Composition and fat grafting using same

Abstract

Provided herein, inter alia, are soft tissue devices, compositions, and uses comprising hydrogel composites for fat grafting and soft tissue regeneration. TIFF2025538204000004.tif98128

Description

[Technical Field]

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 424,345, filed November 10, 2022, which is incorporated herein by reference in its entirety.

[0002] Technical Field Provided herein, inter alia, are soft tissue devices, compositions, and uses comprising hydrogel composites for fat grafting and soft tissue regeneration. [Background technology]

[0003] background Soft tissue defects resulting from trauma, tumor resection, or congenital anomalies have been treated using fat grafting. For example, in 2019, there were 600,000 fat grafting procedures performed on the face worldwide, with substantially more performed on other anatomical regions. Tissue regrowth can require a suitable matrix for cells to attach, migrate, proliferate, differentiate, and organize into new tissue. For example, natural extracellular matrices (ECMs) have been used at repair sites. However, no materials currently exist that can withstand the mechanical and structural loads of cells during healing. Summary of the Invention

[0004] overview In particular, provided herein are compositions for administration (soft tissue devices) comprising hydrogel composites for fat grafting and soft tissue regeneration.

[0005] In one aspect, we provide herein a composition for administration comprising: a) adipocytes or adipose tissue; and b) non-spherical microbeads comprising a hydrogel composite, the hydrogel composite comprising a hyaluronic acid component covalently linked to a fiber component, wherein the weight ratio of the hyaluronic acid component to the fiber component ranges from 1:100 to about 100:1.

[0006] In one aspect, a soft tissue device is provided that includes a biologically active material and non-spherical microbeads containing a hydrogel composite. In one aspect, the hydrogel composite includes a functionalized hyaluronic acid network and an associated fiber or scaffold component. In certain aspects, the hydrogel composition suitably includes 1) a fiber or scaffold component; 2) hyaluronic acid, including functionalized hyaluronic acid; and preferably 3) a cross-linking component.

[0007] In a further aspect, a composition or soft tissue device for administration is provided, the composition or device comprising: a) non-spherical microbeads comprising a hydrogel composite, the hydrogel composite comprising a hyaluronic acid component covalently linked to a fiber component; and b) a population of adipose cells, autologous adipose cells, allogeneic cells, genetically modified allogeneic cells, adipose stromal vascular cells, adipose tissue, autologous adipose tissue, lipoaspirate tissue, derivatives thereof, or combinations thereof, wherein the composition or soft tissue device comprises the non-spherical microbeads in a volume that is about 25% to 75% of the total volume of the soft tissue device, and wherein the weight ratio of the hyaluronic acid component to the fiber component is in the range of 1:100 to about 100:1.

[0008] In certain aspects, the adipocytes of adipose tissue can be in the form of fat particles.As referred to herein, adipocytes or adipose tissue or other similar terms include adipose cells or adipose tissue.In some embodiments, adipocytes or adipose tissue or adipose tissue is liposuction tissue.In some embodiments, adipocytes, adipose tissue or adipose tissue is autologous.Fat particles can exist as a mixture with extracellular matrix protein (ECM) material such as collagen material.

[0009] In one embodiment, suitably, the ratio of the average size of the non-spherical microbeads to the average size of the fat particles is in the range of about 10:1 to 1:10. In one embodiment, suitably, the ratio of the average size of the non-spherical microbeads to the average size of the fat particles is in the range of about 2:8 to 8:2. In one embodiment, suitably, the ratio of the average size of the non-spherical microbeads to the average size of the fat particles is in the range of about 3:7 to 7:3. In one embodiment, suitably, the ratio of the average size of the non-spherical microbeads to the average size of the fat particles is in the range of about 4:6 to 6:4. In one embodiment, suitably, the average size of the non-spherical microbeads is within 5 or 10% of the average size of the fat particles. In aspects, the fat particles are suitably less than 1 millimeter in size (longest dimension), for example, at most 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3 0.3, or 0.1 millimeters in the longest dimension, or less than 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3 0.3, or 0.1 millimeters.

[0010] In one embodiment, the fiber component comprises nonwoven polymeric fibers. In certain embodiments, the polymeric fibers comprise polycaprolactone fibers, such as electrospun polycaprolactone fibers. Optionally, the polymeric fibers comprise synthetic polymeric materials, including, for example, poly(lactic-co-glycolic acid), polylactic acid, and / or polycaprolactone, or combinations thereof. In one embodiment, the composite is formulated to be substantially biocompatible. Optionally, the polymeric fibers comprise biological polymeric materials, including silk, collagen, chitosan, and / or mimetics or combinations thereof. In one aspect, for example, collagen-based peptides, including recombinant peptides, with or without hydroxyproline residues, can be suitable fiber element materials.

[0011] In a preferred aspect, the hydrogel composition comprises a functionalized hyaluronic acid network covalently linked to a plurality of polycaprolactone nanofibers.

[0012] In a preferred aspect, the hydrogel composition comprises a functionalized hyaluronic acid network covalently linked to a plurality of collagen nanofibers. In some embodiments, the collagen nanofibers can be suitably obtained from natural sources or synthesized. In certain embodiments, the collagen nanofibers comprise type I bovine collagen nanofibers or fragments thereof. In certain embodiments, the collagen nanofibers comprise collagen mimetics, such as collagen-like peptide sequences. In certain embodiments, the collagen nanofibers can be obtained from natural sources or made or prepared from a composition (resin composition) containing collagen. For example, the collagen nanofibers can be formed by electrospinning, centrifugal spinning, blow spinning, or a combination thereof. Specifically, the collagen nanofibers are preferably prepared by electrospinning.

[0013] In certain preferred aspects, the non-spherical microbeads have an average size along their longest dimension in the range of about 50 micrometers to about 300 or 400 micrometers, including when the average size along their longest dimension of the non-spherical microbeads is up to about 50, 60, 70, 80, 90, 100, 120, 150, 180, 200, 220, 250, 280, 300, 320, 350, or 400 micrometers along their longest dimension.

[0014] Preferably, the biologically active material (such as adipocytes or adipose tissue) present with the non-spherical microbeads comprising the hydrogel composite comprises a population of adipocytes, autologous adipocytes, allogeneic cells, genetically modified allogeneic cells, stem cells, mesenchymal stem cells, genetically modified stem cells, genetically modified allogeneic induced pluripotent stem (iPS) cells, genetically modified hypoimmunogenic pluripotent stem cells, adipose stromal vascular cells, adipose tissue, autologous adipose tissue, lipoaspirate tissue, derivatives thereof, or combinations thereof.

[0015] Preferably, the soft tissue device comprises non-spherical microbeads in a volume of about 25% to 75% of the total volume of the soft tissue device, including cases where the non-spherical microbeads are present in a volume of at least about 25% and up to 30%, 35%, 40%, 50%, 55%, 60%, or 70% of the total volume of the soft tissue device.

[0016] In one embodiment, in composites including non-spherical microbeads, the composite may include a crosslinker present in a concentration of, for example, about 1 mg / mL to about 25 mg / mL, preferably the microbeads have an average size in the range of about 50 micrometers to about 300 micrometers along their longest dimension, suitably the microbeads are pre-reacted (e.g., the crosslinker has reacted with one or more other components on the microbeads), and / or preferably the microbeads are substantially stable at room temperature for at least about 6 months.

[0017] In one aspect, a method for fat transplantation in a subject is provided. The method comprises administering an effective amount of the composition (soft tissue device) disclosed herein to a subject in need thereof. In one aspect, the composition is suitably implanted into the target tissue of the subject. In one aspect, the composition is suitably injected into the target tissue of the subject.

[0018] Also provided in one aspect are methods for performing cosmetic or reconstructive procedures or for reducing or reversing tissue defects resulting from trauma, surgical intervention, or age-related diseases, disorders, or conditions.

[0019] The method includes administering an effective amount of the composition (soft tissue device) disclosed herein to a subject in need thereof. In one aspect, the composition is suitably implanted into the target tissue of the subject. In one aspect, the composition is suitably injected into the target tissue of the subject.

[0020] Further provided is a kit for preparing to inject the soft tissue device described herein for administration into a target tissue of a subject. The kit includes (i) a first syringe containing microbeads and (ii) a second syringe containing a biologically active material, which may include adipocytes or adipose tissue. The kit further includes (iii) a Luer-Luer union connector having an orifice that allows the microbeads and the biologically active material to pass through for mixing before injection or implantation. Preferably, the microbeads are gelled or hardened before the biologically active material is added to the microbeads.

[0021] The size of the microbeads and fat particles can be readily determined by known methods, including optical methods for measuring bead size and fat particle size, and histological measurements for fat particle size. In particular, dynamic light scattering and laser particle analyzers can be used to measure the size of the microbeads and fat particles referred to herein.

[0022] Fat particles, as referred to herein, can be prepared by various processes, including mechanical processing of adipocytes or adipose tissue. An exemplary fat particle formation process is shown in Example 3 below, and includes forming an admixture of adipose tissue / cells, spreading the admixture into a film layer (optionally cross-linking), and then mechanically processing (e.g., grinding, for example, with a pestle) or cryo-grinding to form particles of the film layer. Alternatively, fat can be obtained from a mammal (e.g., a human, a rodent such as a rat, or other mammal) by, for example, using an aspirator (e.g., a vacuum pump), and the obtained fat can be recovered using a homogenizer to obtain the desired fat particles referred to herein. The collected fat sample can be filtered, and the filtration can be controlled based on the desired particle size. Various commercially available devices, such as the REVOLVE System (trademark) (Abbvie), can be used to prepare fat particles. In another further exemplary approach to obtaining fat particles, adipose tissue can be surgically harvested en bloc from a donor site (either from the same animal / person or from a donor animal / person) and then mechanically disaggregated to produce fat particles with intact cells in an extracellular matrix. As noted above, in certain embodiments, the fat particles can comprise fat cells / tissue along with extracellular matrix (ECM) material, such as collagen material.

[0023] Other aspects of the invention are disclosed below. [Brief explanation of the drawings]

[0024] [Figure 1] Figure 1A shows an exemplary bead comprising nanofibers (101), a crosslinker (102), and hyaluronic acid (103). Figure 1B shows an exemplary soft tissue device comprising exemplary beads and a biologically active material, such as fat. [Figure 2]A scheme is shown for reacting modified hyaluronic acid, surface-functionalized nanofibers, and a cross-linker (polyethylene glycol dithiol) together to form a composite hydrogel structure with covalent bonds between the fiber and hydrogel phases. [Figure 3] 1 shows a scheme for modifying the fiber surface to add functional groups for covalent bonding to a hydrogel phase, according to an exemplary embodiment of the present disclosure. [Figure 4] Figure 4 (including Figures 4A-4C) shows the preparation of nanofiber-hydrogel composite (NHC)-fat mixtures for fat grafting made with polycaprolactone (PCL) nanofibers from Example 2. Figure 4A: Representative images of processed fat fragments. Figure 4B: NHC and processed fat mixed at various volume ratios. Scale bar: 1 cm. Figure 4C: Injection scheme for the fat grafting experiment of Example 4. [Figure 5] Figure 5A shows the storage modulus of prepared NHC fat graft material obtained from Example 2. Comparable G' values ​​around 300 Pa were observed across various combinations of NHC and fat, and no statistically significant differences were detected. n=12; p>0.05. Figure 5B shows the tan delta values ​​of NHC and fat combinations. [Figure 6] Figure 1 shows the distribution of NHCs and processed fat on POD 0, as verified by H&E staining, from Example 4. The processed fat fragments were well mixed with the NHCs, resulting in a uniform distribution of the two components within each type of graft. NHCs are shown in light gray, while processed fat contains pink-stained ECM and adipocytes with vacuolar structures. Scale bars in the full and extracted images are 2.5 mm and 500 μm, respectively. [Figure 7]Figure 7 (including Figures 7A-7C) shows the volume and shape retention of the injected grafts obtained from Example 4. Figure 7A: MRI images were taken every 15 days from POD 0 to POD 90 to estimate the graft volume shown in (B). The graft shape flattened over time in most groups. Figure 7B: The 100% NHC and 50% NHC-50% fat groups had better volume retention than the other three groups by POD 90. n=4. Figure 7C: Representative macroscopic images of various grafts on POD 0, 30, and 90. By POD 90, it was difficult to visualize the 100% fat grafts. Scale bar: 1 cm. [Figure 8] Morphological analysis of the injected grafts from Example 4 is shown. Pure NHC grafts remodeled into capsule-like structures composed primarily of soft tissue. 75% NHC-25% fat grafts underwent early tissue remodeling into neo-soft tissue, with most of the co-injected fat disappearing by POD 30. Grafts composed of 50% or more fat fragments exhibited vacuoles at POD 30, but only the 50% NHC-50% fat group retained healthy vacuoles at POD 90. Meanwhile, enlarged vacuoles were observed in both the 25% NHC-75% fat and 100% fat samples. Nuclei, ECM or cytoplasm, and NHC are shown in purple, pink, and light gray, respectively. Scale bars in the full and extracted images are 2.5 mm and 500 μm, respectively. [Figure 9]Detection of viable adipocytes in the injected grafts from Example 4. Viable adipocytes were visualized by labeling specimens for perilipin. Dying adipocytes were defined as cells with enlarged vacuoles and absent or faded perilipin staining, typically surrounded by infiltrating host cells (DAPI). Abundant perilipin-expressing cells were observed in the 50% NHC-50% fat group, while most adipocytes in the 25% NHC-75% fat and 100% fat groups were nonviable. At POD 90, a few viable adipocytes were found at the periphery of the remodeling soft tissue in the 100% NHC and 75% NHC-25% fat groups. Nuclei and perilipin are shown in blue and red, respectively. Scale bar: 200 μm. [Figure 10] Figure 1 shows blood vessel formation in the injected grafts obtained from Example 4. The specimens were co-labeled for αSMA and RECA-1 to visualize blood vessels. Increased blood vessel formation was observed in the 50% NHC-50% fat group, while the pure fat grafts were poorly vascularized. At POD 90, larger blood vessels were found in the peripheral regions of the constructs in the 100% NHC, 75% NHC-25% fat, and 25% NHC-75% fat groups, while some smaller vessels were also observed toward the center of the specimens. Nuclei, αSMA, and RECA-1 are shown in blue, red, and green, respectively. Scale bar: 200 μm. [Figure 11] 1 shows a scheme of an exemplary method for extracting fat and mixing it with NHCs. [Figure 12] Figure 12 (including Figures 12A-12C) shows the volume retention of injected grafts obtained from Example 7. Figure 12A: Composition of various adipose-NHC (containing collagen fibers prepared in Example 6) grafts. Figure 12B: Macroscopic images of each injected graft after 1 month. Figure 12C: Volume measurements based on macroscopic images of each injected graft. [Figure 13]

[0023] Figure 1 shows morphological analysis of injected grafts based on hemoxylin and eosin staining at POD 30 from Example 7. Samples were stained with hemoxylin and eosin. Nuclei, cytoplasm, and NHCs are shown in purple, pink, and light gray, respectively. [Figure 14] Figure 1 shows the detection of viable adipocytes in injected collagen-based NHC-fat grafts from Example 7. Samples were stained for perilipin to visualize viable adipocytes. Red arrows indicate healthy adipocytes with strong perilipin signal. Yellow arrows indicate nonviable adipocytes with enlarged vacuoles in the absence of perilipin expression. 75% fat-25% NHC grafts and 50% fat-50% NHC grafts showed excellent adipocyte viability at POD 30. DETAILED DESCRIPTION OF THE INVENTION

[0025] Detailed Description The following detailed description is given by way of example and is not intended to limit the invention solely to the particular embodiments described, and may be best understood in conjunction with the accompanying drawings.

