Nanofiber-microsphere composite scaffolds and methods of use thereof

Abstract

Three-dimensional scaffolds comprising microspheres are provided as well as methods of use thereof and methods of making.

Description

[0001] NANOFIBER-MICROSPHERE COMPOSITE SCAFFOLDS AND METHODS OF USE THEREOF

[0002] Jingwei Xie

[0003] This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 63 / 727,731, filed December 4, 2024. The foregoing application is incorporated by reference herein.

[0004] This invention was made with government support under W81XWH-20- 1-0207 awarded by the Defense Health Agency, Medical Research and Development Branch, and R01 GM138552 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0005] FIELD OF THE INVENTION

[0006] This application relates to the field of nanofiber structures. More specifically, this invention provides three-dimensional (3D) scaffolds comprising electrospun nanofibers and microspheres.

[0007] BACKGROUND OF THE INVENTION

[0008] Several publications and patent documents are cited throughout the specification in order to describe the state of the art to which this invention pertains. Each of these citations is incorporated herein by reference as though set forth in full.

[0009] Electrospun nanofibers have found extensive applications across various fields, including filtration, catalysis, energy, photonics, electronics, textiles, wearables, agriculture, and biomedical engineering (Xue, et al. (2019) Chem. Rev., 119:5298; Ji, et al. (2024) Nat. Rev. Method Primers 4: 1; Chen, et al. (2018) Adv. Drug Del. Rev., 132: 188; Wan, et al. (2022) Exploration 2:20210029; Chen, et al. (2022) Adv. Fiber Mater., 4:959; Liang, et al. (2023) Energy Mater., 3:300006; Zhao, et al. (2022) J. Mater. Chem. B 10:6078). In both industries and academic research laboratories, electrospun nanofibers are typically fabricated as two-dimensional (2D) membranes, sheets, or mats deposited on certain electrically conductive substrates such as a piece of aluminum foil or rotating mandrels. These nanofiber mats are generally limited to a thickness of less than several millimeters (Chen, et al. (2020) J. Mater. Chem. B 8:3733). Their densely packed structure and fine nanoscale fibers lead to small pore sizes, which constrains their applicability in fields like wound healing, tissue engineering, and regenerative medicine (Wang, et al. (2024) Acc. Mater. Res., 8:987; Jiang, et al. (2015) ACS Biomater. Sci. Eng., 1 :991; Jiang, et al. (2016) Adv. Healthcare Mater., 5:2993). Thus, a significant need exists to develop advanced methods for creating 3D nanofiber structures that overcome these limitations and unlock broader applications.

[0010] SUMMARY OF THE INVENTION

[0011] In accordance with the instant invention, methods of synthesizing a fibermicrosphere composite scaffolds are provided. In certain embodiments, the fibers are nanofibers. In certain embodiments, the nanofibers are electrospun nanofibers. In certain embodiments, the method comprises a) electrospinning a nanofiber mat with expandable microspheres, and b) thermally treating the electrospun mat of step a). Generally, the thermal treatment causes expansion of the expandable microspheres in the nanofiber mat, thereby resulting in the synthesis of the nanofiber-microsphere composite scaffold. In certain embodiments, the nanofibers comprise at least one polymer. In certain embodiments, the polymer comprises a synthetic polymer, naturally derived polymer, inorganic material, or a combination of thereof. In certain embodiments, the polymer comprises cellulose, cellulose acetate (CA), polyethylene (PE), polypropylene (PP), polystyrene (PS), poly(vinyl chloride) (PVC), poly(vinyl alcohol) (PVA), chitosan, polybutylene succinate (PBS), polyethylene succinate (PESU), poly(anhydrides), polyvinylpyrrolidone (PVP), poly(methyl methacrylate) (PMMA), poly(acrylamide), poly(HEMA) (poly(2 -hydroxyethyl methacrylate)), polyethylene oxide (PEO), polyethylene terephthalate (PET), polyether ether ketone (PEEK), polyurethane (PU), polytetrafluoroethylene (PTFE), polycaprolactone (PCL), polydioxanone (PDO), poly(lactide-co-epsilon-caprolactone) (PLCL), polyglycolic acid (PGA), polylactide (PLA), poly(lactic-co-glycolic) acid (PLGA), or a combination thereof. In certain embodiments, the electrospun nanofibers of the nanofiber mat comprises uniformly aligned fibers or randomly oriented fibers. In certain embodiments, the electrospun nanofibers of the nanofiber mat are aligned radially or laterally. In certain embodiments, the glass transition temperature of the nanofibers of the nanofiber mat is greater than the temperature of the thermal treatment in step b). In certain embodiments, the nanofiber mat of step a) is fixed or fused on one side or a portion thereof prior to thermal treatment in step b). In certain embodiments, step a) comprises electrospinning a solution comprising a polymer and the expandable microspheres. In certain embodiments, step a) comprises electrospinning a first solution comprising a polymer and a second solution comprising the expandable microspheres. In certain embodiments, step a) comprises electrospinning a solution comprising a polymer and electrospraying or gas spraying the expandable microspheres onto the forming electrospun nanofiber mat. In certain embodiments, step a) comprises electrospinning a solution comprising a polymer and the expandable microspheres to form microspheres electrospun nanofiber mats. In certain embodiments, the expandable microspheres increase in volume by at least 5 times after the thermal treatment of step b). In certain embodiments, the thermal treatment of step b) is at least 80°C. In certain embodiments, the method further comprises coating the nanofiber-microsphere composite scaffold with a hydrogel or ECM mimicking molecules (e.g., gelatin, collagen, fibronectin, laminin) to render both mechanical and biological functions. In certain embodiments, the methos further comprises adding cells and / or extracellular matrix to the nanofiber-microsphere composite scaffold. In certain embodiments, the method further comprises adding at least one agent to the nanofiber- microsphere composite scaffold.

[0012] In accordance with another aspect of the instant invention, nanofiber-microsphere composite scaffolds are provided. In certain embodiments, the nanofiber-microsphere composite scaffolds comprise electrospun nanofibers interwoven among microspheres. In certain embodiments, the nanofiber-microsphere composite scaffolds are synthesized by the methods described herein. Compositions comprising a nanofiber-microsphere composite scaffold of the instant invention and a pharmaceutically acceptable carrier are also encompassed. The present invention also encompasses methods of using nanofiber- microsphere composite scaffolds.

[0013] BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 provides a schematic illustrating the process for preparing 3D microsphere-nanofiber composite objects. Expandable Expancel® microspheres were incorporated into a polyvinylpyrrolidone (PVP) nanofiber mat during electrospinning. The composite was then thermally treated.

[0015] Figures 2A-2C show the characterization of expandable Expancel® microspheres. Fig. 2A provides images of expandable microspheres before thermal expansion (left column) and expandable microspheres after thermal expansion (right column). Figs. 2B and 2C provide the size distributions of expandable microspheres before (Fig. 2B) and after (Fig. 2C) thermal expansion. Figure 3 A provides SEM images of cross-sections of electrospun PVP nanofiber mats after being incorporated with different concentrations of expandable Expancel® microspheres. Left column: 1% Expancel®. Center column: 2% Expancel®. Right column: 3% Expancel®. Figure 3B provides photographs of PVP nanofiber mats before (top) and after (bottom) thermal treatment (110°C, 1 minute).

[0016] Figures 4A-4D provide the characterizations of expandable microspheres incorporated electrospun PVP nanofiber mats after thermal treatment. Figure 4A provides images of 3D expanded PVP nanofiber scaffolds incorporated with different amounts of Expancel® microspheres after thermal treatment. Figure 4B provides SEM images of cross-sectional views of 3D expanded PVP nanofiber scaffolds incorporated with different amounts of Expancel® microspheres. Figure 4C provides a graph of the expansion ratios of 3D expanded PVP nanofiber scaffolds incorporated with different amounts of Expancel® microspheres. Figure 4D provides a graph of the compressive strength of gas-foaming expanded 3D PVP nanofiber scaffolds and Expancel® microspheres incorporated 3D PVP nanofiber scaffolds. PVP: gas-foamed 3D PVP nanofiber scaffolds. (** p < 0.1, **** p < 0.0001).

[0017] Figures 5A and 5B show expandable microspheres transform 2D cellulose acetate and polyurethane (PU) nanofiber mats into 3D scaffolds. Figure 5 A provides an image illustrating the expansion of cellulose acetate (CA) nanofiber mats. SEM images are also provided showing the top and cross-sectional views of CA nanofiber mats incorporated with expandable microspheres before and after expansion. Figure 5B provides an image illustrating the expansion of PU nanofiber mats. SEM images are also provided showing the top and cross-sectional views of PU nanofiber mats incorporated with expandable microspheres before and after expansion.

[0018] DETAILED DESCRIPTION OF THE INVENTION

[0019] Numerous studies have focused on developing three-dimensional (3D) nanofiber structures, with gas-foaming technology emerging as a method for transforming 2D nanofiber mats into 3D shapes (Jiang, et al. (2015) ACS Biomater. Sci., 1 :991; Jiang, et al. (2016) Adv. Healthcare Mater., 5:2993; Jiang, et al. (2018) Acta. Biomater., 68:237; Chen, et al. (2019) Nano Lett., 19:2059; Chen, et al. (2020) Appl. Phys. Rev., 7:021406; Chen, et al. (2020) Adv. Mater., 32:2003754; Shariar, et al. (2024) BMEMat 2:el2065). However, this technique has an inherent drawback. After expansion and gas bubble removal, the resulting 3D nanofiber scaffolds struggle to withstand significant compressive loads, requiring additional coatings and cross-linking to maintain structural stability (Jiang, et al. (2015) ACS Biomater. Sci., 1 :991; Jiang, et al. (2016) Adv. Healthcare Mater., 5:2993; Jiang, et al. (2018) Acta. Biomater., 68:237; Chen, et al. (2019) Nano Lett., 19:2059; Chen, et al. (2020) Appl. Phys. Rev., 7:021406; Chen, et al. (2020) Adv. Mater., 32:2003754; Shariar, et al. (2024) BMEMat 2:el2065).

