked elastin and / or fibronectin, and any combination thereof.

14. The composition of claim 3, wherein the carrier is wet or dry.

15. The composition of claim 3, wherein the composition comprises a concentration of the plurality of microparticles in the carrier of 1 mg / ml or more, 300 mg / ml or less, or 1 mg / ml to 300 mg / ml.

16. A method for preparing the plurality of microparticles of claim 1, comprising: (a) mixing a crosslinkable protein solution and a crosslinking agent solution, wherein the crosslinkable protein solution comprises dissolving a crosslinkable protein containing at least one RGD (Arg-Gly-Asp) motif in a liquid; and the crosslinking agent solution comprises dissolving a crosslinking agent in a liquid, the step as described above; (b) forming a crosslinked foam or hydrogel block comprising the mixed crosslinkable protein solution and crosslinking agent solution of (a); (c) removing the crosslinking agent from the crosslinked foam or hydrogel block of (b) to form a crosslinking agent-free foam or hydrogel block. (d) The formed crosslinked foam or hydrogel block of (b), the crosslinking agent-free foam or hydrogel block of (c), or a combination of the formed crosslinked foam or hydrogel block of (b) and the crosslinking agent-free foam or hydrogel block of (c) is reduced in size to form a plurality of microparticles including the size-reduced crosslinked foam of (b) and / or the size-reduced crosslinking agent-free foam of (c). The method as described above includes this step.

17. The formation of the crosslinked foam of (b) is as follows: (b1) While adding the crosslinking agent solution of (a) at 37°C, whipping the crosslinkable protein solution of (a) to form the crosslinked foam of (b), or (b2) Without adding gas, while adding the crosslinking agent solution of (a) at 37°C, mixing the crosslinkable protein solution of (a) to form the crosslinked hydrogel block of (b). The method according to claim 16 includes this step.

18. (e) Freezing the crosslinking agent-free foam or hydrogel block of (c) or the plurality of particles of (d); (f) Lyophilizing the frozen crosslinking agent-free foam or hydrogel block of (e); (g) Further reducing the size of the lyophilized crosslinking agent-free foam or hydrogel block of (f) to form a plurality of crosslinked foams or hydrogel particles. The method according to claim 16 includes this step.

19. A method for treating a subject in need of body shaping, including the step of administering the composition according to claim 2 to a site of the subject in need of body shaping. The method as described above.

20. A method for producing cell-free proteins, comprising: Growing a plurality of protein-producing cells in a cell culture containing the plurality of microparticles according to claim 1 and a culture medium, The growth occurs under conditions that induce protein synthesis, thereby producing cell-free proteins. The method as described above includes this step.

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