[0026] The present invention relates to a pre-reacted beaded composite material comprising a hydrogel and nanostructures for use in methods for soft tissue reconstruction. The present invention also relates to a soft tissue device comprising the beaded composite material for cell and tissue delivery for cosmetic, reconstructive, and cellular treatments.

[0027] The present invention also relates to composite materials capable of recruiting, capturing, encapsulating, binding, and / or embedding specific tissue constituents, including, but not limited to, adipocytes, other mesenchymal cells, or mesenchymal stem cells. The present invention further relates to composite materials capable of recruiting, capturing, encapsulating, binding, and / or embedding specific tissues, including, but not limited to, adipose tissue. The present invention also relates to methods for repairing or reconstructing soft tissue injuries using compositions (e.g., soft tissue devices) comprising a scaffold composite comprising a biomaterial covalently linked to biodegradable fibers. In another aspect, the present invention also relates to methods for making compositions for use in soft tissue reconstruction, the compositions comprising a hydrogel and nanostructures disposed therein. In certain aspects, the present invention also relates to methods for making compositions for use in cell and tissue delivery for cosmetic treatments, reconstructive treatments, and cell therapy.

[0028] The following is a detailed description of the present invention provided to assist those skilled in the art in carrying out the present invention. Those skilled in the art may make modifications and variations to the embodiments described herein without departing from the spirit or scope of the present invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. The terminology used in the description of the present invention herein is only for describing specific embodiments and is not intended to limit the present invention. All publications, patent applications, patents, figures, and other references mentioned herein are expressly incorporated by reference in their entirety.

[0029] Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited.

[0030] Unless otherwise defined, all technical and scientific terms used herein have the meanings that are commonly understood by those skilled in the art to which this invention pertains.The following references, the entire disclosures of which are incorporated herein by reference, provide those skilled in the art with many of the general definitions of the terms used in this invention (unless otherwise defined herein): Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, the HarperCollins Dictionary of Biology (1991).In general, the molecular biology methods and similar procedures described herein or specific herein are common methods used in the art. Such standard techniques can be found in reference manuals such as, for example, Sambrook et al., (2000, Molecular Cloning—A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratories); and Ausubel et al., (1994, Current Protocols in Molecular Biology, John Wiley & Sons, New York).

[0031] The following terms may have the meanings ascribed to them below unless otherwise specified. However, it is understood that other meanings known or understood by those skilled in the art are possible and are within the scope of the present invention. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. Furthermore, the materials, methods, and examples are illustrative only and are not intended to be limiting.

[0032] definition The terms "a" and "an" refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0033] As used herein, "about" can mean plus or minus less than 1, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, or more than 30 percent, depending on the context, as would be known or understood by one of ordinary skill in the art.

[0034] As used herein, a "subject" or "subjects" or "individuals" can include, but is not limited to, mammals, such as humans or non-human mammals, e.g., domesticated, agricultural, or wild animals, as well as birds and aquatic animals. In certain embodiments, a subject is a human patient or an animal that is the subject of medical treatment.

[0035] As used herein, the term "hydrogel" is a type of "gel" and refers to a water-swellable polymeric matrix consisting of a three-dimensional network of macromolecules (e.g., hydrophilic polymers, hydrophobic polymers, or blends thereof) held together by covalent or noncovalent crosslinks that can absorb substantial amounts of water (e.g., 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more per non-water molecular unit) to form an elastic gel. Hydrogels can contain "water-swellable" polymers that absorb at least 50% or more of their weight in water upon immersion in an aqueous medium. The polymeric matrix can be formed from any suitable synthetic or naturally occurring polymeric material. As used herein, the term "gel" refers to a solid three-dimensional network that spans the volume of a liquid medium and retains it via surface tension effects. This internal network structure can result from physical bonds (physical gels) or chemical bonds (chemical gels) and crystallites or other junctions that remain intact within the elongated fluid. Virtually any fluid can be used as the elongated material, including water (hydrogels), oil, and air (aerogels). By weight and volume, gels exhibit densities similar to those of their constituent liquids because they are predominantly fluid in composition. Hydrogels are a type of gel that uses water as the liquid medium.

[0036] In certain embodiments, the hydrogel is a composite or composite material. The term "composite," as used herein, includes any association, bond, or adduct of two or more components. In some embodiments, a "hydrogel composite," as used herein, includes at least a polymer fiber and a hydrogel material. The hydrogel composite contains a polymer fiber (e.g., polycaprolactone) and a hydrogel material (e.g., hyaluronic acid (HA)).

[0037] The term "functional network," as used herein, means that the interaction between components provides a chemical, biochemical, biophysical, physical, or physiological benefit. Additionally, functional networks can include additional components, including cells, biological materials (e.g., polypeptides, nucleic acids, lipids, carbohydrates), therapeutic compounds, synthetic molecules, and the like. In certain embodiments, the scaffold composite promotes tissue growth and cell infiltration when implanted into a target tissue present in a human subject.

[0038] The term "nanofiber," as used herein, refers to a fibrous material having at least one dimension (e.g., length or width) of less than about 999 nm, less than about 900 nm, less than about 800 nm, less than about 700 nm, less than about 600 nm, less than about 500 nm, less than about 400 nm, less than about 300 nm, less than about 200 nm, less than about 100 nm, less than about 50 nm, or less than about 10 nm. In some embodiments, nanofibers can have a length of less than about 999 nm, less than about 900 nm, less than about 800 nm, less than about 700 nm, less than about 600 nm, less than about 500 nm, less than about 400 nm, less than about 300 nm, less than about 200 nm, less than about 100 nm, less than about 50 nm, or less than about 10 nm. In some embodiments, nanofibers can have a width of less than about 999 nm, less than about 900 nm, less than about 800 nm, less than about 700 nm, less than about 600 nm, less than about 500 nm, less than about 400 nm, less than about 300 nm, less than about 200 nm, less than about 100 nm, less than about 50 nm, or less than about 10 nm. The term "nanofiber," as used herein, also includes microfibers, which have at least one dimension less than 10 microns in size, for example, because they can have surface features of sub-micron size and can be made using similar methods and materials.

[0039] The term "microfiber," as used herein, includes fibrous materials having at least one dimension (eg, length or width) that is less than about 10 microns.

[0040] The term "nanofiber-hydrogel composite," as used herein, refers to a composite comprising at least nanofibers (e.g., polymeric fibers) and a hydrogel (e.g., HA) that form a functional network. Furthermore, the terms "nanofiber-hydrogel composite," "hydrogel composite," "composite," or "composite," as used herein, are used interchangeably to refer to such a composite comprising at least nanofibers (e.g., polymeric fibers) and a hydrogel (e.g., HA).

[0041] The term "crosslinked" as used herein refers to a composition containing intramolecular and / or intermolecular crosslinks, whether occurring through covalent or noncovalent bonds, which may be direct or may involve crosslinking agents. "Noncovalent" bonds include both hydrogen bonds and electrostatic (ionic) bonds.

[0042] The term "polymer" includes linear and branched polymer structures, and also encompasses crosslinked polymers and copolymers (which may or may not be crosslinked), and thus includes block copolymers, alternating copolymers, random copolymers, and the like. Compounds referred to herein as "oligomers" are polymers having a molecular weight of less than about 1000 Da, preferably less than about 800 Da. Polymers and oligomers can be naturally occurring or obtained from synthetic sources.

[0043] As used herein, the term "biomaterial" refers to an organic material designed to interact with biological systems. In some embodiments of the present invention, the biomaterial is a hydrogel. In some embodiments, the biomaterial is hyaluronic acid (HA) derived from bacteria.

[0044] As used herein, the term "biodegradable" refers to a material that can be broken down by biological means in a subject.

[0045] As used herein, the term "implantable" means capable of being formulated for implantation into a subject via a syringe.

[0046] As used herein, the term "soft tissue" refers to tissue that connects, supports, or surrounds other structures and organs of the body. Soft tissue includes muscles, tendons, ligaments, fascia, nerves, fibrous tissue, fat, blood vessels, and synovial membranes.

[0047] As used herein, the term "stable" refers to a material that does not decompose at room temperature.

[0048] As used herein, the term "autologous" refers to any material derived from the same individual into which it is subsequently reintroduced.

[0049] As used herein, the term "allogeneic" or alternatively, "allogenic" refers to any material derived from a different animal of the same species as the individual into whom the material is being introduced or from a different patient than the individual into whom the material is being introduced.

[0050] As used herein, the term "lyophilized" refers to a material after it has been subjected to lyophilization, a process used to preserve a material by removing water from it, which involves first freezing the material under vacuum at very low temperatures and then drying it.

[0051] As used herein, the term "functionalized" refers to a material that has been homogeneously or heterogeneously modified (e.g., chemically modified) to have a functional chemical moiety associated therewith. In some cases, the functional chemical moiety has the ability to react to allow for the formation of a covalent or non-covalent bond. In some cases, the functional chemical moiety can impart improved properties to the material.

[0052] Nanofiber-hydrogel composite "Nanofiber-hydrogel composites," "hydrogel composites," or "composites" are provided that are formed by combining hydrogel materials or other biomaterials with polymeric nanofibers. The composites can be formulated to allow for variations in density, gel-to-fiber ratio, and other properties while maintaining sufficient porosity and strength.

[0053] The ratio of polymer nanofibers to hydrogel material can be determined by any means known in the art. For example, the ratio of polymer fiber to hydrogel material can be about 1:100 to about 100:1, such as about 1:50 to about 50:1 or 1:10 to about 10:1, such as 1:5 to about 5:1, or about 1:3 to about 3:1, based on the weight of the components. The ratio of polymer fiber to hydrogel material can also be given on a concentration basis, e.g., a given weight of polymer fiber per volume of hydrogel material. For example, the concentration can be about 1 to 50 mg / mL. The hydrogel material is generally disposed on the polymer fiber, such as by being bonded to the entire outer surface (or one outer surface, depending on the composition and shape) of the polymer fiber. In certain embodiments, a plurality of polymer fibers (e.g., polycaprolactone nanofibers) are present on the surface of the hydrogel composite. In certain embodiments, a plurality of polymer fibers (eg, polycaprolactone nanofibers) are present on the surface of a hydrogel composite bead (eg, a microbead).

[0054] The composite may contain a plurality of pores present on or in the surface of the composite. The presence, size, distribution, frequency, and other parameters of the pores can be adjusted during creation of the composite, hydrogel, or nanofiber. Pore size can be less than about 1 nm up to 100 nm, including 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nm, and the size can be narrowly tailored, for example, so that at least 40% of the pores are of a desired size or within a desired size range, such as 50%, 60%, 70%, 80%, 90%, 95%, or more than 95% of the pores.

[0055] The composites may be suitable for incorporation into the tissues of a human subject and are therefore generally "biocompatible," meaning they have the ability to interact with biological systems (such as those found in a human subject) without inducing a pathophysiological response therein and / or thereby. In some embodiments, the composites are provided to be permanently retained in tissue, e.g., neural tissue. Alternatively, the composites are provided to be capable of transient retention in a human subject and are substantially biodegradable. Preferably, the polymeric fibers or nanofibers in the composite comprise a biocompatible, biodegradable polymer, e.g., a biocompatible, biodegradable polyester. In certain embodiments, the polymeric fibers or nanofibers comprise polycaprolactone. In certain embodiments, the polymeric fibers or nanofibers are polycaprolactone.

[0056] To achieve the fiber reinforcement effect while maintaining high porosity in the hydrogel phase, electrospun fiber-hydrogel composites are provided that offer superior properties compared to other composites. Such composite designs not only allow for greater mechanical reinforcement from the solid fiber component, but also allow for independent tuning of the bulk mechanical properties and the average pore size / porosity of the hydrogel phase, thereby achieving both optimal cell infiltration properties and structural integrity.

[0057] To further achieve the desired effect, in some embodiments, crosslinks are preferably introduced between the nanofibers as well as between the nanofibers and the hydrogel using a PEG crosslinker, which helps extend the durability of the product and allows for adjustment of the crosslink density to achieve optimal other properties.

[0058] Gel / Hydrogel Components In one aspect, the composite comprises a hydrogel having a three-dimensional network of polymers (e.g., hydrophilic polymers, hydrophobic polymers, blends thereof) held together by covalent or non-covalent crosslinks that can absorb a substantial amount of water (e.g., 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than 99% per non-water molecular unit) to form an elastic gel. In some embodiments, the hydrogel can be biodegradable. The hydrogel can include any type of suitable hydrogel components known in the art. The gel and / or hydrogel can be formed from any suitable synthetic or naturally occurring material.

[0059] In some embodiments, the hydrogel material is functionalized, hi certain embodiments, the hydrogel material is functionalized with groups including hydroxyl, amino, carboxyl, thio, acrylate, sulfonate, phosphate, amide, and modified forms thereof, such as activated or protected forms.

[0060] Hyaluronic acid (HA) is preferably used as the hydrogel material. HA is a non-sulfated linear polysaccharide consisting of repeating disaccharide units that form the hydrogel component. HA is also a non-immunogenic natural component of the extracellular matrix in human tissues and is widely used as a dermal filler in cosmetic and reconstructive procedures.

[0061] HA degradation is promoted by natural hyaluronidases, whose expression is increased in areas of tissue injury and inflammation. Importantly, studies have shown that small HA degradation fragments of 3–10 disaccharide units are potent regulators of endothelial cell proliferation, migration, tubule formation, and angiogenesis. These biological functions of HA appear to be mediated through CD44 in a pathway involving Ras and PKC. Blockade of the CD44 / HA interaction using an anti-CD44 antibody reduced the proliferation and migration of human microvascular endothelial cells in vitro. HA hydrogels have been investigated as potential cell delivery matrices in various models of cell and tissue injury. These hydrogels can serve as a protective and supportive scaffold for cells and can also reduce scarring. HA may therefore play a crucial role in promoting tissue regeneration by facilitating cell infiltration and promoting angiogenesis.

[0062] The molecular weight of the hyaluronic acid can affect the overall properties of the composite. In some embodiments, the molecular weight of HA (e.g., HA-SH) is at least about or greater than 10 kDa, at least about or greater than 50 kDa, at least about or greater than 100 kDa, at least about or greater than 200 kDa, at least about or greater than 300 kDa, at least about or greater than 400 kDa, at least about or greater than 500 kDa, at least about or greater than 600 kDa, at least about or greater than 700 kDa, at least about or greater than 800 kDa, at least about or greater than 900 kDa, at least about or greater than 1.0 MDa, at least about or greater than 1.5 MDa, at least about or greater than 2.0 MDa. It may be greater than MDa, at least about 2.5 MDa or greater than 2.5 MDa, at least about 3.0 MDa or greater than 3.0 MDa.

[0063] In some embodiments, the hyaluronic acid is functionalized. In certain embodiments, the hyaluronic acid is functionalized with groups including hydroxyl, amino, carboxyl, thio, acrylate, sulfonate, phosphate, amide, and modified forms thereof, such as activated or protected forms. In certain embodiments, the hydrogel material comprises hyaluronic acid (HA). In certain embodiments, the hydrogel material comprises functionalized hyaluronic acid (HA). In other preferred embodiments, the hydrogel material comprises acrylated hyaluronic acid (HA). In some embodiments, the hydrogel material comprises thiolated hyaluronic acid (HA).

[0064] In some embodiments, the HA of the present invention is sterilized HA, eg, chemically and / or physically sterilized.