[0020] Herein, the present invention describes, generally, three-dimensional (3D) scaffolds (or structures) comprising a composite of fibers, such as nanofibers, and embedded foaming agents, such as microspheres. In certain embodiments, the present invention describes methods of fabricating a two-dimensional (2D) fiber mat embedded with a foaming agent. Upon thermal activation, the 2D mat can be transformed into a 3D scaffold structure (nanofiber-microsphere composite scaffolds). More specifically, the present invention provides a novel foaming that incorporates expandable microspheres as a foaming agent directly into 2D nanofiber mats during electrospinning, followed by thermal treatment. Electrospun nanofiber mats are very densely packed and can be difficult to expand uniformly and without significant damage to the desirable characteristics of the electrospun nanofibers. Herein, it is shown that the thermally induced expansion of electrospun nanofiber mats yields 3D microsphere-nanofiber composite structures with improved compressive strength compared to the gas-foamed nanofiber scaffolds. Specifically, polyvinylpyrrolidone (PVP) nanofiber mats and expandable Expancel® microspheres were used to generate 3D microsphere-nanofiber composite structures. The glass transition temperature of PVP is over 175°C, which is significantly higher than the temperature required for Expancel® microsphere expansion (Haaf, et al. (1985) Polymer J., 17: 143). Figure 1 illustrates a fabrication method of these 3D composites by embedding expandable microspheres within the PVP nanofiber mat during electrospinning, followed by thermal treatment.

[0021] In accordance with the instant invention, 3D scaffolds (also referred to herein as expanded scaffolds) comprising fibers and expandable microspheres and methods of synthesizing the 3D scaffolds are provided. In certain embodiment, 3D scaffolds are expanded nanofiber scaffolds with increased porosity (e.g., compared to non-expanded nanofiber mat). In certain embodiments, the fibers of the 3D scaffold are nanofibers, particularly electrospun nanofibers. As used herein, nanofibers are fibers having a diameter less than about 1 pm (e.g., average diameter), but greater than about 1 nm. In certain embodiments, the nanofibers have an average diameter of about 50 nm to about 750 nm, about 50 nm to about 500 nm or about 100 nm to about 500 nm. In certain embodiments, the nanofibers have an average diameter less than about 900 nm, about 800 nm, about 700 nm, about 600 nm, about 500 nm, about 400 nm, about 300 nm, about 200 nm, or about 100 nm. In certain embodiments, the nanofibers have an average diameter greater than about 10 nm, about 25 nm, about 50 nm, about 75 nm, about 100 nm, about 200 nm, about 300 nm, about 400 nm, or about 500 nm.

[0022] While the fibers of the 3D scaffolds of the instant invention may have a diameter other than that of a nanofiber, the 3D scaffolds will typically comprise nanofibers for most utilities. Accordingly, while the 3D scaffolds of the instant invention are referred to as nanofiber-microsphere composite scaffolds herein, the instant invention also encompasses fiber-microsphere composite scaffolds, wherein the fibers are not nanofibers. In certain embodiments, the fiber-microsphere composite scaffolds comprise microfibers (e.g., having an average diameter about 1 pm to about 10 pm, about 20 pm, about 30 pm, about 40 pm, or about 50 pm or more).

[0023] Methods of synthesizing nanofiber-microsphere composite scaffolds are provided herein. In certain embodiments, the method comprises synthesizing a 2D nanofiber mat (e.g., electrospun nanofiber mat) comprising expandable microspheres and then expanding (e.g., thermal treatment) the 2D nanofiber mat to generate the nanofiber- microsphere composite scaffold. The nanofiber-microsphere composite scaffolds may be fully expanded, partially expanded, or comprising layers wherein some layers may be expanded, and some may not be. The nanofiber-microsphere composite scaffolds may be gradually expanded. In certain embodiments, the nanofiber-microsphere composite scaffolds are fully expanded. In certain embodiments, the nanofiber-microsphere composite scaffolds exhibit improved compressive strength compared to other nanofiber scaffolds such as gas-foamed nanofiber scaffolds. In certain embodiments, the 3D nanofiber-microsphere composite scaffolds exhibit reinforced mechanical stability compared to gas-foaming expanded 3D nanofiber scaffolds.

[0024] The nanofiber-microsphere composite scaffolds of the present invention (e.g., the nanofibers thereof) may comprise any material. In certain embodiments, the nanofibers comprise an inorganic material including but not limited to bioactive glass, metal, metal oxide, hydroxyapatite, or combinations thereof. In certain embodiments, the nanofibers comprise polymers or carbon. For example, the nanofiber-microsphere composite scaffolds may comprise plastics, polymers, ceramic, glass, and / or metal or mixtures thereof. In certain embodiments, the nanofibers comprise silicates / silica (e.g., SiCh). In certain embodiments, the nanofibers comprise metal oxide nanofibers. In certain embodiments, the nanofibers comprise bioactive glass (e.g., bioactive glass nanofibers). Bioactive glasses are biologically compatible synthetic materials comprising varying amounts of silicates / silica (e.g., SiCh), sodium oxides (e.g., NaCh), calcium oxides (e.g., CaO), and / or phosphates / phosphorus pentoxide (e.g., P2O5). In certain embodiments, the bioactive glass comprises silicates / silica (e.g., SiCh), calcium oxides (e.g., CaO), and phosphates / phosphorus pentoxide (e.g., P2O5). In certain embodiments, the molar ratio of Si:P:Ca is 70-90:5-15:5-15, 75-85:7.5-12.5:7.5-12.5, or about 80: 10: 10. In certain embodiments, the nanofiber-microsphere composite scaffolds comprise at least one polymer.

[0025] The nanofibers of the instant invention can be fabricated by any method (e.g., electrospinning, centrifugal spinning, solution blowing, phase separation, and like methods). In certain embodiments, the nanofibers are synthesized by electrospinning. Electrospun nanofibers may be fabricated as 2D membranes, sheets, or mats deposited on electrically conductive substrates, such as metal, aluminum (e.g., aluminum foil), rotating mandrels, or other like substrates. Generally, electrospun nanofiber mats have a thickness of less than several millimeters (e.g., less than 5, less than 3 mm, or less than 2 mm). The nanofibers may be of any orientation. For example, the nanofibers may be orthogonal fibers, aligned fibers (e.g., uniaxially aligned), partially aligned, random fibers, and / or entangled fibers. In certain embodiments, the nanofibers comprise aligned fibers (e.g., uniaxially, radially, laterally, vertically, or horizontally). In certain embodiments, the nanofibers comprise random fibers. In certain embodiments, the nanofibers comprise uniform fibers. In certain embodiments, the nanofibers have a uniform, random, or a combination thereof alignment. In certain embodiments, the nanofibers are radially aligned or laterally aligned. The alignment of the nanofibers may be aligned using the mandrel’s rotation speed. In certain embodiments, the nanofiber- microsphere composite scaffolds comprise woven nanofibers. In certain embodiments, the nanofiber-microsphere composite scaffolds comprise nonwoven nanofibers.

[0026] The nanofibers of the instant invention may comprise any polymer. The nanofibers of the instant invention may comprise one or more polymers. In certain embodiments, the polymer is a synthetic polymer. In certain embodiments, the polymer is a natural or naturally-derived polymer. In certain embodiments, the polymer is biocompatible. In certain embodiments, the polymer is biodegradable. In certain embodiments, the polymer is non-biodegradable. The polymer may be hydrophobic, hydrophilic, or amphiphilic. In certain embodiments, the polymer is hydrophobic. In certain embodiments, the polymer is hydrophilic. The polymer may be, for example, a homopolymer, random copolymer, blended polymer, copolymer, or a block copolymer. Block copolymers are most simply defined as conjugates of at least two different polymer segments or blocks. The polymer may be, for example, linear, star-like, graft, branched, dendrimer based, or hyper-branched (e.g., at least two points of branching). In certain embodiments, the polymer is linear. The polymer of the invention may have from about 2 to about 10,000, about 2 to about 1000, about 2 to about 500, about 2 to about 250, or about 2 to about 100 repeating units or monomers. The polymers of the instant invention may comprise capping termini.

[0027] Examples of hydrophobic polymers include, without limitation: poly(hydroxyethyl methacrylate), poly(N-isopropyl acrylamide), poly(lactic acid) (PLA (or PDLA)), poly(lactide-co-glycolide) (PLG), poly(lactic-co-glycolic acid) (PLGA), polyglycolide or polyglycolic acid (PGA), polycaprolactone (PCL), poly(aspartic acid), polyoxazolines (e.g., butyl, propyl, pentyl, nonyl, or phenyl poly(2-oxazolines)), polyoxypropylene, poly(glutamic acid), polypropylene fumarate) (PPF), poly(trimethylene carbonate), polycyanoacrylate, polyurethane, polyorthoesters (POE), polyanhydride, polyester, polypropylene oxide), poly(caprolactonefumarate), poly(l,2- butylene oxide), poly(n-butylene oxide), poly(ethyleneimine), poly(tetrahydrofurane), ethyl cellulose, polydipyrolle / dicabazole, starch, polyvinylidene fluoride (PVDF), polytetrafluoroethylene (PTFE), polydioxanone (PDO), polyether poly(urethane urea) (PEUU), cellulose acetate, polypropylene (PP), polyethylene terephthalate (PET), nylon (e.g., nylon 6), polycaprolactam, PLA / PCL (PLCL), poly(3-hydroxybutyrate-co-3- hydroxyvalerate) (PHBV), PCL / calcium carbonate, and / or poly(styrene) or combinations thereof.

[0028] Examples of hydrophilic polymers include, without limitation: polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), poly(ethylene glycol) (PEG) and poly(ethylene oxide) (PEO), chitosan, collagen, chondroitin sulfate, sodium alginate, gelatin, elastin, hyaluronic acid, silk fibroin, sodium alginate / PEO, silk / PEO, silk fibroin / chitosan, hyaluronic acid / gelatin, collagen / chitosan, chondroitin sulfate / collagen, and chitosan / PEO or combinations thereof.

[0029] Amphiphilic copolymers or polymer composites may comprise a hydrophilic polymer (e.g., segment) and a hydrophobic polymer (e.g., segment) from those listed above (e.g., gelatin / polyvinyl alcohol (PVA), PCL / collagen, chitosan / PVA, gelatin / elastin / PLGA, PDO / elastin, PHBV / collagen, PLA / hyaluronic acid, PLGA / hyaluronic acid, PCL / hyaluronic acid, PCL / collagen / hyaluronic acid, gelatin / siloxane, PLLA / MWNTs / hyaluronic acid) or combinations thereof.

[0030] Examples of polymers particularly useful for electrospinning are provided in Xie et al. (Macromol. Rapid Commun. (2008) 29:1775-1792; incorporated by reference herein; see e.g., Table 1). Examples of compounds or polymers for use in the fibers of the instant invention, particularly for electrospun nanofibers include, without limitation: natural polymers (e.g., chitosan, gelatin, collagen type I, II, and / or III, elastin, hyaluronic acid, cellulose, silk fibroin, phospholipids (Lecithin), fibrinogen, hemoglobin, fibrous calf thymus Na-DNA, virus Ml 3 viruses), synthetic polymers (e.g., PLGA, PLA, PCL, PHBV, PDO, PGA, poly(L-lactide-co-s-caprolactone) (PLCL), PLLA-DLA, PEUU, cellulose acetate, PEG-b-PLA, EVOH, PVA, PEO, PVP), blended (e.g, PLA / PCL, gelatin / PVA, PCL / gelatin, PCL / collagen, sodium alginate / PEO, chitosan / PEO, Chitosan / PVA, gelatin / elastin / PLGA, silk / PEO, silk fibroin / chitosan, PDO / elastin, PHBV / collagen, hyaluronic acid / gelatin, coll agen / chondroi tin sulfate, collagen / chitosan), and composites (e.g, PDLA / HA, PCL / CaCCh, PCL / HA, PLLA / HA, gelatin / HA, PCL / collagen / HA, collagen / HA, gelatin / siloxane, PLLA / MWNTs / HA, PLGA / HA). In certain embodiments, the nanofiber comprises polymethacrylate, poly vinyl phenol, polyvinylchloride, cellulose, polyvinyl alcohol, polyacrylamide, PLGA, collagen, polycaprolactone, polyurethanes, polyvinyl fluoride, polyamide, silk, nylon, polybennzimidazole, polycarbonate, polyacrylonitrile, polyvinyl alcohol, polylactic acid, polyethylene-co-vinyl acetate, polyethylene oxide, polyaniline, polystyrene, polyvinylcarbazole, polyethylene terephthalate, polyacrylic acid-polypyrene methanol, poly(2-hydroxyethyl methacrylate), polyether imide, polyethylene glycol, poly(ethylene- co-vinyl alcohol), polyacrylnitrile, polyvinyl pyrrolidone, polymetha-phenylene isophthal ami de, gelatin, chitosan, starch, pectin, cellulose, methylcellulose, sodium polyacrylate, starch-acrylonitrile co-polymers, and / or combinations of two or more polymers.

[0031] In certain embodiments, the nanofibers are made from a polymer including but not limited to polymethacrylate, poly vinyl phenol, polyvinylchloride, cellulose, polyvinyl alcohol, polyacrylamide, poly(lactic-co-glycolic) acid (PLGA), poly(glycolide- co-lactide) (PGLA), collagen, polycaprolactone (PCL), poly(lactic acid) (PLA), polydioxanone (PDO), polyurethanes, polyvinyl fluoride, polyamide, silk, nylon, polybennzimidazole, polycarbonate, polyacrylonitrile, polyvinyl alcohol, polylactic acid, polyethylene-co-vinyl acetate, polyethylene oxide, polyaniline, polystyrene, polyvinylcarbazole, polyethylene terephthalate, polyacrylic acid-polypyrene methanol, poly(2-hydroxyethyl methacrylate), polyether imide, polyethylene gricol, polyethylene glycol, poly(ethylene-co-vinyl alcohol), polyacrylnitrile, polyvinyl pyrrolidone, polymetha-phenylene isophthal ami de, gelatin, alginate, chitosan, hyaluronic acid, heparin, starch, pectin, cellulose, methylcellulose, sodium polyacrylate, starchacrylonitrile co-polymers, bioactive glass, and combinations of two or more polymers. Multiple polymers may be mixed to form the nanofibers. In certain embodiments, the nanofibers comprise PCL, PLGA, PGLA, PLA, chitosan, hyaluronic acid, and / or heparin or combinations thereof. The polymers may be mixed evenly or in various ratios depending on the desired properties of the nanofibers. In certain embodiments, the polymer comprises polycaprolactone (PCL), poly(lactide-co-epsilon-caprolactone) (PLCL), polyglycolic acid (PGA), and / or poly(lactic-co-glycolic) acid (PLGA). In certain embodiments, the polymer comprises polycaprolactone (PCL). In certain embodiments, the polymer comprises cellulose, cellulose acetate (CA), polyethylene (PE), polypropylene (PP), polystyrene (PS), poly(vinyl chloride) (PVC), poly(vinyl alcohol) (PVA), chitosan, polybutylene succinate (PBS), polyethylene succinate (PESU), poly(anhydrides), polyvinylpyrrolidone (PVP), poly(methyl methacrylate) (PMMA), poly(acrylamide), poly(HEMA) (poly(2 -hydroxyethyl methacrylate)), polyethylene oxide (PEO), polyethylene terephthalate (PET), polyether ether ketone (PEEK), polyurethane (PU), polytetrafluoroethylene (PTFE), polycaprolactone (PCL), polydioxanone (PDO), poly(lactide-co-epsilon-caprolactone) (PLCL), polyglycolic acid (PGA), polylactide (PLA), poly(lactic-co-glycolic) acid (PLGA), or a combination thereof. In certain embodiments, the polymer comprises cellulose, cellulose acetate (CA), polyvinylpyrrolidone (PVP), polyurethane (PU), polycaprolactone (PCL), poly(lactide-co-epsilon-caprolactone) (PLCL), polyglycolic acid (PGA), polylactide (PLA), polydioxanone (PDO), poly(lactic-co-glycolic) acid (PLGA), or a combination thereof. In certain embodiments, the polymer comprises cellulose, cellulose acetate (CA), polyvinylpyrrolidone (PVP), polyurethane (PU), polycaprolactone (PCL), poly(lactide-co-epsilon-caprolactone) (PLCL), polyglycolic acid (PGA), poly(lactic-co- glycolic) acid (PLGA), or combinations thereof. In certain embodiments, the polymers comprise PVP. In certain embodiments, the polymers comprise CA. In certain embodiments, the polymers comprise PU. In certain embodiments, the polymers comprise PVP and CA. In certain embodiments, the polymers comprise PVP and PU. In certain embodiments, the nanofibers may further comprise at least one surfactant. In certain embodiments, the nanofibers may further comprise at least one amphiphilic block copolymer comprising hydrophilic poly(ethylene oxide) (PEO) and hydrophobic polypropylene oxide) (PPO). In certain embodiments, the nanofibers comprise a poloxamer or an amphiphilic triblock copolymer comprising a central hydrophobic PPO block flanked by two hydrophilic PEO blocks (i.e., an A-B-A triblock structure). In certain embodiments, the amphiphilic block copolymer is selected from the group consisting of Pluronic® L31, L35, F38, L42, L44, L61, L62, L63, L64, P65, F68, L72, P75, F77, L81, P84, P85, F87, F88, L92, F98, L101, P103, P104, P105, F108, L121, L122, L123, F127, 10R5, 10R8, 12R3, 17R1, 17R4, 17R8, 22R4, 25R1, 25R2, 25R4, 25R5, 25R8, 31R1, 31R2, and 31R4. In certain embodiments, the nanofibers comprise poloxamer 188. In certain embodiments, the nanofibers comprise poloxamer 407 (Pluronic® F127). The amphiphilic block copolymer (e.g., poloxamer) may be added in various amounts to the polymer solution during the synthesis process (e.g., electrospinning). In certain embodiments, about 0% to about 20%, about 0% to about 15%, about 0% to about 10%, about 0.1% to about 5%, about 0.5% to about 2%, or about 0.1% to about 1.0% (e.g., w / v) of the polymer solution is an amphiphilic block copolymer (e.g., a poloxamer (e.g., poloxamer 407)). In certain embodiments, about 0.1% to about 50%, about 0.1% to about 40%, about 0.1% to about 30%, about 0.1% to about 25%, about 0.1% to about 20% (e.g., w / v), or about 5% to about 15% of the polymer solution is polymer (e.g., PCL). In certain embodiments, the polymer solution comprises about 10% polymer (w / v) (e.g., PCL) and about 1.0% poloxamer 407 (w / v) (Pluronic® Fl 27). In certain embodiments, the polymer solution comprises PCL and poloxamer 407 in a ratio (e.g., by v / v) of about 200: 1 to about 1 :1, about 100: 1 to about 1 : 1, about 50: 1 to about 1 : 1, about 20: 1 to about 2: 1, about 10:1 to about 2: 1, or about 4: 1.

[0032] In certain embodiments, the nanofibers or the polymers of the nanofibers have a glass transition temperature greater (above) the temperature of the thermal treatment to cause expansion of the expandable microspheres. In certain embodiments, the glass transition temperature of the nanofibers or the polymers of the nanofibers is at least 1°C, at least 3°C, at least 5°C, at least 10°C, at least 15°C, at least 20°C, at least 25°C, at least 30°C, at least 40°C, at least 50°C or at least 60°C higher than the temperature of the thermal treatment to cause expansion of the expandable microspheres. In certain embodiments, the nanofiber has a glass transition temperature of at least 175°C. The nanofiber-microsphere composite scaffolds may be composed of layers wherein each layer may have a different thickness, fiber alignment, and / or porosity. In certain embodiments, the nanofiber-microsphere composite scaffolds comprise one or more regions of different thickness, fiber alignment, and / or porosity.

[0033] The nanofiber-microsphere composite scaffolds of the instant invention may be fabricated into any shape, size, or thickness. The nanofiber-microsphere composite scaffolds may be shaped (e.g., by cutting or shaving) after expansion by thermal treatment. The nanofiber-microsphere composite scaffolds may be shaped before expansion by thermal treatment (e.g., by shaping and / or modifying the electrospun nanofiber mat). The nanofiber-microsphere composite scaffolds may be shaped are during expansion by thermal treatment (e.g., by expanding in a mold or other confined space).

[0034] In certain embodiments of the instant invention, the methods further comprise fixing (e.g., fusing) at least one point, edge, end, or side - or a portion thereof - of a nanofiber mat (e.g., electrospun nanofiber mat) prior to expansion by thermal treatment. The fixation / fusing of a portion of the nanofiber mat allows for rotational expansion and the formation of solids of revolution such as, without limitation: spheres, cones cylinders, hollow spheres, hollow cylinders, and the like.