[0065] Additionally, the polymeric component of the hydrogel may also include cellulose esters such as, for example, cellulose acetate, cellulose acetate propionate (CAP), cellulose acetate butyrate (CAB), cellulose propionate (CP), cellulose butyrate (CB), cellulose propionate butyrate (CPB), cellulose diacetate (CDA), cellulose triacetate (CTA), or the like. In some embodiments, the gel / hydrogel may include other water-swellable polymers, such as acrylate polymers, which are generally formed from acrylic acid, methacrylic acid, methyl acrylate, ethyl acrylate, methyl methacrylate, ethyl methacrylate, and / or other vinyl monomers.

[0066] nanofiber In one aspect, the composite also includes polymer fibers generally having an average diameter of about 10 nm to about 10,000 nm, such as about 100 nm to about 8000 nm, or about 150 nm to about 5,000 nm, or about 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,500, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500, 5,000, 5,500, 6,000, 6,500, 7,000, 7,500, or 8,000. The polymeric fibers generally have an average length of about 10 μm to about 500 μm, such as about 10, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 μm.

[0067] In certain embodiments, the polymeric fibers are nanofibers having an average diameter generally less than about 999 nm, less than about 900 nm, less than about 800 nm, less than about 700 nm, less than about 600 nm, less than about 500 nm, less than about 400 nm, less than about 300 nm, less than about 200 nm, less than about 100 nm, less than about 50 nm, or less than about 10 nm. In some embodiments, the polymeric fibers have a length generally less than about 200 μm, less than about 250 μm, less than about 80 μm, less than about 50 μm, less than about 40 μm, less than about 30 μm, less than about 20 μm, or less than about 10 μm. Nanofiber diameter and length are determined using optical fluorescence microscopy or electron microscopy.

[0068] In some embodiments, the nanofibers are functionalized with groups including hydroxyl, amino, carboxyl, thio, acrylate, sulfonate, phosphate, maleimide, amide, and modified forms thereof, such as activated or protected forms.

[0069] Specifically, the fibers (e.g., nanofibers or microfibers) in the fiber-hydrogel composite comprise one or more extracellular matrix proteins (ECM). In certain embodiments, the fibers (e.g., nanofibers or microfibers) suitably comprise one or more selected from collagen, gelatin, elastin, elastin-like polypeptides, tropoelastin, decellularized matrix, and hyaluronic acid. In certain embodiments, the fibers (e.g., nanofibers or microfibers) comprise one or more of bovine type I collagen, gelatin, or derivatives.

[0070] Preferably, the polymeric fibers or nanofibers in the composite comprise a biocompatible, biodegradable polymer, such as, for example, a biocompatible, biodegradable polyester. In certain embodiments, the polymeric fibers or nanofibers comprise polycaprolactone. In certain embodiments, the polymeric fibers or nanofibers are polycaprolactone.

[0071] In certain embodiments, the fiber-hydrogel composite may include a natural extracellular matrix for the fibers (e.g., nanofibers or microfibers). In certain embodiments, the fiber-hydrogel composite may include a synthetic extracellular matrix for the fibers (e.g., nanofibers or microfibers). In certain embodiments, the one or more ECMs include collagen nanofibers, which may be naturally obtained or synthesized. In certain embodiments, the collagen nanofibers include type I bovine collagen nanofibers or fragments thereof. In certain embodiments, the collagen nanofibers may be obtained from natural sources or may be made or prepared from a composition (resin composition) comprising collagen. For example, the collagen nanofibers may be formed by electrospinning, centrifugal spinning, blow spinning, or a combination thereof. Specifically, the collagen nanofibers are preferably prepared by electrospinning.

[0072] In certain embodiments, the nanofibers are made using microfabrication methods. In various embodiments, the devices of the present disclosure can be assembled and / or manufactured using any suitable microfabrication technique. Such methods and techniques are widely known in the art.

[0073] Nanofibers can also be made by electrostatic spinning (also called electrospinning). The process of electrospinning generally involves introducing a liquid into an electric field, causing the liquid to form fibers. These fibers are generally attracted to a conductor with an attractive potential for collection. During the liquid-to-fiber transformation, the fibers solidify and / or dry. This solidification and / or drying can be caused by cooling of the liquid (i.e., in this case, the liquid is typically a solid at room temperature); by evaporation of a solvent, e.g., dehydration (physically induced solidification); or by a hardening mechanism (chemically induced solidification).

[0074] Electrostatically spun fibers can be produced with very thin diameters. Parameters that affect the diameter, consistency, and uniformity of electrospun fibers include the polymeric and crosslinker concentrations (loadings) in the fiber-forming combination, the solvent composition, the applied voltage, and the needle-to-collector distance.

[0075] Electrospun fibers (e.g., collagen nanofibers) can provide superior properties such as high porosity in the hydrogel phase and mechanical reinforcement from the solid fiber component, which can be beneficial for optimal cell infiltration properties and structural integrity.

[0076] In certain embodiments, collagen fibers are prepared by electrospinning fibers in a solution or suspension containing 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP or HFP) solvent. In other embodiments, collagen fibers are prepared by electrospinning in a solution or suspension containing a different solvent, such as trifluoroethanol (TFE), trifluoroacetic acid (TFA), acetic acid, ethanol, or a phosphate mixture. In certain embodiments, a suitable solvent may include at least one of 1,1,1,3,3,3 hexafluoro-2-propanol (HFIP), 2,2,2-trifluoroethanol (TFE), and a mixture of water and acetic acid. Other solvents that may be used or combined with other solvents in the electrospinning of natural matrix materials such as collagen fibers include acetamide, N-methylformamide, N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), dimethylacetamide, N-methylpyrrolidone (NMP), ethyl acetate, acetonitrile, trifluoroacetic anhydride, 1,1,1-trifluoroacetone, maleic acid, and hexafluoroacetone.

[0077] The choice of solvent depends on the characteristics of the synthetic polymer being electrodeposited, such as secondary forces that stabilize polymer-polymer interactions, and the solvent's ability to replace these with strong polymer-solvent interactions. In the case of polypeptides such as collagen, and in the absence of covalent crosslinks, the primary interchain secondary forces are (1) Coulombic forces resulting from the attraction of fixed charges on the backbone and dictated by the primary structure (e.g., lysine and arginine residues are positively charged at physiological pH, while aspartic acid or glutamic acid residues are negatively charged); (2) dipole-dipole interactions resulting from the interaction of permanent dipoles (hydrogen bonds commonly found in polypeptides are the strongest such interactions); and (3) hydrophobic interactions resulting from the association of nonpolar regions of the polypeptide due to the low tendency of nonpolar species to interact favorably with polar water molecules.

[0078] Therefore, solvents or solvent combinations that can compete favorably for these interactions can dissolve or disperse polypeptides. For example, HFP and TFE possess highly polar hydroxyl groups adjacent to very hydrophobic fluorinated regions. Without wishing to be bound by theory, it is believed that the alcohol moieties can hydrogen bond with peptides and also solvate the backbone charges, thereby reducing intermolecular Coulombic interactions. Furthermore, the hydrophobic moieties of these solvents may interact with hydrophobic domains in polypeptides, helping the latter resist the tendency to aggregate through hydrophobic interactions. Due to their lower overall polarity compared to water, solvents such as HFP and TFE may compete less for intramolecular hydrogen bonds that stabilize secondary structures such as alpha helices. Consequently, alpha helices in these solvents are believed to be stabilized by stronger intramolecular hydrogen bonds. Stabilization of polypeptide secondary structures in these solvents is believed to be desirable, particularly in the case of collagen and elastin, to maintain proper collagen fibril formation during electrospinning. In some embodiments, the solvent is selected based on its tendency to induce helical structure in electrospun protein fibers, thereby facilitating the polymerization of collagen or other protein monomers to form helical polymers that mimic natural collagen fibrils. Examples of such solvents include halogenated alcohols, preferably fluorinated alcohols (HFP and TFE), hexafluoroacetone, chloroalcohols in combination with aqueous solutions of mineral acids, and dimethylacetamide, preferably containing lithium chloride. HFP and TFE are more preferred. In some embodiments, water is added to the solvent.

[0079] In certain embodiments, collagen nanofibers are prepared using vapor-phase glutaraldehyde, an alternative fiber stabilizer. Treatment with glutaraldehyde results in crosslinking of collagen fibers, as the aldehyde groups of glutaraldehyde react with free lysine or hydroxylysine groups on the collagen fibers to form Schiff base structures. For example, a 6-hour vapor-phase glutaraldehyde treatment results in increased tensile strength, elasticity, stretchability, and stability of collagen fibers. In certain embodiments, collagen nanofibers are prepared using an alternative collagen stabilizer or crosslinker, such as D-ribose. As disclosed in U.S. Pat. No. 4,971,954, the entire contents of which are incorporated herein, D-ribose can crosslink collagen fibers to provide a nontoxic and nonimmunogenic matrix.

[0080] In certain embodiments, the EDC and the fiber (e.g., nanofiber or microfiber) can be crosslinked (e.g., via a crosslinking moiety or directly linked). For example, the mode of interaction between the EDC and the fiber (e.g., nanofiber or microfiber) can be effective in introducing a bond (e.g., a covalent bond) between them.

[0081] Nanofibers may include, but are not limited to, nanofibers, nanotubes, nanofilaments, mesh sections, branched filaments, or networks. Nanofibers may also include any suitable chemical functional groups to facilitate covalent or non-covalent crosslinking between the nanofibers and the polymers of the hydrogels of the present invention.

[0082] Preferably, the nanofibers have a diameter ranging from about 10 nm to about 10,000 nm. In some embodiments, the nanofibers have a diameter ranging from about 10 nm to about 1000 nm. Additionally, the nanofibers can have an aspect ratio ranging from at least about 10 to about at least 200. Because the fiber diameter is so small, it will be appreciated that the fibers have a high surface area per unit of mass. This high surface area:mass ratio allows the fiber-forming solution or liquid to be transformed from a liquid or solvated fiber-forming material into solid nanofibers in a fraction of a second.

[0083] In certain embodiments, HA can be covalently bonded to fibers (e.g., nanofibers or microfibers). For example, HA can be covalently bonded to fragments of type I bovine collagen nanofibers. In certain embodiments, a crosslinker generates an interfacial bond between the collagen nanofiber and HA. Due to bonds and interactions (e.g., covalent, non-covalent, or ionic), the collagen nanofibers can be held within or within the interior space of the fiber-hydrogel composite (e.g., within the composite network). In certain embodiments, the crosslinker can react with the hydroxyl groups of HA and the amino groups of the collagen nanofiber to form a composite network. For example, the interfacial bond between the collagen nanofiber and HA can increase the stiffness of the composite, even at relatively low fiber loading densities.

[0084] Due to interfacial bonding and composite network formation, the fiber-hydrogel composite can have increased cell permeability and / or maintain a storage modulus. In certain embodiments, the storage modulus of the fiber-hydrogel composite is at least about 10 Pa, at least about 20 Pa, at least about 30 Pa, at least about 40 Pa, at least about 50 Pa, at least about 60 Pa, at least about 70 Pa, at least about 80 Pa, at least about 90 Pa, at least about 100 Pa, at least about 150 Pa, at least about 200 Pa, at least about 250 Pa, at least about 300 Pa, at least about 400 Pa, or at least about 500 Pa. In certain embodiments, the storage modulus of the fiber-hydrogel composite is in the range of about 1 to about 1,000 Pa, about 20 to about 800 Pa, about 100 to about 500 Pa, or about 150 to about 500 Pa. In certain embodiments, the storage modulus of the fiber-hydrogel composite ranges from about 0.5 to about 30 kPa.

[0085] In certain embodiments, alternative hydrogel phases such as collagen, chitosan, alginate, PVA, gelatin, PEG, cellulose, or cellulose derivatives may be used in place of HA.

[0086] Crosslinking A preferred form of interaction for a composite containing polymer fibers and a hydrogel includes cross-linking moieties generally present in an amount effective to introduce bonds between the polymer fibers and the hydrogel material, e.g., an amount effective to induce cross-linking between polycaprolactone fibers and hyaluronic acid.

[0087] For certain applications, particularly where high cohesive strength is desired, the polymers of the gel / hydrogels of the present invention may be covalently crosslinked. The present disclosure contemplates that crosslinking may be desirable not only between polymers of the gel / hydrogel component, but also between the polymers of the gel / hydrogel component and the nanostructured component of the composite materials of the present invention. The present invention contemplates any suitable means for crosslinking polymers to each other and gel / hydrogel polymers to the nanostructured component of the present invention. Gel / hydrogel polymers may be covalently crosslinked to other polymers or nanostructures, either intramolecularly, intermolecularly, or via covalent bonds. In the former case, there are no covalent bonds connecting the polymers to each other or to the nanostructure, while in the latter case, there are covalent crosslinks connecting the polymers to each other or to the nanostructure. Crosslinks may be formed using any suitable means, including the use of heat, radiation, or chemical curing (crosslinking) agents. The degree of crosslinking should be sufficient to eliminate or at least minimize cold flow under compression. Crosslinking also involves the use of a "crosslinker," which is a third molecule utilized in the crosslinking process.

[0088] The "cross-linking substance" or "cross-linking agent" may suitably be selected from the group of, for example, poly(ethylene glycol) PEG, such as thiolated poly(ethylene glycol), poly(ethylene glycol) diacrylate (PEGDA), or derivatives thereof. Examples of other cross-linking agents that may be suitable include DEO (diepoxyoctane), BDDE (1,4-butanediol diglycidyl ether), and DVS (divinyl sulfone).

[0089] For thermal crosslinking, a free radical polymerization initiator is used, which can be any of the known free radical-generating initiators conventionally used in vinyl polymerization. Preferred initiators are organic peroxides and azo compounds, which are commonly used in amounts of about 0.01 wt. % to 15 wt. %, preferably 0.05 wt. % to 10 wt. %, more preferably about 0.1 wt. % to about 5%, and most preferably about 0.5 wt. % to about 4 wt. % of the polymerizable material. Suitable organic peroxides include dialkyl peroxides such as t-butyl peroxide and 2,2-bis(t-butylperoxy)propane, diacyl peroxides such as benzoyl peroxide and acetyl peroxide, peresters such as t-butyl perbenzoate and t-butyl per-2-ethylhexanoate, perdicarbonates such as dicetyl peroxydicarbonate and dicyclohexyl peroxydicarbonate, ketone peroxides such as cyclohexanone peroxide and methyl ethyl ketone peroxide, and hydroperoxides such as cumene hydroperoxide and tert-butyl hydroperoxide. Suitable azo compounds include azobis(isobutyronitrile) and azobis(2,4-dimethylvaleronitrile). The thermal crosslinking temperature depends on the actual components and can be easily estimated by one skilled in the art, but typically ranges from about 80°C to about 200°C.