[0035] In certain embodiments, a whole or entire side of the nanofiber mat is fixed. In certain embodiments, one or more sections or portions of the nanofiber mat is fixed (e.g., the top and bottom comers on one side may be fixed). The nanofiber mat may be fixed by any means. For example, the nanofiber mat may be thermally fixed or chemically fixed. In certain embodiments, the nanofiber mat is thermally fixed.

[0036] In certain embodiments, the nanofiber mat is fixed by exposing at least one point, edge, end, or side - or a portion thereof - of the nanofiber mat to elevated temperatures (e.g., thermally fixing or thermally welding). In certain embodiments, only one side of the nanofiber mat is fixed. In certain embodiments, the nanofiber mat is a rectangle. In certain embodiments, the nanofiber mat is a rectangle and only one long side of the rectangle is fixed. In certain embodiments, the nanofiber mat is a rectangle and only one short side of the rectangle is fixed.

[0037] In certain embodiments, the nanofiber mat is exposed to temperatures at or above the melting temperature of the nanofibers. In certain embodiments, the nanofiber mat is fixed by exposing at least one point, edge, end, or side - or a portion thereof - of the nanofiber mat to a temperature of at least about 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, 90°C, 95°C, 100°C, or higher. In certain embodiments, the nanofiber mat is fixed by exposing at least one point, edge, end, or side - or a portion thereof - of the nanofiber mat to a temperature of at least about 3 °C, 5°C, 8°C, 10°C, or more, higher than the melting temperature of the nanofiber. To avoid excess fixation and / or damage to the remainder of the nanofiber mat and / or avoid undesired or premature expansion of the nanofiber mat, the exposure to elevated temperatures should be localized to essentially the desired location of fixation (e.g., fusion). To avoid excess fixation and / or damage to the remainder of the nanofiber mat and / or avoid undesired or premature expansion of the nanofiber mat, the exposure to elevated temperatures may be brief (e.g., less than 10 seconds, less than 5 seconds, for about 3 seconds, or for about 1 second). In certain embodiments, the heat is applied perpendicularly to the nanofiber mat. In certain embodiments, the thermal fixing comprises exposing at least one point, edge, end, or side - or a portion thereof - of a nanofiber mat to about 75°C to about 95°C, particularly about 85°C (e.g., for less than 5 seconds, particularly about 1-3 seconds).

[0038] In certain embodiments, the nanofiber mat is chemically fixed (e.g., fused), for example, by exposure to a chemical, solvent, or crosslinker. In certain embodiments, a chemical or solvent based method is used to fix the nanofiber mat. The chemical or solvent can be, without limitation: di chloromethane (DCM), dimethylformamide (DMF), N,N-dichloroformamide, acetone, and other organic solvents. In certain embodiments, the nanofiber mat is fixed by exposure to a crosslinker. In certain embodiments, the nanofiber mat is chemically fixed by exposing at least one point, edge, end, or side - or a portion thereof - of the nanofiber mat to a chemical, solvent, or crosslinker with minimal or no exposure the remainder of the nanofiber mat to the chemical, solvent, or crosslinker.

[0039] The nanofiber mat may be cut, trimmed, or shaped prior to fixation and / or expansion. The nanofiber mat (if fixed) may be cut, trimmed, or shaped prior to fixation or cut, trimmed, or shaped after fixation. In certain embodiments, the nanofiber mat is cut, trimmed, or shaped under cryogenic or frozen conditions (e.g., in liquid nitrogen or on dry ice). The nanofiber mat can be cut, trimmed, or shaped into any desired shape such as, without limitation: rectangles, squares, triangles, quadrangles, pentagons, hexagons, circles, ovals, semicircles, L’s, C’s, O’s, U’s, and arches. In certain embodiments, an aligned nanofiber mat is cut such that the length of the cut (or resultant strips or structures) is aligned with the direction of the nanofibers of the mat (e.g., to generate laterally expanded scaffolds). As explained hereinabove, the nanofiber mats comprise expandable microspheres. The expandable microspheres may be added to the nanofiber mats by any means. For example, the expandable microspheres may be added by electrospinning, centrifugal spinning, solution blowing, electrospray, gas spray, other like methods, or a combination thereof. In certain embodiments, the expandable microspheres are added during formation of the nanofiber mat. In certain embodiments, the expandable microspheres are added during formation of the nanofiber mat by electrospinning. In certain embodiments, the expandable microspheres are in the solution used for electrospinning (e.g., polymeric solution). In certain embodiments, the expandable microspheres are added during electrospinning via electrospraying (e.g., onto the forming nanofiber mat). In certain embodiments, the expandable microspheres are added during electrospinning via gas spraying (e.g., onto the forming nanofiber mat). In certain embodiments, the expandable microspheres are added during electrospinning via electrospinning (e.g., onto the forming nanofiber mat), such as via a second spinneret. In certain embodiments, the expandable microspheres are added during electrospinning via a dual or double spinneret (e.g., a polymer solution in one stream and a solution of the expandable microspheres in another stream).

[0040] In certain embodiments, the nanofiber mat is expanded by thermal treatment (e.g., heating) to expand the expandable microspheres. In certain embodiments, the nanofiber mat is expanded radially and / or around a fixed axis (e.g., as defined by the fixed portion of the nanofiber mat, if present). In certain embodiments, the nanofiber mat is expanded in the third dimension (e.g., the z-axis for a nanofiber mat). Generally, the greater the amount or concentration of expandable microspheres, the greater the thickness and / or porosity of the expanded nanofiber structure increases. The nanofiber mat may also be expanded within a mold (e.g., a metal, plastic, or other material that does not expand in the presence of the thermal treatment) to assist in the formation of a desired shape. The nanofiber mat may be treated with air plasma prior to exposure to thermal treatment (e.g., to increase hydrophilicity). The nanofiber mat may be exposed to thermal treatment more than once (e.g., repeatedly). A negative pressure could be applied to facilitate the expansion.

[0041] Generally, the thermal treatment results in the expansion of the expandable microspheres. The thermal treatment may lead to a rapid increase in the thickness of the initially 2D nanofiber mats containing the expandable microspheres, transforming such mats into nanofiber-microsphere composite scaffolds. In certain embodiments, the thermal treatment causes the rapid vaporization of the encapsulated blowing agent within the expandable microsphere and produces a large volume of gas to expand the expandable microsphere. The expansion of the expandable microsphere upon thermal treatment may occur fully or partially. The expansion of the expandable microsphere upon thermal treatment may occur uniformly or irregularly. In certain embodiments, the thermal treatment is performed only on certain regions of the nanofiber mat. In certain embodiments, the thermal treatment is performed evenly across the entirety of the nanofiber mat.

[0042] Expandable microspheres are known in the art. Generally, expandable microspheres comprise a sphere (e.g., polymeric shell or thermoplastic shell) which contains a blowing agent in the core wherein thermal treatment (heat) causes the blowing agent to expand and the shell to soften to allow expansion of the microsphere. Without being bound by theory, the ability to expand is governed by the sphere’s (e.g., polymer’s) glass transition temperature. Below the glass transition temperature, the sphere is small and rigid. The blowing agent is selected to match the glass transition temperature of the polymer so that when the shell starts to soften, the internal pressure increases due to gas expansion and the sphere expands. When the spheres cool down, they retain their expanded shape and harden. In certain embodiments, the expandable microsphere is an Expancel® expandable microsphere. The concentration of the expandable microspheres may range from 0.1% to 99.9% of the total concentration (e.g., of the nanofiber mat or the electrospun solution). Generally, the expansion of the expandable microsphere after thermal treatment positively correlates with the amount or concentration of expandable microspheres comprised in the scaffold.

[0043] In certain embodiments, the expandable microsphere increases in volume about 2 to about 60 times, about 5 to about 60 times, about 10 to about 60 times, about 20 to about 60 times, about 30 to about 60 times, or about 40 to about 60 times after thermal treatment. In certain embodiments, the expandable microsphere increases in volume at least about 2 times, at least about 5 times, at least about 10 times, at least about 20 times, at least about 30 times, at least about 40 times, or at least about 50 times after thermal treatment.

[0044] In certain embodiments, the expandable microspheres have a diameter (e.g., average diameter) from about 1 pm to about 50 pm, about 1 pm to about 35 pm, about 1 pm to about 30 pm, about 5 pm to about 30 pm, about 5 pm to about 25 pm, about 5 pm to about 20 pm, about 10 pm to about 20 pm, about 10 pm to about 15 pm, or about 12 pm prior to expansion. In certain embodiments, the expandable microspheres have a diameter (e.g., average diameter) of at least about 1 pm, at least about 5 pm, or at least about 10 pm prior to expansion. In certain embodiments, the expandable microspheres have a diameter (e.g., average diameter) less than about 40 pm, less than about 35 pm, less than about 30 pm, less than about 25 pm, less than about 20 pm, or less than about 15 pm prior to expansion.

[0045] In certain embodiments, the expandable microspheres have a diameter (e.g., average diameter) from about 30 pm to about 120 pm, about 30 pm to about 100 pm, about 40 pm to about 100 pm, about 30 pm to about 90 pm, about 40 pm to about 90 pm, about 50 pm to about 80 pm, about 60 pm to about 70 pm, or about 65 pm after expansion. In certain embodiments, the expandable microspheres have a diameter (e.g., average diameter) of at least about 30 pm, at least about 35 pm, at least about 40 pm, at least about 45 pm, at least about 50 pm, at least about 55 pm, or at least about 60 pm after expansion. In certain embodiments, the expandable microspheres have a diameter (e.g., average diameter) less than about 120 pm, less than about 110 pm, less than about 100 pm, less than about 90 pm, less than about 80 pm, or less than about 70 pm after expansion.

[0046] The expandable microsphere prior, during, and / or after thermal treatment may have a smooth surface topography. In certain embodiments, the expandable microsphere prior, during, and / or after thermal treatment may have an irregular surface topography. While described as a sphere, the expandable microsphere may take the form of other shapes (e.g., when expanded).

[0047] Any amount of expandable microspheres may be added to the nanofiber mat. In certain embodiments, the concentration of the expandable microspheres may range from 0.1% to 99.9% of the solution (e.g., solutions used for electrospinning) or the nanofiber mat (e.g., by weight). In certain embodiments, the expandable microspheres comprise at least 0.1%, at least 0.5%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25% or more. In certain embodiments, the expandable microspheres comprise less than 75%, less than 50%, less than 25%, less than 20%, less than 15%, less than 10%, or less than 5%. In certain embodiments, the expandable microspheres comprise about 0.5% to about 10% or about 1% to about 5%.