[0090] Crosslinking can also be achieved using radiation, typically in the presence of a photoinitiator. The radiation can be ultraviolet, alpha, beta, gamma, electron beam, and x-ray radiation, with ultraviolet radiation being preferred. Useful photosensitizers are "hydrogen abstraction" type triplet sensitizers, including benzophenones and substituted benzophenones and acetophenones such as benzyl dimethyl ketal, 4-acryloxybenzophenone (ABP), 1-hydroxycyclohexylphenyl ketone, 2,2-diethoxyacetophenone, and 2,2-dimethoxy-2-phenylacetophenone; substituted alpha-ketols such as 2-methyl-2-hydroxypropiophenone; benzoin ethers such as benzoin methyl ether and benzoin isopropyl ether; and anisoin methyl ether. These include substituted benzoin ethers of the formula (I), aromatic sulfonyl chlorides such as 2-naphthalenesulfonyl chloride, photoactive oximes such as 1-phenyl-1,2-propanedione-2-(O-ethoxycarbonyl)-oxime, thioxanthones including alkyl- and halogen-substituted thioxanthones such as 2-isopropylthioxanthone, 2-chlorothioxanthone, 2,4-dimethylthioxanone, 2,4-dichlorothioxanone, and 2,4-diethylthioxanone, and acylphosphine oxides. Radiation having wavelengths of 200-800 nm, preferably 200-500 nm, is preferred for use herein; in most cases, low-intensity UV light is sufficient to induce crosslinking. However, with hydrogen abstraction-type photosensitizers, higher-intensity UV exposure may be required to achieve sufficient crosslinking. Such exposure can be provided by mercury lamp processors such as those available from PPG, Fusion, Xenon, and others. Crosslinking can also be induced by gamma or electron beam irradiation. Appropriate irradiation parameters, i.e., the type and dose of radiation used to induce crosslinking, will be apparent to those skilled in the art.

[0091] Suitable chemical curing agents, also referred to as chemical crosslinking "accelerators," include, but are not limited to, 2,2-dimercaptodiethyl ether, dipentaerythritol hexa(3-mercaptopropionate), ethylene bis(3-mercaptoacetate), pentaerythritol tetra(3-mercaptopropionate), pentaerythritol tetrathioglycolate, polyethylene glycol dimercaptoacetate, polyethylene glycol di(3-mercaptopropionate), trimethylolethane tri(3-mercaptopropionate), trimethylolethane trithioglycolate, trimethylolpropane tri(3-mercaptopropionate), trimethylolpropane trithioglycolate, dithioethane, dithiopropane, or trithiopropane, and polymercaptans such as 1,6-hexanedithiol. Adding a cross-linking promoter to a non-cross-linked hydrophilic polymer promotes its covalent cross-linking, or adding a cross-linking promoter to a blend of a non-cross-linked hydrophilic polymer and a complementary oligomer provides cross-links between the two components.

[0092] The polymer and / or nanostructure may be crosslinked before mixing with the complementary oligomer. In such cases, it may be preferable to synthesize the polymer in crosslinked form by mixing and copolymerizing a monomer precursor with the polymer along with a multifunctional comonomer. Polymerization can be carried out in bulk, suspension, solution, or emulsion. Solution polymerization is preferred, with polar organic solvents such as ethyl acetate and lower alkanols (e.g., ethanol, isopropyl alcohol, etc.) being particularly preferred.

[0093] In some embodiments, a chemical crosslinker is used, preferably in an amount such that the weight ratio of crosslinker to hydrophilic polymer is in the range of about 1:100 to 1:5, and chemical crosslinking can be combined with radiation curing, if desired, to achieve higher crosslink densities.

[0094] In some embodiments, the cross-linking agent comprises poly(ethylene glycol) diacrylate (PEGDA) or a derivative thereof. In certain embodiments, when the functionalized hyaluronic acid comprises thiolated hyaluronic acid, the cross-linking agent comprises poly(ethylene glycol) diacrylate (PEGDA) or a derivative thereof.

[0095] microbeads As discussed, in various aspects, it may be preferable to form the composite / hydrogel into a microparticulate formulation, thereby allowing for the use of higher concentrations of each component and enhanced stability. In a preferred aspect, a micronization system may be used, in which a preformed hydrogel-nanofiber composite is physically conditioned, such as by forcing it through one, two, three, or more mesh screens to create a population of non-spherical beads that are relatively similar in shape and size. This two-screen system allows for tight control over bead size, allowing the user to adjust the size as needed. Such non-spherical microbeads are disclosed in US 2020 / 0069846 and US Patent 11,771,807.

[0096] activator Any of the gel / hydrogel compositions described herein can be utilized to contain an active agent, thereby acting as an active agent delivery system when applied to a body surface (e.g., a tissue repair site) in such a manner that the active agent is delivered thereto. Release of the active agent "loaded" into the hydrogel or composite typically involves both water absorption and desorption of the agent via a swelling-controlled diffusion mechanism. For example, active agent-containing hydrogel compositions can be used in, for example, transdermal drug delivery systems, wound dressings, topical pharmaceutical formulations, implantable drug delivery systems, oral dosage forms, and the like.

[0097] Suitable active agents that can be incorporated into the hydrogel compositions of the present invention and delivered systemically (e.g., using transdermal, oral, or other dosage forms suitable for systemic administration of drugs) include analgesics; anesthesia agents; anti-arthritic agents; respiratory medications, including anti-asthmatic agents; anti-cancer medications, including antineoplastic agents; anticholinergics; anticonvulsants; antidepressants; antidiabetic agents; antidiarrheals; anthelmintics; antihistamines; anti-dyslipidemic agents; anti-hypertensive agents; anti-infective agents, such as antibiotics and antivirals; anti-inflammatory agents; anti-migraine preparations; antiemetics; anti-Parkinson's agents; antipruritics; antipsychotics; antipyretics; antispasmodics; anti-tuberculosis agents; antiulcer agents; antivirals; anti-anxiety agents; appetite suppressants; attention deficit disorder (ADD) and attention deficit hyperactivity disorder (ADHD) medications; calcium channel blockers, anti-anginal agents, central nervous system (CNS) These include, but are not limited to, cardiovascular preparations, including CNS agents, beta-blockers, and antiarrhythmics; central nervous system stimulants; cold preparations, including decongestants; diuretics; genetic materials; herbal medicines; hormonelytics; hypnotics; hypoglycemic agents; immunosuppressants; leukotriene inhibitors; antimitotic agents; muscle relaxants; anesthetic antagonists; nicotine; nutritional supplements, such as vitamins, essential amino acids, and fatty acids; eye drops, such as antiglaucoma agents; parasympatholytics; peptide drugs; psychostimulants; sedatives; steroids, including progestogens, estrogens, corticosteroids, androgens, and anabolic agents; smoking cessation agents; sympathomimetics; tranquilizers; and vasodilators, including general coronary, peripheral, and cerebral. Specific active agents useful in combination with the adhesive compositions of the present invention include, but are not limited to, anabasine, capsaicin, isosorbide dinitrate, aminostigmine, nitroglycerin, verapamil, propranolol, cilabolin, foridone, clonidine, cytisine, phenazepam, nifedipine, fluacizin, and salbutamol.

[0098] For topical drug administration and / or medicated cushions (eg, medicated foot pads), suitable active agents include, by way of example, the following:

[0099] Bacterial Growth Inhibitors and Bactericides: Suitable bacterial growth inhibitors and bactericides include, by way of example, iodine, iodopovidone complex (i.e., a complex of PVP and iodine, also known as "povidine" and available from Purdue Frederick under the trademark Betadine), iodide salts, chloramine, chlorhexidine, and halogen compounds such as sodium hypochlorite; sulfadiazine, silver protein acetyltannate; oxidizing agents such as hydrogen peroxide and potassium permanganate; arylmercury compounds such as phenylmercuric borate or merbromine; alkylmercury compounds such as thiomersal; phenols such as thymol, o-phenylphenol, 2-benzyl-4-chlorophenol, hexachlorophene, and hexylresorcinol; and organic nitrogen compounds such as 8-hydroxyquinoline, chlorquinaldol, clioquinol, ethacridine, hexetidine, chlorhexedine, and ambazone.

[0100] Antibiotic Agents: Suitable antibiotic agents include, but are not limited to, the lincomycin family of antibiotics (referring to a class of antibiotic agents first recovered from Streptomyces lincolnensis), members of the tetracycline family (referring to a class of antibiotic agents first recovered from Streptomyces aureofaciens), and sulfur-based antibiotics, i.e., sulfonamides. Exemplary antibiotics of the lincomycin family include lincomycin, clindamycin, related compounds, and their pharmacologically acceptable salts and esters. Exemplary antibiotics of the tetracycline family include tetracycline itself, chlortetracycline, oxytetracycline, tetracycline, demeclocycline, rolitetracycline, methacycline, and doxycycline, and their pharmaceutically acceptable salts and esters, particularly acid addition salts such as hydrochlorides. Exemplary sulfur-based antibiotics include, but are not limited to, the sulfonamides sulfacetamide, sulfabenzamide, sulfadiazine, sulfadoxine, sulfamerazine, sulfamethazine, sulfamethizole, sulfamethoxazole, and pharmacologically acceptable salts and esters thereof, e.g., sulfacetamide sodium.

[0101] Pain relievers: Suitable pain relievers include acetamidoeugenol, alphadolone acetate, alphaxalone, amucaine, amoranone, amylocaine, benoxinate, betoxicaine, biphenamine, bupivacaine, burethamine, butacaine, butaben, butanilicaine, butarital, butoxicaine, carticaine, 2-chloroprocaine, cinchocaine, cocaethylene, cocaine, Caine, cyclomethycaine, dibucaine, dimethisoquin, dimethocaine, diperadon, dyclonine, ecgonidine, ecgonine, ethylaminobenzoate, ethyl chloride, etidocaine, etoxadrol, beta-eucaine, euprosin, fenalcomine, fomocaine, hexobarbital, hexylcaine, hydroxydione, hydroxyprocaine, hydroxytetracaine, isobutyl p-aminobenzoate, Kentamine, leucinocaine mesylate, levoxadrol, lidocaine, mepivacaine, meprylcaine, metabutoxycaine, methohexital, methyl chloride, midazolam, myrtecaine, naepain, octacaine, orthocaine, oxethazaine, parethoxycaine, phenacaine, phencyclidine, phenol, piperocaine, pyridocaine, polidocanol, pramoxine, prilocaine, procaine, pro Local anesthetics, including but not limited to, panidid, propanocaine, proparacaine, propipocaine, propofol, propoxycaine, pseudococaine, pyrrocaine, lisocane, salicylic alcohol, tetracaine, thialbarbital, thimylal, thiobutabarbital, thiopental, tricaine, trimecaine, zolamine, and combinations thereof. Tetracaine, lidocaine, and prilocaine are referred to herein as pain relievers.

[0102] Other topical agents that can be delivered using the hydrogel compositions of the present invention as a drug delivery system include: antifungals such as undecylenic acid, tolnaftate, miconazole, griseofulvin, ketoconazole, ciclopirox, clotrimazole, and chloroxylenol; keratolytic agents such as salicylic acid, lactic acid, and urea; vesicants such as cantharidin; organic peroxides (e.g., benzoyl peroxide), retinoids (e.g., retinoic acid, adapalene, and tazarotene), sulfonamides (e.g., sodium sulfacetamide), resorcinol, corticosteroids (e.g., triamcinolone), alpha-hydroxy acids (e.g., lactic acid and glycolic acid), alpha-keto acids (e.g., glyoxylic acid), and azelaic acid, clindamycin, erythromycin, meclocycline, minocycline, nadifloxacin, cephalexin, doxycycline, and ofloxacin anti-acne agents, such as antibacterial agents specifically formulated for the treatment of acne, including: skin lightening agents and bleaching agents such as hydroquinone, kojic acid, glycolic acid, and other alpha-hydroxy acids, artocarpin, and certain organic peroxides; agents for treating warts, including salicylic acid, imiquimod, dinitrochlorobenzene, dibutylsqualic acid, podophyllin, podophyllotoxin, cantharidin, trichloroacetic acid, bleomycin, cidofovir, adefovir, and analogs thereof; and anti-inflammatory agents such as corticosteroids and nonsteroidal anti-inflammatory drugs (NSAIDs include ketoprofen, flurbiprofen, ibuprofen, naproxen, fenoprofen, benoxaprofen, indoprofen, pirprofen, carprofen, oxaprozin, pranoprofen, suprofen, alminoprofen, butibufen, fenbufen, and tiaprofenic acid).

[0103] For wound dressings, suitable active agents are those useful in treating wounds, including, but not limited to, bacterial growth inhibitory and bactericidal compounds, antibiotics, pain relievers, vasodilators, tissue healing enhancers, amino acids, proteins, proteolytic enzymes, cytokines, and polypeptide growth factors.

[0104] For topical and transdermal administration of some active agents in wound dressings, it may be necessary or desirable to incorporate a permeation enhancer into the hydrogel composition to enhance the rate of permeation of the agent into or through the skin. Suitable enhancers include, for example, sulfoxides such as dimethyl sulfoxide (DMSO) and decyl methyl sulfoxide; ethers such as diethylene glycol monoethyl ether (commercially available as Transcutol) and diethylene glycol monomethyl ether; surfactants such as sodium laurate, sodium lauryl sulfate, cetyltrimethylammonium bromide, benzalkonium chloride, poloxamer (231, 182, 184), Tween (20, 40, 60, 80), and lecithin (U.S. Pat. No. 4,783,450); 1-substituted azacycloheptan-2-ones, specifically 1-n-dodecylcycloaza-cycloheptan-2-one (Nelson Research & Development Co., Irvine, Calif. under the trademark Azone; see U.S. Patent Nos. 3,989,816, 4,316,893, 4,405,616, and 4,557,934); alcohols such as ethanol, propanol, octanol, decanol, benzyl alcohol, and the like; fatty acids such as lauric acid, oleic acid, and valeric acid; fatty acid esters such as isopropyl myristate, isopropyl palmitate, methyl propionate, and ethyl oleate; propylene glycol, ethylene glycol, glycerin, Polyols and esters thereof, such as butanediol, polyethylene glycol, and polyethylene glycol monolaurate (PEGML; see, e.g., U.S. Pat. No. 4,568,343); amides and other nitrogen compounds, such as urea, dimethylacetamide (DMA), dimethylformamide (DMF), 2-pyrrolidone, 1-methyl-2-pyrrolidone, ethanolamine, diethanolamine, and triethanolamine; terpenes; alkanones; and organic acids, specifically, salicylic acid and salicylates, citric acid, and succinic acid. Mixtures of two or more enhancers may also be used.

[0105] In certain other embodiments, composite compositions comprising hydrogel components and nanofibers may also contain additional optional additive components. Such components are known in the art and may include, for example, fillers, preservatives, pH adjusters, softening agents, viscosity enhancers, pigments, coloring agents, refractive particles, stabilizers, reinforcing agents, adhesion-reducing agents, pharmaceutical agents (e.g., antibiotics, angiogenesis promoters, antifungals, immunosuppressants, antibodies, and the like), and permeation enhancers. Such additives and their amounts are selected so that they do not significantly interfere with the desired chemical and physical properties of the hydrogel composition.

[0106] When the adhesive is applied to the skin or other body surfaces, absorbent fillers can be advantageously incorporated to control the degree of hydration.Such fillers can include microcrystalline cellulose, talc, lactose, kaolin, mannitol, colloidal silica, alumina, zinc oxide, titanium oxide, magnesium silicate, magnesium aluminum silicate, hydrophobic starch, calcium sulfate, calcium stearate, calcium phosphate, calcium phosphate dihydrate, woven and nonwoven paper, and cotton material.Other suitable fillers are inert, i.e., substantially non-absorbent, and include, for example, polyethylene, polypropylene, polyurethane polyetheramide copolymer, polyester and polyester copolymer, nylon, and rayon.

[0107] The composition may also include one or more preservatives, examples of which include p-chloro-m-cresol, phenylethyl alcohol, phenoxyethyl alcohol, chlorobutanol, 4-hydroxybenzoic acid methyl ester, 4-hydroxybenzoic acid propyl ester, benzalkonium chloride, cetylpyridinium chloride, chlorhexidine diacetate or chlorhexidine gluconate, ethanol, and propylene glycol.