[0048] In certain embodiments, the shell comprises a polymer, particularly a thermoplastic polymer. In certain embodiments, the thermoplastic polymer is a biodegradable polymer. Examples of thermoplastic polymers include, without limitation: acrylic polymers, acrylic copolymers, cellulose, cellulose ester (optionally with acetate and or propionate substituents), carboxylate-functionalized cellulose, vinylidene chloride / acrylonitrile copolymer, ethylene vinylacetate, polymethyl methacrylate (PMMA), polyvinylidene chloride (PVDC), polyacrylonitrile, polyacrylic ester (PAE), polyvinyl acetate (PVA), acrylonitrile, vinyl acetate ethylene, poly(s- caprolactone) (PCL), poly(lactide-co-glycolide) (PLGA), and poly(acrylonitrile-co- methacrylonitrile).

[0049] Generally, the shell thickness (e.g., average thickness) decreases with thermal treatment and expansion. In certain embodiments, the shell thickness is about 0.5 pm to about 10 pm, about 1 pm to about 5 pm, about 1 pm to about 3 pm, or about 2 pm prior to heat expansion. In certain embodiments, the shell thickness is at least about 0.5 pm, at least about 1 pm, at least about 1.5 pm, or at least about 2 pm prior to heat expansion. In certain embodiments, the shell thickness is less than about 10 pm, less than about 8 pm, less than about 5 pm, or less than about 3 pm prior to heat expansion. In certain embodiments, the shell thickness is about 0.01 pm to about 1 pm, about 0.05 pm to about 0.5 pm, about 0.05 pm to about 0.2 pm, or about 0.1 pm after heat expansion. In certain embodiments, the shell thickness is at least about 0.01 pm after heat expansion. In certain embodiments, the shell thickness is less than about 1 pm, less than about 0.5 pm, less than about 0.3 pm, or less than about 0.2 pm after heat expansion.

[0050] The blowing agent within the expandable microsphere can be a liquid or a gas. In certain embodiments, the blowing agent is a gas. In certain embodiments, the blowing agent is a liquid with a low boiling point (e.g., a low-boiling-point substance that vaporizes when heated, creating pressure inside the shell). In certain embodiments, the blowing agent is a hydrocarbon, particular a saturated hydrocarbon, aliphatic hydrocarbon or halogenated hydrocarbon, gas or low boiling point liquid hydrocarbon. Examples of hydrocarbon gases include without limitation: isopentane, isobutane, n- butane, and 2,2-dimethylpropane (neopentane). In certain embodiments, the hydrocarbon is a C4 - Cs hydrocarbon or a C4 - Ce hydrocarbon. The hydrocarbons can be linear or branched.

[0051] The temperature of the thermal treatment to expand the expandable microsphere depends on the shell and the blowing agent as explained herein. In certain embodiments, the thermal treatment is between about 50°C and about 200°C. In certain embodiments, the thermal treatment is between about 80°C and about 200°C. In certain embodiments, the thermal treatment is between about 94°C and about 164°C. In certain embodiments, the thermal treatment is between about 50°C and about 150°C. In certain embodiments, the thermal treatment is between about 80°C and about 120°C. In certain embodiments, the thermal treatment is between about 50°C and about 120°C. In certain embodiments, the thermal treatment is between about 100°C and about 110°C. In certain embodiments, the thermal treatment temperature is at least about 50°C, at least about 60°C, at least about 70°C, at least about 80°C, at least about 90°C, or at least about 100°C. In certain embodiments, the thermal treatment temperature is less than about 200°C, less than about 150°C, less than about 125°C, less than about 120°C, less than about 110°C, less than about 100°C, less than about 90°C, less than about 80°C, less than about 70°C, or less than about 60°C.

[0052] The length of time of the thermal treatment can be adjusted for the desired amount of expansion of the nanofiber mat. Generally, the longer the exposure to the thermal treatment, the greater the expansion of the nanofiber mat until a maximum is reached. In certain embodiments, the thermal treatment is for at least about 5 seconds, at least about 10 seconds, at least about 15 seconds, at least about 20 seconds, at least about 30 seconds, at least about 45 seconds, at least about 1 minute, at least about 2 minutes, at least about 3 minutes, at least about 4 minutes, at least about 5 minutes or longer. In certain embodiments, the thermal treatment is for less than about 5 minutes, less than about 4 minutes, less than about 3 minutes, less than about 2 minutes, less than about 90 seconds, less than about 1 minute, less than about less than about 30 seconds, or less than about 15 seconds.

[0053] The nanofiber-microsphere composite scaffolds of the instant invention may be coated with a coating material such as gelatin. The coating may be applied before or after expansion. The methods of the instant invention may further comprise coating the nanofiber-microsphere composite scaffolds with a coating material such as gelatin. While gelatin is described herein as the coating material, other coating materials may be used (e.g., coating materials with adhesive properties). For example, the scaffolds may be coated with a hydrogel, collagen, a proteoglycans, elastin, a glycosaminoglycan (e.g., hyaluronic acid, heparin, chondroitin sulfate, or keratan sulfate), gelatin, alginate, chitosan, chitin, starch, pectin, cellulose, methylcellulose, sodium polyacrylate, starchacrylonitrile co-polymers, a glue (bioadhesive) (e.g., fibrin glue), and / or other natural or synthetic hydrogels, and derivatives thereof (e.g., del Valle et al., Gels (2017) 3:27). As used herein, a hydrogel is a polymer matrix able to retain water, particularly large amounts of water, in a swollen state. In certain embodiments, the coating material is present at 0.01% to 10%, 0.01% to 1%, 0.05% to 0.5%, or about 0.1%. In certain embodiments, the coating material is gelatin, chitosan, collagen, cellulose, chitin, a hydrogel, or a glue (bioadhesive) (e.g., fibrin glue). In certain embodiments, the coating material is gelatin. In certain embodiments, the coating material is a ECM mimicking molecule (e.g., gelatin, collagen, fibronectin, laminin) which can render both mechanical and biological functions.

[0054] Prior to coating, the nanofiber-microsphere composite scaffolds may also be modified (e.g., physically and / or chemically) to enhance the coating process. In certain embodiments, the modification increases the hydrophilicity of the scaffold. In certain embodiments, the scaffold undergoes plasma treatment (e.g., air plasma and / or oxygen plasma). Plasma treatment will generate negatively charged groups (e.g., carboxyl groups) that enhance the interaction between the scaffold and the coating.

[0055] The term “coat” refers to a layer of a substance / material on the surface of a scaffold and / or the fibers of the scaffold. Coatings may, but need not, also impregnate the scaffold (e.g., form a layer on the nanofibers of the scaffold). Further, while a coating may cover 100% of the scaffold, a coating may also cover less than 100% of the surface of the scaffold (e.g., at least about 50%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or more of the surface may be coated).

[0056] The coating material may be applied to the scaffold by any method (e.g., vapor deposition). For example, the coating material may be applied to the nanofiber- microsphere composite scaffold by immersing or soaking the scaffold in a solution or suspension comprising the coating material, spraying (e.g., electrospraying) the scaffold with a solution or suspension comprising the coating material, and / or physically applying (e.g., painting) a solution or suspension comprising the coating material onto the scaffold. In certain embodiments, the coating material is applied to the nanofiber- microsphere composite scaffold by immersing or soaking the scaffold in a solution or suspension comprising the coating material.

[0057] The coating material and optionally the nanofibers may be crosslinked (e.g., with the scaffold). Crosslinking may be done using a variety of techniques including thermal crosslinking, chemical crosslinking, UV-crosslinking, and photo-crosslinking. For example, the scaffold of the instant invention may be crosslinked with a crosslinker such as, without limitation: formaldehyde, paraformaldehyde, acetaldehyde, glutaraldehyde, a photocrosslinker, genipin, and natural phenolic compounds (Mazaki, et al., Sci. Rep. (2014) 4:4457; Bigi, et al., Biomaterials (2002) 23 :4827-4832; Zhang, et al., Biomacromolecules (2010) 11: 1125-1132; incorporated herein by reference). The crosslinker may be a bifunctional, trifunctional, or multifunctional crosslinking reagent. In certain embodiments, the crosslinker is glutaraldehyde.

[0058] The nanofiber-microsphere composite scaffolds may be washed or rinsed in water and / or a desired carrier or buffer (e.g., a pharmaceutically or biologically acceptable carrier). The washing may occur after expansion and / or after coating (if present). The nanofiber-microsphere composite scaffolds may also be stored in a cold solution, lyophilized, frozen in a vacuum, and / or freeze-dried (e.g., after expansion and / or after coating, if present).

[0059] The nanofiber-microsphere composite scaffolds of the instant invention may also be sterilized. Sterilization may occur after expansion and / or after coating (if present). For example, the nanofiber-microsphere composite scaffolds can be sterilized using various methods (e.g., by treating with ethylene oxide gas, gamma irradiation, or 70% ethanol). In certain embodiments, the nanofiber-microsphere composite scaffolds are sterilized by treating with ethylene oxide.

[0060] The methods of the instant invention may further comprise culturing cells on and / or within the nanofiber-microsphere composite scaffolds. In certain embodiments, the cells are seeded on and / or in the nanofiber-microsphere composite scaffold prior to culturing. In certain embodiments, the cells secrete and / or produce extracellular matrix. In certain embodiments, the cells are human. In certain embodiments, the cells are autologous (e.g., from a subject to be treated with a nanofiber-microsphere composite scaffolds of the instant invention. Different cells or combinations of cells may be seeded on the nanofiber-microsphere composite scaffolds to produce different types of extracellular matrix. Cells that may be used in the present invention include, without limitation, fibroblasts, osteoblasts, Schwann cells, endothelial cells, epithelial cells, muscle cells, adipocytes, adipose-derived stem cells, tenocytes, chondrocytes, bone marrow stem cells, mesenchymal stem cells, neural progenitor cells, human induced pluripotent stem cells, embryonic stem cells, as well as genetically engineered cells (e.g., Crispr-Cas9, viral vectors, electroporation, lipofection, transposon systems, microinjection, homologous recombination, mRNA transfection) of various origins, or combinations thereof. Cell types also include, without limitation: embryonic stem cells, adult stem cells, bone marrow stem cells, induced pluripotent stem cells, progenitor cells (e.g., neural progenitor cells), embryonic like stem cells, mesenchymal stem cells, CAR- T cells, immune cells (including but not limited to T cells, B cells, NK cells, macrophages, neutrophils, dendritic cells and modified forms of these cells and various combinations thereof), cell based vaccines, and cell lines expressing desired therapeutic proteins and / or genes. In certain embodiments, a co-culture of at least two or more different types of cells on the nanofiber-microsphere composite scaffolds is used. In certain embodiments, the cells are fibroblasts, osteoblasts, or chondrocytes. In certain embodiments, the cells are fibroblasts. In certain embodiments, the cells are dermal fibroblasts.

[0061] The cells may be cultures in the nanofiber-microsphere composite scaffolds as long as desired. In certain embodiments, the cells are cultured at least long enough to produce ECM throughout the nanofiber-microsphere composite scaffolds. Culturing of the cells may include one or more media changes. In certain embodiments, the media is changed at least every 2-3 days. In certain embodiments, the cells are cultured in the in the nanofiber-microsphere composite scaffold for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, or more days. In certain embodiments, the cells are cultured in the in the nanofiber- microsphere composite scaffold for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more weeks. In certain embodiments, the cells or tissue may be cultured within the nanofiber-microsphere composite scaffolds in differentiation media. In certain embodiments, the cells or tissue may be cultured within the nanofiber-microsphere composite scaffolds in the presence of different growth factors (e.g., TGF-beta) which can modulate secretion of ECM.

[0062] In certain embodiments, the methods of the instant invention further comprise decellularization of the nanofiber-microsphere composite scaffolds after the culturing of the cells has produced the ECM. In certain embodiments, the decellularization results in the elimination of viable cells from the nanofiber-microsphere composite scaffolds. Decellularization methods are known in the art and include, without limitation: thermal shock, freeze-thawing, detergent treatment (e.g., ionic detergents such as SDS), osmatic shock, ultrasonication, mechanical disruption, and enzymatic action (e.g., trypsin and / or collagenase). In certain embodiments, the decellularization does not degrade or negatively impact the ECM in a significant manner. In certain embodiments, the decellularization process further comprises contacting the nanofiber-microsphere composite scaffold with DNase (e.g., DNase I). In certain embodiments, the ECM comprises one or more (or all) of elastin, glycosaminoglycans, collagen, fibronectin, elastin, and growth factors (e.g., VEGF, bFGF, TGF-pi, and EGF).

[0063] In certain embodiments, the decellularization is perform by freeze-thaw cycling. In certain embodiments, freeze-thaw cycling comprises about 1 to about 15 or about 1 to about 10, or about 1 to about 8 cycles of freeze-thaws. In certain embodiments, the freeze-thaw cycling occurs 10 or fewer times, 9 or fewer times, 8 or fewer times, 7 or fewer times, 6 or fewer times, 5 or fewer times, 4 or fewer times, 3 or fewer times, 2 or fewer times, or 1 time. In certain embodiments, the freezing is performed at -20°C or lower, -40°C or lower, -60°C or lower, or -80°C or lower. In certain embodiments, the thawing is performed at room temperature to about 37°C.

[0064] In certain embodiments, the decellularization is performed by detergent treatment. In certain embodiments, the decellularization is performed by ionic detergent treatment. In certain embodiments, the decellularization is performed by SDS. In certain embodiments, the detergent is present at about 0.01% to about 1% (by wt), about 0.05% to about 0.5%, or about 0.1%. In certain embodiments, the detergent treatment is performed at room temperature. In certain embodiments, the detergent treatment is performed with agitation and / or shaking. In certain embodiments, the detergent treatment is performed for at least 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more hours. In certain embodiments, the detergent treatment is performed for 10 hours or less, 10 hours or less, 9 hours or less, 8 hours or less, 7 hours or less, 6 hours or less, 5 hours or less, 4 hours or less, 3 hours or less, 2 hours or less, or 1 hour or less.

[0065] The nanofiber-microsphere composite scaffolds may be washed or rinsed in water and / or a desired carrier or buffer (e.g., a pharmaceutically or biologically acceptable carrier) after decellularization. The nanofiber-microsphere composite scaffolds may also be stored in a cold solution, lyophilized, frozen in a vacuum, and / or freeze-dried after decellularization.

[0066] The nanofiber-microsphere composite scaffolds of the instant invention may also be sterilized after decellularization. For example, the nanofiber-microsphere composite scaffolds can be sterilized using various methods (e.g., by treating with ethylene oxide gas, gamma irradiation, or 70% ethanol). In certain embodiments, the nanofiber- microsphere composite scaffolds are sterilized by treating with ethylene oxide.

[0067] In certain embodiments, the nanofiber-microsphere composite scaffolds of the instant invention may further comprise at least one agent, particularly a bioactive agent, biologic, cells, cell based therapy, tissue based therapy, and / or drug. The methods of the instant invention may further comprise adding at least one agent, particularly a bioactive agent, biologic, cell based therapy, tissue based therapy, and / or drug to the nanofiber- microsphere composite scaffolds. The agent may be added before, during or after expansion. In certain embodiments, the agent is added after expansion. The agent may be added uniformly throughout the scaffold or added differently (e.g., different agents and / or different concentrations of agents) to different regions and / or layers of the scaffold. The agent may be applied to the scaffold by any method. For example, the agent may be applied to the scaffold by immersing or soaking the scaffold in a solution or suspension comprising the agent, spraying (e.g., electrospraying) the scaffold with a solution or suspension comprising the agent, and / or physically applying (e.g., painting), contacting, or injecting a solution or suspension comprising the agent onto or into the scaffold. In certain embodiments, the agent is applied to the scaffold by immersing or soaking the scaffold in a solution or suspension comprising the agent. In certain embodiments, the agent is applied to the scaffold by incorporating the agent during electrospinning.

[0068] Biologies include but are not limited to small molecules, proteins, peptides, antibodies, antibody fragments, nucleic acid, DNA, RNA, and other known biologic substances, particularly those that have therapeutic use. In a particular embodiment, the agent is a drug or therapeutic agent (e.g., a small molecule) (e.g., analgesic, growth factor, anti-inflammatory, signaling molecule, growth factor, cytokine, antimicrobial (e.g., antibacterial, antibiotic, antiviral, and / or antifungal), hormone (e.g., insulin), ephrins, hemostatic agent (e.g., blood clotting agent, factor, or protein), pain medications (e.g., anesthetics), etc.). In a particular embodiment, the agent enhances tissue regeneration, tissue growth, and wound healing (e.g., growth factors). In a particular embodiment, the agent treats / prevents infections (e.g., antimicrobials such as antibacterials, antivirals and / or antifungals). In a particular embodiment, the agent is an antimicrobial, particularly an antibacterial. In a particular embodiment, the agent enhances wound healing and / or enhances tissue regeneration (e.g., bone, tendon, cartilage, skin, nerve, and / or blood vessel). Such agents include, for example, growth factors, cytokines, chemokines, immunomodulating compounds, and small molecules. Growth factors include, without limitation: platelet derived growth factors (PDGF), vascular endothelial growth factors (VEGF), epidermal growth factors (EGF), neuregulins, fibroblast growth factors (FGF; e.g., basic fibroblast growth factor (bFGF)), insulin-like growth factors (IGF-1 and / or IGF-2), bone morphogenetic proteins (e.g., BMP -2, BMP-7, BMP-12, BMP-9; particularly BMP -2 fragments, peptides, and / or analogs thereof), transforming growth factors (e.g., TGFa, TGFP, TGFP3), tumour Necrosis Factor alpha (TNF alpha), nerve growth factors (NGF), neurotrophic factors, stromal derived factor-1 (SDF-1), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), granulocyte-colony stimulating factor (G-CSF), erythropoietin (EPO), glial cell -derived neurotrophic factors (GDNF), hepatocyte growth factors (HGF), keratinocyte growth factors (KGF), neurotrophin, and / or growth factor mimicking peptides (e.g., VEGF mimicking peptides). In a particular embodiment, the growth factor is bFGF. Chemokines include, without limitation: CCL21, CCL22, CCL2, CCL3, CCL5, CCL7, CCL8, CCL13, CCL17, CXCL9, CXCL10, and CXCL11. Cytokines include without limitation IL-2 subfamily cytokines, interferon subfamily cytokines, IL- 10 subfamily cytokines, IL-1, 1- 18, IL-17, tumor necrosis factor, and transforming-growth factor beta superfamily cytokines. Examples of small molecule drugs / therapeutic agents include, without limitation, simvastatin, kartogenin, retinoic acid, paclitaxel, vitamins (e.g., vitamin D3), etc. In a particular embodiment, the agent is a blood clotting factor such as thrombin or fibrinogen. In a particular embodiment, the agent is a bone morphogenetic protein (e.g., BMP -2, BMP-7, BMP- 12, BMP-9; particularly human; particularly BMP -2 fragments, peptides, and / or analogs thereof). In a particular embodiment, the agent is a BMP -2 fragment (e.g., up to about 25, about 30, about 35, about 40, about 45, about 50 amino acids, or more of BMP -2) comprising the knuckle epitope (e.g., amino acids 73-92 of BMP -2). In a particular embodiment, the BMP-2 peptide is linked to a peptide of acidic amino acids (e.g., Asp and / or Glu; particularly about 3-10 or 5-10 amino acids such as E7, E8, D7, D8) and / or bisphosphonate (e.g., at the N-terminus).