[0108] The composition may also include a pH adjusting compound. Compounds useful as pH adjusting substances include, but are not limited to, glycerol buffer, citrate buffer, borate buffer, phosphate buffer, or citrate-phosphate buffer, which may also be included to ensure that the pH of the hydrogel composition is compatible with that of an individual's body surface.

[0109] The composition can also contain a suitable softening agent.Suitable softening substances include citric acid esters such as triethyl citrate or acetyltriethyl citrate, tartaric acid esters such as dibutyl tartrate, glycerol esters such as glycerol diacetate and glycerol triacetate; phthalic acid esters such as dibutyl phthalate and diethyl phthalate; and / or hydrophilic surfactants, preferably hydrophilic nonionic surfactants such as partial fatty acid esters of sugars, polyethylene glycol fatty acid esters, polyethylene glycol fatty alcohol ethers, and polyethylene glycol sorbitan fatty acid esters.

[0110] Composition can also contain thickener.Preferred thickener herein is naturally occurring compound or its derivative, for example, includes collagen; galactomannan; starch; starch derivatives and hydrolysates; cellulose derivatives such as methylcellulose, hydroxypropylcellulose, hydroxyethylcellulose and hydroxypropylmethylcellulose; colloidal silicic acid; and sugars such as lactose, saccharose, fructose and glucose.Synthetic thickeners such as polyvinyl alcohol, vinylpyrrolidone-vinyl acetate copolymer, polyethylene glycol and polypropylene glycol can also be used.

[0111] In certain embodiments, the hydrogel composites of the present invention, comprising a hydrogel and nanofibers, further comprise a component that promotes angiogenesis. Prior to the present invention, a challenge in achieving clinically relevant soft tissue regeneration was that the regenerated tissue should preferably be revascularized. Therefore, any material that promotes soft tissue regeneration should preferably also promote angiogenesis. One way to achieve this is through the use of heparin-containing hydrogel components, which can serve as growth factor binding sites to enrich and retain growth factors that promote angiogenesis and tissue formation.

[0112] In one embodiment, the composition further comprises and delivers an antibody. The term "antibody" is used herein in its broadest sense and includes a specific type of immunoglobulin molecule that contains one or more antigen-binding domains that specifically bind to an antigen or epitope. Antibodies include, in particular, intact antibodies (e.g., intact immunoglobulins), antibody fragments, and multispecific antibodies.

[0113] In some aspects, the antibody comprises an antibody. In some aspects, the antibody is a monoclonal antibody. In some aspects, the antibody is a chimeric antibody. In some aspects, the antibody is a humanized antibody. In some aspects, the antibody is a human antibody. In some aspects, the antibody comprises an antibody fragment. In some aspects, the antibody comprises an alternative scaffold.

[0114] In one aspect, the compositions provided herein further comprise cells for delivery. In some aspects, the cells are derived from the subject to which they are administered. In some aspects, the cells are derived from a source other than the subject to which they are administered. In some aspects, the cells are derived from a cell line. In some aspects, the cells are derived from a human source. In some aspects, the cells are derived from a humanized animal source.

[0115] In some aspects, the cells provided are stem cells, hi some aspects, the cells provided are neural cells.

[0116] In some embodiments, the compositions provided herein further comprise a small molecule for delivery, wherein the small molecule is a biologically active material. In some embodiments, the small molecule can cause pharmacological activity or another direct effect in the diagnosis, cure, mitigation, treatment, or prevention of disease, or can affect the structure or function of the body.

[0117] The gel / hydrogel / nanofiber composites of the present invention can also contain tissue repair agents such as several growth factors, including epidermal growth factor (EDF), PDGF, and nerve growth factor (NGF). For example, the composition can contain EGF. Epidermal growth factor (EGF) was discovered after the observation that skin wounds in laboratory mice seemed to heal faster when licked by the mice. This was not simply due to some bactericidal agent in saliva (such as lysozyme). A specific growth factor, now known as EGF, was shown to be responsible. EGF is identical to urogastrone and has angiogenic properties. Transforming growth factor-alpha (TGFα) is very similar, binds to the same receptor, and is even more effective at stimulating epithelial cell regeneration (epithelialization).

[0118] Thus, hydrogels containing EGF / TGF may be advantageously used in accelerating wound healing and reducing keloid scar formation (especially for burns), skin survival dressings, and treating chronic leg ulcers.

[0119] Tissue repair agents useful in the present invention include several growth factors, including epidermal growth factor (EDF), PDGF, and nerve growth factor (NGF). Generally, growth-promoting hormones affect one to four tissues. Many of the products developed from such proteins target some type of wound repair, although other indications exist. Some of the most important tissue growth factors are further described below.

[0120] The gel / nanofiber compositions of the present invention may also include one or more growth factors that may be useful in the tissue repair methods and other applications of the present invention.

[0121] The hydrogel / nanofiber compositions of the present invention may also contain VEGF to promote angiogenesis. Vascular endothelial growth factor (VEGF—also known as vascular permeability factor) is another vascular growth factor that is a multifunctional angiogenic cytokine. It contributes to angiogenesis (blood vessel growth) both indirectly and directly by stimulating the proliferation and migration of endothelial cells at the microvascular level, as well as by altering their overall expression. VEGF also contributes to angiogenesis by causing regional changes in these endothelial cells, causing them to enter a hyperpermeable state, which allows them to release plasma proteins outside the vascular lumen.

[0122] The compositions of the present invention may also contain FGF. Fibroblast growth factors (FGFs) are actually a family of at least 19 different 14-18 kD peptides belonging to the heparin-binding growth factor family and are mitogenic for cultured fibroblasts and vascular endothelial cells. They are also angiogenic in vivo, and this angiogenicity is enhanced by TNF. FGFs may be used in a manner similar to EGF. bFGF, also known as FGF-2, is involved in regulating human megakaryocytopoiesis, and FGFs have been shown to be effective in stimulating endothelial cell formation and supporting connective tissue repair.

[0123] The hydrogel / nanofiber composition may also include keratinocyte growth factor (KGF), also known as FGF-7, for use in wound healing and other disorders involving epithelial cell destruction.

[0124] Transforming growth factors (TGFs) have the ability to transform various cell lines, conferring, for example, the ability to grow in culture beyond a limited number of generations, growth in multilayers rather than monolayers, and the acquisition of abnormal karyotypes. There are at least five members of the TGF family, with the two most widely studied being TGF-alpha and TGF-beta. The former is mitogenic for fibroblasts and endothelial cells, angiogenic, and promotes bone resorption. Compositions may also contain TGFs. TGF-beta is a general mediator of cell regulation and a potent inhibitor of cell growth, inhibiting the proliferation of many cell types. TGF-beta can antagonize the mitogenic effects of other peptide growth factors and can also inhibit the growth of many tumor cell lines. TGF-beta also has angiogenic effects and promotes collagen formation in fibroblasts.

[0125] The hydrogel / nanofiber compositions of the invention may usefully include, for example, free, uncrosslinked collagen, which in this form is unlikely to perform a useful structural function but may instead serve primarily as a sacrificial protein, for example, when proteolytic activity is unnecessarily high, thereby helping to prevent dissociation of healthy tissue.

[0126] The hydrogel / nanofiber composition may also contain certain enzymes. Enzymes are used in debridement of both acute and chronic wounds. Debridement is the removal of non-viable tissue and foreign material from the wound and is a naturally occurring event in the wound repair process. During the inflammatory phase, neutrophils and macrophages digest and remove "spent" platelets, cellular debris, and avascular injured tissue from the wound area. However, with the accumulation of significant amounts of damaged tissue, this natural process becomes overwhelmed and insufficient. The resulting accumulation of necrotic tissue places a significant phagocytic demand on the wound, delaying wound healing. Consequently, debridement of necrotic tissue is a targeted local treatment and a key component of optimal wound management.

[0127] Enzymes can be incorporated into the hydrogels of the present invention, for example, for topical use to provide an alternative method of wound debridement. Suitable enzymes can be obtained from a variety of sources, such as krill, crab, papaya, bovine extracts, and bacteria. Suitable commercially available enzymes include collagenase, papain / urea, and a combination of fibrinolysin and deoxyribonuclease.

[0128] Enzymes for use in the present invention generally function in one of two ways: by directly digesting components of carrion (e.g., fibrin, bacteria, leukocytes, cellular debris, serous exudates, DNA) or by dissolving the collagen "anchors" that secure avascular tissue to the underlying wound bed.

[0129] The hydrogels of the present invention may generally contain Dakin's solution to provide antimicrobial effects and odor control, if desired. As a debriding agent, Dakin's solution is nonselective due to its cytotoxic properties. Dakin's solution denatures proteins, making them easier to remove from the wound. Loosening the carrion also facilitates debriding by other methods. Hydrogels containing Dakin's solution may be changed twice daily if the purpose is debriding. Protection of the skin around the wound should generally be provided, for example, with an ointment, a liquid skin barrier film dressing, or a solid skin barrier wafer.

[0130] The gels of the present invention may be delivered by any suitable method, such as via a syringe or bellows pack (single dose delivery systems), or via a multi-dose system, such as delivery via a pressurized delivery system or a "bag in the can" type system.

[0131] Thus, the present invention also extends to single-dose delivery systems comprising a gel according to the present invention for the treatment of wounds. The present invention also extends to pressurized delivery systems comprising a gel according to the present invention, and pressurized hydrogels according to the present invention in aerosol containers capable of forming a spray upon release of pressure therefrom. The use of such delivery means allows the gel to be delivered to areas of a patient that are otherwise difficult to reach by direct application.

[0132] In certain embodiments, it may be advantageous to render the hydrogel compositions of the present invention electrically conductive for use in biomedical electrodes and other electrotherapeutic settings, i.e., for attaching electrodes or other electrically conductive members to body surfaces. For example, the hydrogel compositions may be used to attach transcutaneous nerve stimulation electrodes, electrosurgical return electrodes, or EKG electrodes to a patient's skin or mucosal tissue. Such applications involve modifying the hydrogel compositions to contain conductive species. Suitable conductive species are ionically conductive electrolytes, particularly those commonly used in the manufacture of conductive adhesives for application to skin or other body surfaces, including ionizable inorganic salts, organic compounds, or a combination of both. Examples of ionically conductive electrolytes include, but are not limited to, redox couples such as ammonium sulfate, ammonium acetate, monoethanolamine acetate, diethanolamine acetate, sodium lactate, sodium citrate, magnesium acetate, magnesium sulfate, sodium acetate, calcium chloride, magnesium chloride, calcium sulfate, lithium chloride, lithium perchlorate, sodium citrate, and potassium chloride, as well as mixtures of ferric and ferrous salts (such as sulfate and gluconate). Preferred salts are potassium chloride, sodium chloride, magnesium sulfate, and magnesium acetate, with potassium chloride being most preferred for EKG applications. While virtually any amount of electrolyte can be present in the adhesive compositions of the present invention, any electrolyte is preferably present at a concentration within the range of about 0.1 to about 15 wt. % of the hydrogel composition. The techniques described in U.S. Patent No. 5,846,558 to Nielsen et al. for fabricating biomedical electrodes may be adapted for use with the hydrogel compositions of the present invention, the disclosure of which is incorporated by reference for manufacturing details. Other suitable fabrication techniques may also be used, as will be understood by those skilled in the art.

[0133] Non-spherical beaded formulations The composite / hydrogel can be formed into a particulate formulation (e.g., beads, particles, or microbeads), which allows for the use of higher concentrations of each component, facilitates gel delivery, improves mixing with biologically active ingredients, and enhances stability. In the related art, some commercial hydrogel-based fillers can be blended using blades or similar to form beads. While this method is not ideal, it offers little control over the size and shape of the beads. Furthermore, nanoparticle-hydrogel composites are formed as bulk composite gels or solidified gels, which generally do not result in a uniform, solid material.

[0134] Improvements are provided, including introducing the composite as a beaded gel. This allows users to vary the bead properties to achieve desired results and improve the storage modulus of the composite. In certain embodiments, preformed hydrogel-nanofiber composites are physically conditioned to form microparticles, such as by forcing them through one, two, three, or more than three mesh screens to create a population of non-spherical beads that are relatively similar in shape and size. This multiple screen system allows for tight control over the size of the beads, allowing users to adjust the size as needed.

[0135] Pre-reacted composite Preferably, the composite or hydrogel composite can be reacted before injection or storage. If the components of the formulation are mixed by the end user immediately before injection into a subject, application of the composite can involve human factors complications, such as the required preparation and handling by the user, and the mixing and waiting time can change the reaction time, which can significantly alter the stiffness of the composite. For example, the gel may be too stiff or not stiff enough to be injected via a syringe, which can create undesirable properties when injected into a subject. To address this issue, the inventors developed a pre-reacted composition, in which the reaction (e.g., gelation) occurs during manufacture before storage.

[0136] In certain embodiments, the hydrogel composite is gelled or cured and then micronized or screened to form beads, particles, or microbeads. In certain embodiments, after gelling, the hydrogel composite is converted into beads by forcing the gel through a mesh or screen. Bead size was varied by varying the mesh size of the screen used in the beading process. Preferably, the mesh or screen has an opening size ranging from about 90 μm to about 250 μm, from about 95 μm to about 200 μm, or from about 100 μm to about 150 μm.

[0137] Because the resulting beads must have at least one dimension sized to be smaller than the inner diameter of the syringe needle, bead screen sets with minimum openings larger than 250 μm may be inappropriate for typical injection situations. Needles commonly used for dermal filler applications range from 25 gauge to 30 gauge, with inner diameters ranging from 260 μm to 160 μm. The practicality of smaller screen sizes may be limited by the size of the fiber component, as there is a risk of fiber-gel rupture if the length of individual fibers exceeds the screen opening. For this reason, meshes with openings larger than 20 microns are used.

[0138] Freeze drying For example, hydrogel compositions such as beads (e.g., microbeads) or particles can be processed by freeze-drying before storage. The introduction of freeze-drying allows the product to be stored at room temperature for extended periods without loss of function and allows flexibility for fluid rehydration. Preferably, the beaded product is freeze-dried in an isotonic solution of sucrose, trehalose, and sodium chloride. These variables protect the microstructure during the drying process and extend the shelf life of the product. Freeze-dried gel beads (e.g., microbeads) can be reconstituted with water after storage, making them ready for injection within seconds.

[0139] The mechanical properties of the nanofiber phase of a fiber-hydrogel composite remain substantially unchanged in both the dry and frozen states, in contrast to most hydrogel components. Thus, during freezing or lyophilization, the fiber portion can help maintain the overall composite microstructure. With the correct lyophilization cycle and formulation, the composite can be lyophilized while still remaining as discrete beads (e.g., microbeads) upon rehydration.

[0140] Soft Tissue Devices Provided herein is a soft tissue device or implant comprising a biologically active material and non-spherical microbeads comprising a hydrogel composite, the hydrogel composite comprising a functionalized hyaluronic acid network covalently linked to a plurality of polycaprolactone nanofibers described herein.

[0141] Preferably, the non-spherical microbeads have an average size along their longest dimension in the range of about 50 micrometers to about 300 micrometers.

[0142] Preferably, the biologically active material may be suitable for fat grafting, which may differentiate into soft tissue such as adipose tissue when supported with an appropriate matrix microenvironment, for example. In certain aspects, the biologically active material comprises a population of adipose cells, autologous adipose cells, allogeneic cells, genetically modified allogeneic cells, stem cells, mesenchymal stem cells, genetically modified stem cells, genetically modified allogeneic induced pluripotent stem (iPS) cells, genetically modified hypoimmunogenic pluripotent stem cells, adipose stromal vascular cells, adipose tissue, autologous adipose tissue, lipoaspirate tissue, derivatives thereof, or combinations thereof.

[0143] In some embodiments, the biologically active material comprises adipose tissue. In some embodiments, the adipose tissue is lipoaspirate tissue. In some embodiments, the adipose tissue is autologous.

[0144] In certain embodiments, a soft tissue device or implant may suitably contain non-spherical microbeads in a volume that is about 25% to 75% of the total volume of the soft tissue device. In certain embodiments, a soft tissue device or implant may suitably contain non-spherical microbeads in a volume that is about 30% to 70% of the total volume of the soft tissue device. In certain embodiments, a soft tissue device or implant may suitably contain non-spherical microbeads in a volume that is about 35% to 65% of the total volume of the soft tissue device. In certain embodiments, a soft tissue device or implant may suitably contain non-spherical microbeads in a volume that is about 40% to 60% of the total volume of the soft tissue device. In certain embodiments, a soft tissue device or implant may suitably contain non-spherical microbeads in a volume that is about 50% of the total volume of the soft tissue device.

[0145] In certain embodiments, a soft tissue device or implant may suitably comprise non-spherical microbeads in an amount of about 10% to 90% of the total weight of the soft tissue device. In certain embodiments, a soft tissue device or implant may suitably comprise non-spherical microbeads in an amount of about 20% to 80% of the total weight of the soft tissue device. In certain embodiments, a soft tissue device or implant may suitably comprise non-spherical microbeads in an amount of about 30% to 70% of the total weight of the soft tissue device. In certain embodiments, a soft tissue device or implant may suitably comprise non-spherical microbeads in an amount of about 40% to 60% of the total weight of the soft tissue device.

[0146] In certain embodiments, the biological material is formed into fat particles. Preferably, the ratio of the average size of the non-spherical microbeads to the average size of the fat particles ranges from about 10:1 to about 1:10. In some embodiments, the ratio of the average size of the non-spherical microbeads to the average size of the fat particles is about 10:1, about 3:1, about 1:1, about 1:2, about 1:3, or about 1:10.

[0147] Fat particles can be prepared using any non-limiting method for clinical use. For example, fat particles can be processed by forming a fat film and digesting the film using mechanical forces such as shearing, micronization, or grinding. In certain embodiments, fat particles can be obtained from animal body fat (e.g., fish oil or mammalian fat) using an aspirator or vacuum suction. Animal fat can be further processed by using physical forces such as recovery, micronization, or grinding to create homogenized fat particles or to obtain a uniform particle size distribution. In certain embodiments, the size or diameter (e.g., average diameter) of fat particles can be controlled using a filter or filtration device. In certain embodiments, the size or diameter (e.g., average diameter) of the fat particles can be less than about 1,000 μm, less than about 900 μm, less than about 800 μm, less than about 700 μm, less than about 600 μm, less than about 500 μm, less than about 400 μm, less than about 300 μm, less than about 200 μm, less than about 100 μm, or preferably in the range of 10-90 μm, or specifically in the range of 20-80 μm.

[0148] In certain embodiments, the microbeads are gelled or cured before the biologically active material is added to the microbeads. The microbeads may be gelled by chemical reaction (e.g., cross-linking) or UV irradiation so that the polymer cures or polymerizes to form nanofibers.

[0149] In certain embodiments, the device or implant may comprise a hydrogel composition made from thiolated hyaluronic acid and a crosslinker selected from poly(ethylene glycol) diacrylate (PEGDA) or its derivatives. In certain embodiments, the device or implant may comprise a hydrogel composition made from acrylated hyaluronic acid and a crosslinker selected from thiolated poly(ethylene glycol) or its derivatives.

[0150] In certain embodiments, the plurality of polycaprolactone fibers is made by electrospinning. The plurality of polycaprolactone fibers suitably comprises electrospun fibers.

[0151] In some embodiments, the soft tissue device or implant may further comprise a compound selected from the group consisting of growth factors, angiogenesis-stimulating compounds, immunomodulators, inflammation-inhibiting substances, and combinations thereof. In some embodiments, the soft tissue device may further comprise one or more compounds having therapeutic, angiogenic, anti-angiogenic, anti-inflammatory, antibacterial, antihistamine effects, and combinations thereof.

[0152] Preferably, the soft tissue device has a tan delta value of less than about 0.27. Tan delta is the rheological loss modulus divided by the storage modulus, meaning that a lower tan delta number corresponds to a more "solid-like" as opposed to "liquid-like" material. Tan delta can also represent a rheological property, which can vary based on the oil or fat content of a material or substance.

[0153] In certain embodiments, the microbeads are substantially stable at room temperature for at least about 1 week, at least about 2 weeks, at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, or at least about 6 months.

[0154] In certain embodiments, the soft tissue device is stable at a temperature of 37°C.

[0155] The implant soft tissue device may be used for implantation or injection administration near the target tissue.

[0156] Fiber-hydrogel composite combined with adipose tissue Provided herein is a nanofiber-hydrogel composite (composite-adipose) combined with adipose tissue for use in a medical device that is incorporated into the tissue of a human subject to which the composite is administered, for example, by injection or implantation. The composite-adipose can be prepared by any means known in the art. Various ratios of fat and fiber-hydrogel composite can be combined to achieve optimal results for the desired outcome. In a preferred embodiment, the adipose tissue is lipoaspirate tissue.

[0157] In certain embodiments, the nanofiber-hydrogel composite can be allowed to gel for a certain amount of time, such as about 1 hour, 3 hours, 5 hours, 8 hours, 12 hours, 24 hours, 36 hours, 48 ​​hours, or between about 1 hour and 24 hours, before adding the adipose tissue. In certain embodiments, the nanofiber-hydrogel composite is allowed to gel and stored (in hydrated form or lyophilized) for an indefinite amount of time before adding a biologically active component, such as adipose tissue.

[0158] After combining hydrogel composite (microbeads) and lipoaspirate, superior mechanical integrity can be obtained compared with lipoaspirate alone.Microbeads and lipoaspirate can be administered (e.g., injected) into human subjects to promote fat graft retention and vascularization, and act as a tissue scaffold that mimics the natural extracellular matrix.This approach offers the hope of larger volume reconstruction without the risk of implant failure and fibrosis.Composite-lipoaspirate has improved mechanical properties that are more similar to in vivo fat compared with the processed lipoaspirate currently used clinically.

[0159] Depending on the ratio of fat (e.g., lipoaspirate) to composite, the storage modulus (G') of the combined materials can be increased in a synergistic manner. This is important because storage modulus is a measure of how deformable a material is; an ideal tissue scaffold would have properties / strength similar to native adipose tissue. The higher the storage modulus and the lower the tan delta, the less deformable the fat / composite combination will be, and therefore, the stronger and less susceptible to shear forces. Therefore, having a high storage modulus in the fat-composite combination may be associated with improved adipocyte and fat graft survival in vivo. This has strong clinical implications, as it translates into reduced morbidity and fewer procedures for patients undergoing fat grafting for soft tissue reconstruction.

[0160] Composite-fat has the potential to exhibit superior rates of angiogenesis and vascular ingrowth compared to lipoaspirate tissue. Adequate blood supply is essential for the survival of fat grafts. This is why the increased angiogenesis and vascular ingrowth provided by composite-fat is necessary to improve long-term adipocyte survival. Furthermore, composite-fat does not prevent adipocytes from accessing cell culture media for survival. Composite materials provide adipocytes with access to angiogenic growth factors required for long-term survival. On the other hand, lipoaspirate tissue alone has a lipid layer surrounding the sample, which prevents adipocytes from accessing cell culture media.

[0161] How to use Also provided are methods of fat grafting in a subject by implanting or injecting a soft tissue device or implant described herein.

[0162] The present disclosure further provides a method for performing cosmetic or reconstructive procedures, or for reducing or reversing tissue defects resulting from trauma, surgical intervention, or age-related diseases, disorders, or conditions.The method comprises the step of implanting or injecting the soft tissue device described herein into the target tissue of a subject.Preferably, the biologically active material has the ability to perform at least one of the following in the subject: i) recruit host cell infiltration, ii) promote tissue growth, and / or iii) cell regeneration or tissue regeneration, and the soft tissue device has the ability to be implanted or injected into the target tissue of a subject in need thereof.

[0163] Additionally, a kit is provided for preparing the soft tissue device described herein for injection into a target tissue of a subject. For example, the hydrogel composite (microbeads) and the biologically active material can be injected simultaneously or separately into the target tissue of a subject in need thereof. In certain embodiments, the kit can include (i) a first syringe containing microbeads and (ii) a second syringe containing the biologically active material, where the microbeads are gelled or hardened before the biologically active material is added to the microbeads. In certain embodiments, the microbeads can be pre-reacted and freeze-dried. In certain embodiments, the microbeads and the biologically active material (e.g., fat or adipose tissue) are not gelled in situ. For example, the biologically active material (e.g., fat or adipose tissue) for fat transplantation can be applied onto or inside a pre-reacted / beaded hydrogel composite.

[0164] In some embodiments, the kit further comprises an additional syringe or vial containing water, saline, or a fluid suitable for reconstituting the dehydrated microbeads.

[0165] Although specific examples and uses for the hydrogel / nanostructured composites of the present invention are described herein, such specific uses are not intended to be limiting. The hydrogel / nanostructured composites of the present invention can be used in any application commonly used for known hydrogels, and are particularly useful for the repair and / or regeneration of soft tissue anywhere in the body. [Example]

[0166] The compositions, methods, and systems of the present invention incorporate technical improvements for enhancing the survival and function of grafted fat by blending it with a fiber-hydrogel composite. Fat grafting is a procedure for soft tissue restoration and volume augmentation following soft tissue loss due to trauma, surgery, disease, or aging. Approximately 600,000 fat grafting procedures were performed on the face worldwide in 2019, with substantially more procedures performed on other anatomical regions [ISAPS 2019 International Survey]. However, fat grafting is limited by its variable and incomplete graft survival rates, ranging from 34% to 82% in the breast and 30% to 83% in the facial region [Yu, Nan-Ze et al. "A Systemic Review of Autologous Fat Grafting Survival Rate and Related Severe Complications," Chinese Medical Journal, Vol. 128, 9 (2015): 1245-51]. Results from animal studies have shown that survival rates after 15 weeks in nude mice range from 38.3% to 52.5% and after one year in rabbits range from 14.00% to 14.56%, and can be even more severe [Yu, Nan-Ze et al. "A systematic review of autologous fat grafting survival rate and related severe complications." Chinese medical journal vol. 128,9 (2015): 1245-51]. This can reduce surgical accuracy, patient satisfaction, and require repeated surgical procedures.

[0167] The compositions, methods, and systems of the present invention improve the survival and volume expansion of fat grafts by blending processed fat with a fiber-hydrogel composite gel immediately prior to injection. In one aspect, the gel inclusions mimic the body's own extracellular matrix structure, thereby providing mechanical support for the processed fat globules, protection from shear forces during the injection procedure, a reduced tan delta (and therefore increased solid-like characteristics) to resist unintended implant migration, adhesion sites for cells after implantation, immunomodulation and protection for the transplanted cells, and vascularization to support the survival of the transplanted cells. In one aspect, the morphology of the fiber-hydrogel composite of the present invention is such that fragmented-length individual polymer fibers are uniformly dispersed throughout the hydrogel phase, with interfacial bonding between the fibers and the hydrogel phase. The gel is fully reacted and disintegrated into individual microbeads approximately 150 microns in diameter before blending with the processed fat. By combining fat with the composite, better outcomes can be achieved than with fat grafting alone.

[0168] While such a combination of fat and fiber-hydrogel composites has been described in previous disclosures, this is the first disclosure of a pre-reacted "beaded" form. Previous work was performed using an in situ gelling form of the composite. The use of a beaded form of the composite gel in this disclosure highlights various optimal ratios of the concentrations and sizes of the two components (fiber-hydrogel composite microbeads and fat particles) to result in enhanced soft tissue regeneration.

[0169] Example 1: Nanofiber-Hydrogel Composite Bead Preparation A nanofiber-hydrogel composite (Lot 0026-093020-1) was obtained using the method described in patent application PCT / US2019 / 031638. Briefly, the gel consists of polycaprolactone (PCL) fiber fragments uniformly dispersed within a hyaluronic acid (HA) hydrogel with polyethylene glycol dithiol (PEG-SH) as a crosslinker. PEG-SH reacts with the acrylate groups functionalized on the HA molecules and the maleimide groups on the PCL fiber surface to form a three-dimensional network hydrogel with strong covalent interfacial bonds between the hydrogel and polymer fiber phases. The concentration of PEGSH (molecular weight 5 kDa) was set at a 1:1 molar stoichiometry between the thiol groups of PEG-SH and the acrylate groups of HA (degree of acrylate 9%) to maximize crosslinking and minimize pendant crosslinking groups. The composition was 8 mg / mL HA, 30 mg / mL PCL fiber, 4.6 mg / mL PEG-SH, and 3 mg / mL lidocaine hydrochloride. The gel was formulated in phosphate-buffered saline (DPBS), allowed to fully react, and then disintegrated into injectable non-spherical beads approximately 150 microns in size.

[0170] Example 2: Rodent Fat Processing and Preparation of NHC-Adipose Graft Material Intact adipose tissue was extracted from the fat pads in the groin of female Lewis rats (approximately 10 cc was collected per rat). The collected tissue was minced into small pieces using surgical scissors. Three milliliters of the minced fat was then transferred to a single Telfa™ pad and spread evenly on it to remove oily components. A total of two Telfa™ treatments were applied to the adipose pieces. The processed fat, shown in Figure 4A, was collected in a syringe and ready to be mixed with the NHC from Example 1.

[0171] As demonstrated in Example 3, the fat may be further processed to form fat particles. The fat for each experiment was pooled and mixed together before being distributed to each NHC mixture group. Approximately 20 cc of fat from two rats was pooled for mechanical testing, and approximately 40 cc from four rats was pooled for in vivo studies.

[0172] Three different combinations of NHC and processed rodent fat were prepared by manually mixing the two parts in a syringe at the specified volume ratios (Table 1 and Figure 4B). Two control groups—pure NHC and pure fat samples—were also included.

[0173] Table 1. Combinations of NHC and processed fat for fat grafting TIFF2025538204000002.tif33156

[0174] The prepared implant materials were then examined in an ARG2 rheometer to determine the storage modulus (G'). Briefly, samples were first trimmed into 8 mm diameter, 2 mm thick disks and sandwiched between two parallel plates. Amplitude sweep tests were performed over a strain range of 0.01 to 10% (at 1 Hz) to identify the optimal amplitude value for the sample, followed by frequency sweep tests (0.1 to 10 Hz at 1% strain). The storage modulus was calculated by averaging the values ​​within the linear range of the frequency sweep curve. Tangent delta values ​​(loss modulus divided by storage modulus) were calculated over the same range.

[0175] Example 3: Fat particle formation Animal fat (e.g., fish oil or rat fat) was thickened using heat and enzymes until the desired viscosity was reached. The thickened oil was spread onto a PTFE-coated plate using an adjustable casting knife set, UV-treated, and then heated to obtain a crosslinked fatty acid-derived biomaterial film. The film can be converted to particles by placing it in a mortar, covering it with liquid nitrogen, and crushing it into particle form using a pestle, or by cryo-grinding it into particle form.