[0069] Antimicrobials may include, without limitation, small molecules, peptides, proteins, DNA, RNA, and other known biologic substances. In a particular embodiment, the antimicrobial is a small molecule. In a particular embodiment, the antimicrobial is an antiviral, antifungal, antibiotic or antibacterial, particularly an antibiotic or antibacterial. Examples of antimicrobials include, without limitation, antibiotics such as beta-lactams (e.g., penicillin, ampicillin, oxacillin, cioxacillin, methicillin, cephalosporin, etc.), monobactams (e.g., aztreonam, tigemonam, nocardicin A, tabtoxin, etc.), carbapenems (e.g., imipenem, meropenem, ertapenem, doripenem, etc.), cephalosporins (e.g., cefdinir, cefaclor, cephalexin, cefixime, cefepime, etc.), carbacephems, cephamycins, macrolides (e.g., erythromycin, clarithromycin, azithromycin etc.), quinolones or fluoroquinolones (e.g., ciprofloxacin, levofloxacin, ofloxacin, delafloxacin, etc.), tetracyclines (e.g., tetracycline, doxycycline etc.), sulfonamides (e.g., sulfamethoxazole, sulfafuraxole, etc.), aminoglycosides (e.g., gentamicin, neomycin, tobramycin, kanamycin, etc.), oxazolidinones (e.g., linezolid, posizolid, tedizolid, radezolid, contezolid, etc.), lipopeptides (e.g., daptomycin), glycylcyclines (e.g., tigecycline), moenomycins, aminocoumarins (e.g., novobiocin), co-trimoxazoles (e.g., trimethoprim and sulfamethoxazole), lincosamides (e.g., clindamycin and lincomycin), polypeptides (e.g., colistin), and glycopeptides (e.g., vancomycin); silver containing compounds (e.g., silver ions, silver nitrate, silver nanoparticles, colloidal silver, etc.), gallium containing compounds (e.g., gallium ions, gallium nitrate, gallium nanoparticles, colloidal gallium, etc.), and antimicrobial peptides. Examples of antifungals include, without limitation, amphotericin B, pyrimethamine, thiazoles, allylamines, flucytosine, caspofungin acetate, fluconazole, griseofulvin, terbinafine, amorolfme, imidazoles, triazoles (e.g., voriconazole), flutrimazole, cilofungin, echinocandines, pneumocandin omoconazole terconazole, nystatin, natamycin, griseofulvin, ciclopirox, naftifine, and itraconazole. In a particular embodiment, the antimicrobial is an antibiotic. In a particular embodiment, the antimicrobial is an antimicrobial peptide. In a particular embodiment, the scaffold comprises an antimicrobial peptide and at least one other antimicrobial (e.g., antibiotic). Antimicrobial peptides may be therapeutically effective against one or more bacteria. Examples of antimicrobial peptides are provided in the Antimicrobial Peptide Database (aps.unmc.edu / AP / main.php). Examples of antimicrobial peptides are also disclosed in U.S. Patent No. 7,465,784, U.S. Patent No. 9,580,472, U.S. Patent No. 10,144,767, U.S. Patent Application Publication No. 20090156499, U.S. Patent Application Publication No. 20150259382, U.S. Patent Application Publication No. 20140303069, and PCT / US2019 / 039792, each incorporated by reference herein. In a particular embodiment, the antimicrobial peptide has fewer than about 50 amino acids, fewer than about 25 amino acids, fewer than about 20 amino acids, fewer than about 17 amino acids, fewer than about 15 amino acids, fewer than 12 amino acids, fewer than 10 amino acids, or fewer than 9 amino acids. In a particular embodiment, the antimicrobial peptide has more than about 6 amino acids, particularly more than about 7 amino acids.

[0070] The nanofiber-microsphere composite scaffolds synthesized by the methods of the instant invention are also encompassed herein. Compositions comprising the nanofiber-microsphere composite scaffolds described herein and a carrier (e.g., a pharmaceutically acceptable carrier) are also encompassed herein.

[0071] The nanofiber-microsphere composite scaffolds of the instant invention can be used to create tissue architectures for a variety of application including, without limitation: wound healing, tissue engineering, tissue growth, tissue repair, tissue regeneration, and engineering 3D in vitro tissue models. Tissues include, without limitation: lung, liver, kidney, and heart tissues. Some examples of uses for the three- dimensional scaffolds of the instant invention include, but are not limited to: use as tissue structures (in vitro or in vivo), hemostatic bandages, tissue repair structures, and tissue regeneration structures.

[0072] The nanofiber-microsphere composite scaffolds can also be combined with a variety of hydrogels or biological matrices / cues to form 3D hybrid structures that can release biologically functional agents. The tissue constructs can be used for regeneration of many tissue defects (e.g., skin, bone, cartilage) and healing of various wounds (e.g., injuries, diabetic wounds, venous ulcer, pressure ulcer, bums). The nanofiber- microsphere composite scaffolds may be used ex vivo to generate tissue or tissue constructs / models. The nanofiber-microsphere composite scaffolds may also be used in vivo in patients (e.g., human or animal) for the treatment of various diseases, disorders, and wounds. In a particular embodiment, the nanofiber-microsphere composite scaffold stimulates the growth of existing tissue and / or repair of a wound or defect when applied in vivo. The nanofiber-microsphere composite scaffolds can be used for engineering, growing, and / or regenerating a variety of tissues including but not limited to skin, bone, cartilage, muscle, nervous tissue, and organs (or portions thereof).

[0073] In accordance with the instant invention, the nanofiber-microsphere composite scaffolds may be used in inducing and / or improving / enhancing wound healing and inducing and / or improving / enhancing tissue regeneration. The nanofiber-microsphere composite scaffolds of the present invention can be used for the treatment, inhibition, and / or prevention of any injury or wound. In a particular embodiment, the method comprises administering a nanofiber-microsphere composite scaffold, optionally comprising an agent, as described herein. The nanofiber-microsphere composite scaffolds of the instant invention can be loaded with different agents as necessary for regeneration of various tissues. In a particular embodiment, the nanofiber-microsphere composite scaffold comprises blood clotting factors (e.g., for accelerating blood clot formation and / or preventing blood loss). For example, the nanofiber-microsphere composite scaffold can be used to induce, improve, or enhance wound healing associated with surgery (including non-elective (e.g., emergency) surgical procedures or elective surgical procedures). Elective surgical procedures include, without limitation: liver resection, partial nephrectomy, cholecystectomy, vascular suture line reinforcement and neurosurgical procedures. Non-elective surgical procedures include, without limitation: severe epistaxis, splenic injury, liver fracture, cavitary wounds, minor cuts, punctures, gunshot wounds, and shrapnel wounds. The nanofiber-microsphere composite scaffold of the present invention can also be incorporated into delivery devices that allow for their injection / delivery directly into a desired location (e.g., a wound). The nanofiber- microsphere composite scaffold also may be delivered directly into a cavity (such as the peritoneal cavity) (e.g., using a pressurized cannula).

[0074] In accordance with the instant invention, the nanofiber-microsphere composite scaffolds of the present invention can be used in medicine. The nanofiber-microsphere composite scaffolds of the present invention can be used for a number of purposes including, but not limited to, cell expansion (e.g., in vitro, such as in bioreactors), 3D cell culture, tissue scaffold, tissue modeling, wound healing, and tissue regeneration. The nanofiber-microsphere composite scaffolds of the instant invention may also be used as grafts. The nanofiber-microsphere composite scaffolds of the instant invention may also be used for wound healing, myocardial infarction repair, spinal cord injury repair, and whole organ development and repair. In certain embodiments, the nanofiber- microsphere composite scaffolds of the present invention can be used to treat and / or prevent a variety of diseases and disorders. Examples of diseases and / or disorders include but are not limited to wounds, ulcers, infections, hemorrhage, tissue injury, tissue defects, tissue damage, bone fractures, bone degeneration, cartilage damage, cancer (e.g., the use of docetaxel and curcumin for the treatment of colorectal cancer (Fan, et al., Sci. Rep. (2016) 6:28373)), neurologic diseases (e.g., Alzheimer’s and Parkinson’s), ischemic diseases, inflammatory diseases and disorders, heart disease, myocardial infarction, and stroke. Methods for inducing and / or improving / enhancing wound healing in a subject are also encompassed by the instant invention. Methods of inducing and / or improving / enhancing tissue regeneration (e.g., blood vessel growth, neural tissue regeneration, and bone and / or cartilage regeneration) in a subject are also encompassed by the instant invention. Methods of inducing and / or improving / enhancing hemostasis in a subject are also encompassed by the instant invention. The methods of the instant invention comprise administering or applying nanofiber-microsphere composite scaffolds of the instant invention to the subject (e.g., at or in a wound). In a particular embodiment, the method comprises administering nanofiber-microsphere composite scaffolds comprising an agent and / or cell as described herein. The nanofiber- microsphere composite scaffolds of the instant invention can be loaded with agents as necessary for regeneration of various tissues. In a particular embodiment, the nanofiber- microsphere composite scaffolds comprise blood clotting factors (e.g., for accelerating blood clot formation and / or preventing blood loss). In a particular embodiment, the method comprises administering nanofiber-microsphere composite scaffolds to the subject and an agent as described herein (i.e., the agent is not contained within the nanofiber-microsphere composite scaffold). When administered separately, the nanofiber-microsphere composite scaffold may be administered simultaneously and / or sequentially with the agent. The methods may comprise the administration of one or more nanofiber-microsphere composite scaffold. When more than one nanofiber- microsphere composite scaffold is administered, the nanofiber-microsphere composite scaffolds may be administered simultaneously and / or sequentially.

[0075] The nanofiber-microsphere composite scaffolds of the present invention may also have various pore sizes (e.g., by altering length of time of expansion and / or the number of times expanding the nanofiber mat and / or the amount or concentration of expandable microspheres and / or using expandable microspheres which expand larger or smaller compared to other expandable microspheres). Different pore sizes may be beneficial depending on the type of tissue desired for repair or regeneration.

[0076] The nanofiber-microsphere composite scaffolds can also be used to expand and increase cell numbers (e.g., stem cell numbers) in culture. In a particular embodiment, microtissues can be grown in situ by prolonged culture of cell laden nanofiber- microsphere composite scaffolds (e.g., in confined microfluidic channel devices). These microtissues are injectable or transplantable into a tissue defect to promote wound healing in a subject (e.g., the nanofiber-microsphere composite scaffolds comprise autologous cells).

[0077] The nanofiber-microsphere composite scaffolds may also be employed for cell detection, separation, and / or isolation of cell populations in a mixture. For example, structures conjugated to specific antibodies can be used for the isolation, separation, and / or expansion of different cell types from their mixtures (Custodio, et al., Biomaterials (2015) 43:23-31). Further, nanofiber-microsphere composite scaffolds can be used for the in vitro adhesion, proliferation, and / or maturation of chondrocytes as well as in vivo cartilage formation and osteochondral repair induced by nanofiber- microsphere composite scaffolds when together with chondrocytes (Liu, et al., Nat. Mater. (2011) 10:398-406).

[0078] The nanofiber-microsphere composite scaffolds of the present invention may be administered by any method. The nanofiber-microsphere composite scaffolds described herein may be administered to a subject or a patient as a pharmaceutical composition. The compositions of the instant invention comprise a nanofiber-microsphere composite scaffold and a pharmaceutically acceptable carrier. The term “patient” as used herein refers to human or animal subjects. These compositions may be employed therapeutically, under the guidance of a physician.