[0176] Alternatively, fat may be obtained from a mammal (e.g., a rat or a human) by using an aspirator (e.g., a vacuum pump), and the obtained fat may be recovered using a homogenizer. The collected fat sample may be filtered, and the filtration may be controlled based on the desired particle size. It is understood that REVOLVE (trademark) (Abbvie) can be a device for preparing fat particles.

[0177] Alternatively, adipose tissue can be surgically harvested en bloc from a donor site (either from the same animal / person or from a donor animal / person) and then mechanically disaggregated to generate fat particles with intact cells in the extracellular matrix.

[0178] Example 4: Fat Grafting and Graft Evaluation The prepared graft material from Example 2 (500±50 μL / injection) was injected subcutaneously into the back of each Lewis rat using a disposable 14G intravenous catheter. Each rat received up to four grafts, and the injection scheme is summarized in Figure 4C. Pure fat injections were performed in separate animals from the NHC-containing samples to avoid systemic inflammation, which may affect the outcome of the pure fat grafts.

[0179] Magnetic resonance imaging (MRI) was performed immediately after injection and every 15 days thereafter to track the volume of each injected graft individually over a 90-day time course. Volume retention was determined by normalizing the volume of each specimen at a particular time point to its corresponding initial value on day 0 (postoperative day 0, or POD 0).

[0180] Injected grafts were harvested for histological and immunofluorescence analysis at POD 0, 30, and 90. Initial assessment of graft morphology was performed by hematoxylin and eosin staining followed by immunolabeling with (i) perilipin and (ii) α-smooth muscle actin (αSMA) and rat endothelial cell antigen (RECA-1) to visualize viable adipocytes and newly formed blood vessels, respectively.

[0181] result Comparable storage modulus and uniform graft morphology across various combinations of NHC and adipose fragments NHC and processed fat fragments were mixed in various volume ratios, and the prepared implant materials were subjected to mechanical testing. Rheological results suggest that both NHC and processed fat exhibited similar mechanical strength, with G' values ​​near 300 Pa. Therefore, when combined together in various proportions, the resulting storage moduli were comparable across the various groups, as shown in Figure 6. Tan delta values ​​were lower for the composite gel (0.13) than for the fat (0.36), with the mixtures having intermediate values ​​that correlated with their relative composition (0.17 for the 75% composite, 0.19 for the 50% composite, and 0.27 for the 25% composite). This demonstrates that blending processed fat with a fiber hydrogel composite can be used to lower the tan delta of the mixture (thus making it more "solid-like" and better maintaining its intended implant shape and resisting flow or migration after injection).

[0182] The injected grafts were retrieved immediately after injection to evaluate the distribution of NHC and processed fat under various mixing conditions. Our H&E staining results (Figure 6) demonstrate that, although homogeneity was incomplete, NHC and processed fat were evenly distributed within the various grafts, indicating good mixing between the two components.

[0183] The 50% NHC-50% fat graft achieved superior volume retention over 3 months in vivo compared with other combinations of NHC and fat. MRI was performed over a 90-day time course (Figure 7A) to determine the volume and shape retention of each injected graft over time. Specifically, graft swelling was observed only in the pure NHC group within the first 2 weeks after injection, while the volume of all other graft types continued to decrease over time from POD 0 (Figure 7B). From POD 45 onwards, graft volume in most groups began to stabilize, and with the exception of the pure NHC injection, the rate of volume loss also slowed. By POD 90, 100% NHC grafts showed the best volume retention, followed by the 50% NHC-50% fat group, which retained 49.49 ± 3.77% and 40.37 ± 6.27% of their initial values, respectively (Figure 7B and Table 2); the difference between the two groups was not statistically significant (p = 0.056). Conversely, volume retention for all other graft types was significantly impaired, being 20% ​​or less when compared to the corresponding POD 0 values.

[0184] Table 2: Volume retention of injected grafts at POD 90, n = 4. TIFF2025538204000003.tif30156

[0185] Furthermore, based on MRI and macroscopic images (Figures 7A and 7C), the injected grafts exhibited a bolus-like shape at POD 0 in all groups but flattened over time, particularly in the 75% NHC-25% fat, 25% NHC-75% fat, and 100% fat groups. Furthermore, pure fat grafts continued to be absorbed by the host and became less visible by POD 90. In the 50% NHC-50% fat graft, abundant vacuoles and viable adipocytes remained after 3 months in vivo.

[0186] Morphologically, pure NHC grafts developed into capsule-like structures with an NHC core surrounded by an outer ring of cells in close proximity to host tissue and cells. As a result, tissue remodeling into neo-soft tissue occurred first in this region and gradually progressed toward the center of the construct (Figure 8). At POD 30, 75% NHC-25% fat grafts contained few or virtually no vacuoles, a characteristic morphology of native adipose tissue, indicating early tissue remodeling in this group. By POD 90, these constructs had completely remodeled into soft-tissue-like structures. In the other three groups (50% NHC-50% fat, 25% NHC-75% fat, and 100% fat), a hybrid composed of both adipose tissue and remodeled soft tissue was observed early in the fat grafting process. On the other hand, after 90 days in vivo, only the 50% NHC-50% fat combination had a large number of vacuoles with appropriate or healthy sizes in the 50-100 μm range, whereas the majority of the 25% NHC-75% fat grafts had remodeled into soft tissue with a few enlarged vacuoles, and the pure fat grafts contained extremely large vacuoles.

[0187] The presence of viable adipocytes within each type of graft was detected by staining specimens for perilipin, a lipid droplet-associated protein. Nonviable adipocytes were defined as cells with enlarged vacuole-like structures with little or no perilipin staining, typically surrounded by infiltrating host cells (Figure 9). Specifically, perilipin staining was not observed in pure NHC and 75% NHC-25% fat grafts at POD 30. Nevertheless, at POD 90, several viable adipocytes were found in the peripheral areas of the remodeling tissue in both groups, which were absent at earlier time points. These adipocytes were very close to the host interface, and therefore most likely migrated from the host into the injected graft. On the other hand, early in the fat grafting process, many nonviable or dying adipocytes were detected in both the 25% NHC-75% fat and 100% fat groups. By POD 90, the 25% NHC-75% fat samples contained minimal perilipin staining, while the pure fat grafts consisted primarily of nonviable adipocytes. Notably, the 50% NHC-50% fat constructs displayed a mixed morphology of abundant viable adipocytes and remodeled soft tissue at both early and late time points. This finding suggests that the 50% NHC-50% fat combination may provide superior conditions for maintaining or improving the viability of processed fat injected with NHCs, as verified by increased adipocyte viability within the graft.

[0188] After 3 months in vivo, a higher degree of vascularization was achieved in 50% NHC-50% fat grafts. The grafts were harvested and co-stained for αSMA and RECA-1 to detect angiogenesis within the various grafts (Figure 10). Our data demonstrate that enhanced angiogenesis was observed in the 50% NHC-50% fat construct compared with other fat-containing samples, potentially contributing to the improved survival of the co-injected fat fragments in this group. Conversely, pure fat grafts remained poorly vascularized throughout the 90-day fat grafting process. Furthermore, in the 100% NHC, 75% NHC-25% fat, and 25% NHC-75% fat groups, most of the blood vessels were identified at the edges of the constructs, while by POD 90, some small vessels were found toward the interior of the remodeled soft tissue. However, their overall vascularity remained inferior to that of the 50% NHC-50% fat group.

[0189] conclusion Injection of pure NHC led to the highest volume retention over 90 days in vivo, with the injected grafts developing into a core-shell-like structure with an NHC core and an outer cellular ring that slowly infiltrated with cells and underwent remodeling.

[0190] The 75% NHC-25% fat graft underwent rapid soft tissue remodeling, resulting in only few or virtually no adipose fragments remaining in the construct at POD 30, and fully developed soft tissue with only a few detectable viable adipocytes at the periphery by POD 90. The composite gel component degraded much more rapidly than the 100% NHC group, and volume retention was significantly lower than the 100% NHC and 50% NHC groups.

[0191] Although volume retention in the 50% NHC-50% fat group was slightly lower than that in the 100% NHC group at the end of the fat grafting process, the two values ​​were not significantly different. This may result from increased vascularization of these grafts, which potentially contributes to improved survival of the fat fragments injected with NHC and further contributes to better volume retention compared to other fat-containing constructs. The 50% NHC-50% fat group had superior cellular infiltration, promoting the survival or growth of mature adipocytes throughout the implant to a much greater extent than either of the other groups, and had greater blood vessel density and vascularity.

[0192] Although the 25% NHC-75% fat grafts exhibited a mixed adipose and soft tissue morphology early in the fat grafting process, most of the adipocytes in the constructs were non-viable or dying, and as a result, by POD 90, the majority of the grafts had remodeled into neo-soft tissue with a few enlarged vacuoles.

[0193] At both time points, the pure fat grafts were poorly vascularized, leading to loss of viability of the injected adipocytes and poor volume retention.

[0194] Benefits of beaded morphology The performance of these groups is superior to that of the gels in a previous patent application (US 2020 / 00304996) that discussed the combination of fat and composite hydrogels. Previous forms of composite gels used with fat grafting were not in microbeaded form. They were in the form of gel components that were mixed together (and with processed fat) before gelling. Thus, the mixture was injected in a partially gelled state (after 3 hours of incubation at 38°C in Example 18), which required in situ gelling, and was therefore mixed with processed fat in a pre-gelled state for in situ gelling in the body after injection.

[0195] Irregular shapes and porosity for reconstruction / shape retention The microbead morphology is important in distinguishing it from prior art features. The Martin et al. application described a continuous, intact gel that would be impossible to inject (when conforming to the dimensions of a syringe, the intact gel would be much larger than the inner diameter of the syringe needle, resulting in the gel having to be broken down by the process of forcing it through the needle, a process that significantly increases the force required). Other microparticle-based gels in this product category, such as Radiesse, Sculptra, or Ellanse, are composed of spherical, nonporous particles in a gel carrier, and a smooth, spherical surface is desirable to achieve their mechanism of action (permanent volume increase caused by collagen deposition on the particle surface). The smooth, spherical design minimizes the surface area:volume ratio, which is important because increased surface area of ​​nonporous materials in the body is associated with an undesirable inflammatory foreign body response by the body's immune system.

[0196] The mechanism of action of such devices differs from that of the present invention, which achieves permanent volume expansion by acting as a substrate or sponge that encourages cell infiltration into the microbeads. Because the microbeads themselves are porous, the surface of the beads does not elicit the same immune response, and there is no need to minimize the surface area:volume ratio. Therefore, irregular, non-spherical microbead shapes have a larger surface area:volume ratio, which increases the contact area between the microbeads and increases the desired cohesion of the injected gel. Individual microbeads in this composition also have multiple fibers present on all surfaces of the microbead, creating greater friction and mechanical entanglement at the interface between two microbeads. Because microbead gel particles readily adhere to each other, the increased interbead contact area creates a gel that better maintains its shape after injection and becomes more solid-like, better mimicking natural soft tissue and resisting forces from surrounding tissue that could cause the gel to migrate from the intended augmentation area. Indeed, the decrease in tan delta when bulk gels were formed into microbeads (both 150 and 250 micron sizes) indicates that the microbeads may be more solid-like than bulk gels. The irregular, non-spherical shape of soft, porous microbeads also allows for more efficient packing (less interparticle void space) compared to spherical beads in the carrier, which in turn improves gel cohesion. The size, shape, and flexibility of the beads allow for good mixing with biologically active materials (e.g., adipose tissue), and the gel material is generally located within hundreds of microns of the implanted tissue at all points. This provides mechanical support to the tissue, protecting it during implantation, preventing migration after implantation, and providing the necessary blood supply to keep the implanted tissue viable for longer periods (because the gel phase can promote vascularization).

[0197] Potential mechanisms for the importance of volume ratios Processed fat poses challenges for use in implantation processes: adipocytes are fragile (prone to adipocyte lysis due to shear forces during liposuction, processing, and implantation) and have a relatively high tan delta (more "liquid-like," making them prone to spreading / moving in response to pressure from surrounding tissues after implantation, limiting surgical control over fixation and ultimately minimizing the intended volumetric effect). Once implanted, only approximately 50% of the fat volume is typically maintained over time due to many factors, including lack of blood supply and local ischemia due to immune system-derived inflammation or clearance. When adipocytes die (either due to mechanical forces, an inflammatory response, or ischemia), they can release oil, which can trigger inflammation and phagocytic degradation, as opposed to remodeling. This leads to oil cysts, which ultimately result in persistent volume gain and a lack of tissue remodeling (such as angiogenesis and ECM deposition). This is evident in the 100% fat group, with insufficient volume gain, large vacuoles, and minimal remodeling throughout 90 days. The composite hydrogel itself, while effective at maintaining volume, does not fully replicate the native subcutaneous tissue. This is evident in the 100% NHC, where adipogenesis is nearly nonexistent, vascularization is slowed, and only a portion of the implant site is remodeled over 90 days. Combining processed fat with composite gel enhances the gel response by providing adipocytes with support for cells, native fat structure, and growth factors. Combining processed fat with composite gel enhances the fat graft response by providing mechanical support, shear protection, sites for cell adhesion, immunomodulation to limit inflammatory or phagocytic macrophage responses, regenerative polarization of macrophages, enhanced angiogenesis to limit ischemia, and reduced tan delta, improving volume gain and resisting implant migration within the body. The ideal ratio of fat to composite gel that simultaneously maximizes volume retention, adipocyte proliferation and survival, and vascularization is unknown. Too little composite (e.g., 75% fat, 25% composite) may be insufficient to prevent the negative cycle of necrosis, oil accumulation, and inflammation.Too little fat (e.g., 25% fat, 75% composite) may not be ideal because the rate of vascularization through the gel phase may be too slow to prevent ischemia and necrosis of implanted cells in the center of the injected fat-gel implant, resulting in the same negative cycle of necrosis.

[0198] Potential Expansion Due to the limited amount available in the rat model, allogeneic fat transfer was used in these examples. The procedure requires minimal manipulation of the fat, making it a good candidate for autologous fat transfer. The fat is removed from the patient (such as by liposuction), processed, mixed with a composite gel, and then injected back into the patient. The transferred cells and / or tissues can consist of whole fat transfer, stromal vascular fraction (SVF), or stem cell transfer (mesenchymal stem cells or adipose stem cells) harvested either from the patient or from a cell bank.

[0199] Composite gels can also be made from formulations with increased or decreased constituent concentrations or alternative fiber morphologies (such as collagen fibers or cellulose fibers) or alternative hydrogel phases (either containing different crosslinkers such as BDDE or DVS, or different gel-forming molecules such as collagen, PEG, carboxymethylcellulose, chitosan, alginate, chondroitin sulfate, or PVA).

[0200] Fat survival and implantation function can be further regulated by incorporating drugs. This can be selectively tailored for burst release (soluble, non-encapsulated drugs), sustained release (drugs loaded into hydrogel-phase nanoparticles, or co-spun into fibers, or covalently linked in the hydrogel phase), or drugs surface-bound to the fibers via covalent or other methods. Such bound drug presentations may have biological effects different from those seen in the free, soluble form (such as VEGF). Included drugs may consist of growth factors (e.g., bFGF, IGF-1, PDGF, VEGF, HGF), vitamins, antioxidants, anti-inflammatory agents, nutraceuticals / extracts, pain relievers, drugs, surfactants (such as Pluronic F68), or others.

[0201] Example 5: Collagen fiber production Bovine type I collagen solution was purchased from Advanced Biomatrix. After first lyophilizing the bovine collagen solution overnight to obtain collagen powder, type I collagen solution (8% w / v) was prepared in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) at room temperature for approximately 6 hours to produce a viscous, cloudy electrospinning solution. Electrospinning was performed using the following parameters: a flow rate of 5 mL / h; an applied voltage of 20–25 kV to a 22-G metal needle; a collection distance of 12.5 cm; and a rotation speed of the metal collector of 900 rpm. These parameters resulted in an average fiber diameter of approximately 600 nm. Using carbodiimide chemistry, fibers were immersed in an ethanol solution (95% v / v%) containing 50 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 20 mM N-hydroxysuccinimide (NHS) for 24 hours. After crosslinking, the fibers were washed three times for 5 minutes in 0.75% glycine solution to remove excess reagent and quench the activated fiber surface. The collagen fibers were then disassembled into fragments using cryomilling (Freezer / Mill 6770, SPEX SamplePrep). The fragments were filtered through various cell strainers (40 and 100 μm) to achieve a relatively uniform fiber length.

[0202] Example 6: Preparation of collagen fiber-HA hydrogel composite The preferred NHC construct is composed of three components: a hyaluronic acid (HA) network, bovine type I collagen nanofibers, and a divinyl sulfone (DVS) crosslinker. Prior to incorporating collagen nanofibers (obtained in Example 5 above) into the HA network during crosslinking, we optimized the crosslinking conditions for the HA gel phase alone.

[0203] Sodium hyaluronate (MW 1.5 MDa) was purchased from LifeCore. HUVEC and vascular endothelial cell culture media were purchased from Lonza. All other chemical reagents were purchased from Sigma-Aldrich. All other cell culture reagents and additives were obtained from Invitrogen. HA was dissolved in distilled water at a stock concentration of 25 mg / mL. DVS concentrations were calculated as a ratio to hydroxyl groups in HA (e.g., 1.17 w / v%, 2.34 w / v%, and 4.68 w / v%). The stock HA solution was diluted to 2 w / v% using distilled water and sodium hydroxide to obtain four different pH values ​​(12.4, 12.7, 13.0, and 13.3), with other parameters kept the same (2 w / v% HA, 37°C, 3 h reaction time). Multiple samples were prepared and the reaction time or gelation kinetics was performed by measuring the mechanical properties at various time points (30 min, 1 h, 2 h, 3 h, 4 h, 8 h, and 16 h) to determine the time point at which stiffness plateaued. NHC crosslinking was performed under the same conditions as for HA hydrogels, and various fiber densities (0, 1, and 3 w / v%) were added to the mixed precursors to test the gelation kinetics of NHCs. After gelation of the hydrogels and NHCs, dialysis was performed against pH 7.4 phosphate buffer using a dialysis membrane (6000-8000 MWCO, Spectrum) for 48 h to remove unreacted DVS, balance the pH, and allow the samples to swell for further study. Mechanical properties were then measured again after swelling. Microgels were then generated using stainless steel wire cloth discs, as previously reported, to reach gel particle sizes of approximately 100 μm.

[0204] We found that cross-linking HA networks at a pH of 12.7 using DVS chemistry was effective in forming strong cross-linked hydrogels while minimizing degradation of HA molecules and collagen fibers during gelation. The reaction pH had a significant effect on the resulting gels in the pH range of 12 to 13.3. While other parameters (37°C, 2% HA w / v concentration, 2.93% DVS w / v concentration) were kept the same, the reaction pH was set at 12.4, 12.7, 13.0, and 13.3 using various NaOH concentrations (0.001 M, 0.01 M, 0.1 M, 1 M). Reactions at pH 13.3 and 13.0 showed dramatic degradation after 2 hours of reaction, resulting in two unstable hydrogels with poor reproducibility.

[0205] The crosslinking time was optimized around 2 hours to reach the maximum storage modulus and limit degradation. After adjusting the DVS chemistry, we introduced nanofibers into the HA network while crosslinking to generate interfacial bonds between the HA network and the nanofibers. We observed a reinforcement effect by comparing HA and NHC at similar crosslink densities. Furthermore, the composites could easily pass through a 27-gauge needle after crosslinking. To further quantitatively investigate this reinforcement effect, we measured the storage modulus G0' of the HA hydrogel phase (normalized to the storage modulus control), G' of the total NHC, and G' of the hydrogel-nanofiber blend without interfacial bonding. In rheological tests with fiber loading densities of 1-3 w / v%, the G' of the composites ranged from 1.5-4 times higher than those without interfacial bonding, and the G' difference increased with increasing fiber loading and crosslinker concentration. Additionally, we investigated the effect of fiber length on stiffness enhancement by filtering out large fiber fragments using various cell strainers (40 μm, 100 μm, no filtering). A counterintuitive result was that gels with fiber fragments between 40 μm and 100 μm in length produced the greatest stiffness enhancement, although this relative enhancement was minimized at the highest crosslinking concentration.

[0206] As a result, this preparation process allowed us to generate NHCs with post-crosslinking G' values ​​ranging from 450 Pa to 1500 Pa and post-swelling G' values ​​ranging from 150 Pa to 1000 Pa. To mimic the soft tissue microenvironment, HA control (G' = 100 Pa and G' = 250 Pa) and composite (G' = 250 Pa and G' = 100 Pa) were generated and micronized into microgels with diameters around 100 μm. The storage moduli of all three groups were measured, and these storage moduli were not significantly different from the initial crosslinked bulk gel. Finally, before utilizing the prepared materials for subsequent in vitro and in vivo studies, we autoclaved the hydrogels and composites and observed that the G' measurements did not decrease significantly after terminal sterilization, indicating the transfer potential of this material. Autoclaving was performed to sterilize the hydrogels and NHCs after gelation. Briefly, the gels were subjected to an autoclave cycle at 118°C with a 5-minute sterilization step. The entire sterilization cycle took 30 minutes to complete. After sterilization, the mechanical properties of each gel were measured again using rheological testing. The thermal stability during autoclaving also indicates that the gels have excellent storage stability at the much lower temperatures required for ambient storage.

[0207] Example 7: Fat lattice by co-injecting engineered adipose tissue particles with collagen fiber-HA hydrogel composite (Matrix C) Prepared implant materials composed of various volumetric ratios of collagen fiber-HA hydrogel composites of the type prepared in Example 6 above (this collagen fiber-HA hydrogel composite is referred to as Matrix C in this example) and processed fat particles were subcutaneously injected into the backs of Sprague Dawley (SD) rats using a disposable 14-G intravenous catheter in a total volume of 500 ± 50 μL per injection ( FIG. 11 ). Thirty days after implantation, the injected implants were retrieved for volumetric, histological, and immunofluorescent evaluation. Volumetric analysis was based on macroscopic images and caliper measurements. Histological evaluation involved hematoxylin and eosin staining followed by immunofluorescent imaging with perilipin to detect viable adipocytes.

[0208] result: Matrix co-injection improved fat graft volume retention in vivo over 1 month After the rats were sacrificed on day 30, caliper-based macroscopic imaging measurements were performed to determine the volume and shape retention of each injected graft over time. Specifically, with increasing volume fraction of Matrix C, the retained volume of adipose tissue also increased (Figure 12), with the best volume retention achieved with the injection of 100% Matrix C, likely due to the slow degradation of the hydrogel material. This demonstrates that the inclusion of a matrix can modestly increase the viability of fat grafts.

[0209] Superior adipocyte survival was observed in 75% matrix C / 25% fat grafts versus 50% matrix C / 50% fat grafts The grafts were harvested and stained with hemoxilin and eosin for histological analysis. Specifically, at 30 days, pure fat grafts showed enlarged vacuoles in the subcutaneous space, indicating adipocyte necrosis. Pure matrix C showed signs of host cell infiltration, angiogenesis, and neo-soft tissue formation. Both 50% matrix C-50% fat and 25% matrix C-75% fat maintained some adipocytes without enlarged vacuoles, but the number of healthy adipocytes was limited. 25% matrix C-75% fat was the only group at this time point that had a large number of adipocyte vacuoles in the healthy size range, which were distributed throughout the injected graft (Figure 13).

[0210] To further detect and distinguish between viable and nonviable adipocytes, perilipin-1 staining was performed to mark the lipid droplet-associated protein normally present in viable adipocytes (Figure 14). Specifically, by day 30, both the 25% matrix C-75% and 50% matrix C-50% fat grafts demonstrated excellent adipocyte survival. However, in the 50% matrix C-50% fat graft, several enlarged vacuoles lacking perilipin expression were observed at the periphery, indicating the presence of nonviable adipocytes. Beyond these two compositional ranges, very limited viable adipocytes were observed in the other test materials. This finding suggests that the composition of the 25% matrix C-75% to 50% matrix C-50% fat grafts may provide a superior microenvironment that can improve the survival of processed fat fragments co-injected with matrix C.

[0211] equivalent It will be understood that the detailed examples and embodiments described herein are given by way of example only for illustrative purposes and are not to be construed as limiting the invention in any manner. Various modifications or variations therein will occur to those skilled in the art and are considered to be within the spirit and scope of this application and within the scope of the appended claims. For example, the relative amounts of components may be varied to optimize a desired effect, additional components may be added, and / or one or more of the described components may be substituted with similar components. Additional advantageous features and functionality associated with the systems, methods, and processes will be apparent from the appended claims. Moreover, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

1. adipocytes or adipose tissue; non-spherical microbeads comprising a hydrogel composite, the hydrogel composite comprising a hyaluronic acid component covalently linked to a fiber component, the weight ratio of the hyaluronic acid component to the fiber component being in the range of 1:100 to about 100:1; A composition for administration comprising:

2. The composition of claim 1, wherein the adipocytes or adipose tissue are adipose cells or microparticles of adipose tissue.

3. 3. The composition of claim 1 or 2, comprising particles comprising adipocytes or adipose tissue.

4. 4. The composition of claim 2 or 3, wherein the ratio of the average size of the non-spherical microbeads to the average size of the fat particles is in the range of about 10:1 to 1:

10.

5. 4. The composition of claim 2 or 3, wherein the ratio of the average size of the non-spherical microbeads to the average size of the fat particles is in the range of about 2:8 to 8:

2.

6. 4. The composition of claim 2 or 3, wherein the ratio of the average size of the non-spherical microbeads to the average size of the fat particles is in the range of about 3:7 to 7:

3.

7. 4. The composition of claim 2 or 3, wherein the ratio of the average size of the non-spherical microbeads to the average size of the fat particles is in the range of about 4:6 to 6:

4.

8. 4. The composition of claim 2 or 3, wherein the average size of the non-spherical microbeads is within 5 or 10% of the average size of the fat particles.

9. 9. The composition of claim 1, wherein the microbeads are gelled or hardened before the biologically active material is added to the microbeads.

10. 10. The composition of claim 2, wherein the average size of the fat particles is less than about 1,000 μm, less than about 500 μm, or less than about 300 μm, or less than about 100 μm.

11. 10. The composition of any one of claims 1 to 9, wherein a plurality of nanofibers are present on the surface of the microbead.

12. 12. The composition of any one of claims 1-11, wherein the hydrogel composite is formed by combining functionalized hyaluronic acid, polymeric nanofibers, and a crosslinker.

13. 13. The composition of claim 12, wherein the functionalized hyaluronic acid comprises thiolated hyaluronic acid and the cross-linking agent comprises poly(ethylene glycol) diacrylate (PEGDA) or a derivative thereof.

14. 13. The composition of claim 12, wherein the functionalized hyaluronic acid comprises acrylated hyaluronic acid and the crosslinker comprises thiolated poly(ethylene glycol) or a derivative thereof.

15. The composition of any one of claims 1 to 14, wherein the polymeric fiber component comprises polycaprolactone fibers.

16. The composition of any one of claims 1 to 14, wherein the polymer fiber component comprises collagen.

17. 17. The composition of any one of claims 1 to 16, wherein the weight ratio of the hyaluronic acid component to the fiber component ranges from 10:90 to about 90:

10.

18. 17. The composition of any one of claims 1 to 16, wherein the weight ratio of the hyaluronic acid component to the fiber component ranges from 20:80 to about 80:

20.

19. 17. The composition of any one of claims 1 to 16, wherein the weight ratio of the hyaluronic acid component to the fiber component ranges from 30:70 to about 70:

30.

20. 17. The composition of any one of claims 1 to 16, wherein the weight ratio of the hyaluronic acid component to the fiber component ranges from 40:60 to about 60:

40.

21. 17. The composition of any one of claims 1 to 16, wherein the weight ratio of the hyaluronic acid component to the fiber component ranges from 45:55 to about 55:

45.

22. 22. The composition of any one of claims 1-21, wherein the soft tissue device further comprises a compound selected from the group consisting of a growth factor, an angiogenesis stimulating compound, an immunomodulator, an inflammation inhibitor, and combinations thereof.

23. 23. The composition of any one of claims 1-22, wherein the soft tissue device further comprises one or more compounds having a therapeutic effect, an angiogenic effect, an anti-angiogenic effect, an anti-inflammatory effect, an antibacterial effect, an antihistamine effect, and combinations thereof.

24. 24. The composition of any one of claims 1-23, wherein the soft tissue device has a tan delta value of less than about 0.

27.

25. 24. The composition of any one of claims 1 to 23, comprising adipose tissue.

26. 24. The composition of claim 23, wherein the adipose tissue is lipoaspirate tissue.

25. 25. The composition of claim 23 or 24, wherein the adipose tissue is autologous.

26. (a) non-spherical microbeads comprising a hydrogel composite, the hydrogel composite comprising a hyaluronic acid component covalently linked to a fiber component, the weight ratio of the hyaluronic acid component to the fiber component being in the range of 1:100 to about 100:1; (b) a population of adipose cells, autologous adipose cells, allogeneic cells, adipose stromal vascular cells, adipose tissue, autologous adipose tissue, lipoaspirate tissue, derivatives thereof, or combinations thereof; A composition for administration comprising:

27. 27. A method of fat transplantation in a subject, comprising administering to a subject in need thereof an effective amount of the composition of any one of claims 1 to 26.

28. 27. A method for performing a cosmetic or reconstructive procedure, or for reducing or reversing tissue loss resulting from trauma, surgical intervention, or an age-related disease, disorder, or condition, comprising administering to a subject in need thereof an effective amount of the composition of any one of claims 1-26.

29. 29. The method of claim 27 or 28, wherein the composition is implanted into a target tissue of the subject.

30. 30. The method of any one of claims 27 to 29, wherein the composition is injected into a target tissue of the subject.

31. 27. A kit for preparing soft tissue injection according to any one of claims 1 to 26 for administration into a target tissue of a subject, the kit comprising: (i) a first syringe containing the microbeads; and (ii) a second syringe containing a biologically active material, wherein the microbeads have been gelled or hardened before the biologically active material is added to the microbeads.

32. 32. The kit of claim 31, further comprising: (iii) a Luer-Luer union connector having an orifice that allows the passage of the microbeads and the biologically active material for mixing prior to injection or implantation.

33. 33. The kit of claim 31 or 32, wherein the biologically active material is adipocytes or adipose tissue.

34. 34. The kit of claim 33, wherein the adipocytes or adipose tissue are adipose cells or microparticles of adipose tissue.

35. 35. The kit of claim 33 or 34, wherein the adipocytes or adipose tissue are present as fat particles.

36. 36. The kit of any one of claims 31 to 35, wherein the biologically active material comprises particles comprising adipocytes or adipose tissue.

37. 37. The kit of claim 36, wherein the fatty particles comprise ECM material.

38. 37. The kit of claim 36, wherein the fat particles comprise a collagen material.

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