[0079] The compositions of the instant invention may be conveniently formulated for administration with any pharmaceutically acceptable carrier(s). For example, the agents may be formulated with an acceptable medium such as water, buffered saline, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like), dimethyl sulfoxide (DMSO), oils, detergents, suspending agents or suitable mixtures thereof. Except insofar as any conventional media or agent is incompatible with the agents to be administered, its use in the pharmaceutical preparation is contemplated.

[0080] Compositions of the instant invention may be administered by any method. For example, the compositions of the instant invention can be administered, without limitation, parenterally, subcutaneously, orally, topically (ex. using a cream or spray), pulmonarily, rectally, vaginally, intravenously, intraperitoneally, intrathecally, intracerbrally, epidurally, intramuscularly, intradermally, intratumoral, intracarotidly, or by direct injection (e.g., a localized injection into a specific tissue or organ). Selection of a suitable pharmaceutical preparation will also depend upon the mode of administration chosen. For example, the compositions of the invention may be administered parenterally. In this instance, a pharmaceutical preparation comprises the nanofiber- microsphere composite scaffolds dispersed in a medium that is compatible with the parenteral injection.

[0081] Pharmaceutical compositions containing an agent of the present invention as the active ingredient in intimate admixture with a pharmaceutically acceptable carrier can be prepared according to conventional pharmaceutical compounding techniques.

[0082] In a particular embodiment of the instant invention, methods for modulating (increasing) hemostasis; inhibiting blood or cartilage loss; and / or treating hemorrhage are provided. In a particular embodiment, the method comprises administering the nanofiber-microsphere composite scaffolds to the wound or site of bleeding. In a particular embodiment, the nanofiber-microsphere composite scaffolds comprise a blood clotting factor such as thrombin and / or fibrinogen.

[0083] In a particular embodiment of the instant invention, methods for stimulating bone and / or cartilage regeneration and / or treating bone and / or cartilage loss are provided. In a particular embodiment, the method comprises administering the nanofiber-microsphere composite scaffolds to the site of bone and / or cartilage loss. In a particular embodiment, the site of bone and / or cartilage loss is periodontal. In a particular embodiment, the nanofiber-microsphere composite scaffolds are mineralized. In a particular embodiment, the nanofiber-microsphere composite scaffolds comprise a bone growth stimulating growth factor such as a bone morphogenic protein or fragment or analog thereof. In a particular embodiment, the agent is a bone morphogenetic protein (e.g., BMP-2, BMP-7, BMP-12, BMP-9; particularly human; particularly BMP -2 fragments, peptides, and / or analogs thereof). In a particular embodiment, the agent is a BMP -2 fragment (e.g., up to about 25, about 30, about 35, about 40, about 45, about 50 amino acids, or more of BMP - 2) comprising the knuckle epitope (e.g., amino acids 73-92 of BMP -2). In a particular embodiment, the BMP-2 peptide is linked to a peptide of acidic amino acids (e.g., Asp and / or Glu; particularly about 3-10 or 5-10 amino acids such as E7, E8, D7, D8) and / or bisphosphonate (e.g., at the N-terminus).

[0084] In accordance with the instant invention, antimicrobial (e.g., antibiotic)-loaded nanofiber-microsphere composite scaffolds are provided. In a particular embodiment, the antimicrobial (e.g., antibiotic)-loaded nanofiber-microsphere composite scaffold is in the form of a wound dressing. The antimicrobial (e.g., antibiotic)-loaded nanofiber- microsphere composite scaffolds may be in any form including, without limitation, a wound dressing, bandage, gauze, covering, suture, thread, ligature, hemostasis material, or coating for biomedical device or implant. In a particular embodiment, the antimicrobial (e.g., antibiotic)-loaded nanofiber-microsphere composite scaffold is in a wound dressing.

[0085] The nanofiber-microsphere composite scaffolds of the instant invention can also be used in non-medical methods and have industrial utility. In certain embodiments, the nanofiber-microsphere composite scaffolds can be used for various applications including, but not limited to: environmental, filtration, catalysis, energy, photonics, electronics, textile, wearables, agriculture, and biomedical engineering. In certain embodiments, the nanofiber-microsphere composite scaffold is used as insulation. In certain embodiments, the nanofiber-microsphere composite scaffold is used as a filter. In certain embodiments, the nanofiber-microsphere composite scaffold is used for filtration.

[0086] Definitions

[0087] The singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

[0088] As used herein, the term “electrospinning” refers to the production of fibers (i.e., electrospun fibers), particularly micro- or nano-sized fibers, from a solution or melt using interactions between fluid dynamics and charged surfaces (e.g., by streaming a solution or melt through an orifice in response to an electric field). Forms of electrospun nanofibers include, without limitation, branched nanofibers, tubes, ribbons and split nanofibers, nanofiber yams, surface-coated nanofibers (e.g., with carbon, metals, etc.), nanofibers produced in a vacuum, and the like. The production of electrospun fibers is described, for example, in Gibson et al. (1999) AlChE J., 45: 190-195.

[0089] “Pharmaceutically acceptable” indicates approval by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

[0090] A “carrier” refers to, for example, a diluent, adjuvant, preservative (e.g., Thimersol, benzyl alcohol), anti-oxidant (e.g., ascorbic acid, sodium metabisulfite), solubilizer (e.g., polysorbate 80), emulsifier, buffer (e.g., TrisHCl, acetate, phosphate), water, aqueous solutions, oils, bulking substance (e.g., lactose, mannitol), excipient, auxiliary agent or vehicle with which an active agent of the present invention is administered. Suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E.W. Martin (Mack Publishing Co., Easton, PA); Gennaro, A. R., Remington: The Science and Practice of Pharmacy, (Lippincott, Williams and Wilkins); Liberman, et al., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y.; and Kibbe, et al., Eds., Handbook of Pharmaceutical Excipients (3rd Ed.), American Pharmaceutical Association, Washington.

[0091] As used herein, the term “polymer” denotes molecules formed from the chemical union of two or more repeating units or monomers. The term “block copolymer” most simply refers to conjugates of at least two different polymer segments, wherein each polymer segment comprises two or more adjacent units of the same kind. “Hydrophobic” designates a preference for apolar environments (e.g., a hydrophobic substance or moiety is more readily dissolved in or wetted by non-polar solvents, such as hydrocarbons, than by water). In certain embodiments, hydrophobic polymers may have aqueous solubility less than about 1% wt. at 37°C. In certain embodiments, polymers that at 1% solution in bi-distilled water have a cloud point below about 37°C, particularly below about 34°C, may be considered hydrophobic.

[0092] As used herein, the term “hydrophilic” means the ability to dissolve in water. In a particular embodiment, polymers that at 1% solution in bi-distilled water have a cloud point above about 37°C, particularly above about 40°C, may be considered hydrophilic.

[0093] As used herein, the term “amphiphilic” means the ability to dissolve in both water and lipids / apolar environments. Typically, an amphiphilic compound comprises a hydrophilic portion and a hydrophobic portion.

[0094] The term “antimicrobials” as used herein indicates a substance that kills or inhibits the growth of microorganisms such as bacteria, fungi, viruses, or protozoans.

[0095] As used herein, the term “antiviral” refers to a substance that destroys a virus and / or suppresses replication (reproduction) of the virus. For example, an antiviral may inhibit and or prevent: production of viral particles, maturation of viral particles, viral attachment, viral uptake into cells, viral assembly, viral release / budding, viral integration, etc.

[0096] As used herein, the term “antibiotic” refers to antibacterial agents for use in mammalian, particularly human, therapy. Antibiotics include, without limitation, betalactams (e.g., penicillin, ampicillin, oxacillin, cioxacillin, methicillin, and cephalosporin), carbacephems, cephamycins, carbapenems, monobactams, aminoglycosides (e.g., gentamycin, tobramycin), glycopeptides (e.g., vancomycin), quinolones (e.g., ciprofloxacin), moenomycin, tetracyclines, macrolides (e.g., erythromycin), fluoroquinolones, oxazolidinones (e.g., linezolid), lipopetides (e.g., daptomycin), aminocoumarin (e.g., novobiocin), co-trimoxazole (e.g., trimethoprim and sulfamethoxazole), lincosamides (e.g., clindamycin and lincomycin), polypeptides (e.g., colistin), and derivatives thereof.

[0097] As used herein, an “anti-inflammatory agent” refers to compounds for the treatment or inhibition of inflammation. Anti-inflammatory agents include, without limitation, non-steroidal anti-inflammatory drugs (NSAIDs; e.g., aspirin, ibuprofen, naproxen, methyl salicylate, diflunisal, indomethacin, sulindac, diclofenac, ketoprofen, ketorolac, carprofen, fenoprofen, mefenamic acid, piroxicam, meloxicam, methotrexate, celecoxib, valdecoxib, parecoxib, etoricoxib, and nimesulide), corticosteroids (e.g., prednisone, betamethasone, budesonide, cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, tramcinolone, and fluticasone), rapamycin, acetaminophen, glucocorticoids, steroids, beta-agonists, anticholinergic agents, methyl xanthines, gold injections (e.g., sodium aurothiomalate), sulphasal azine, and dapsone.

[0098] As used herein, the term “analgesic” refers to an agent that lessens, alleviates, reduces, relieves, or extinguishes pain in an area of a subject's body (i.e., an analgesic has the ability to reduce or eliminate pain and / or the perception of pain).

[0099] As used herein, the term “small molecule” refers to a substance or compound that has a relatively low molecular weight (e.g., less than 2,000). Typically, small molecules are organic, but are not proteins, polypeptides, or nucleic acids.

[0100] As used herein, the term “subject” refers to an animal, particularly a mammal, particularly a human.

[0101] As used herein, the term “prevent” refers to the prophylactic treatment of a subject who is at risk of developing a condition resulting in a decrease in the probability that the subject will develop the condition.

[0102] The term “treat” as used herein refers to any type of treatment that imparts a benefit to a patient afflicted with a disease, including improvement in the condition of the patient (e.g., in one or more symptoms), delay in the progression of the condition, etc.

[0103] The term “hydrogel” refers to a water-swellable, insoluble polymeric matrix (e.g., hydrophilic polymers) comprising a network of macromolecules, optionally crosslinked, that can absorb water to form a gel.

[0104] As used herein, a linker is generally a chemical moiety comprising a covalent bond or a chain of atoms that covalently attaches two compounds. The linker can be linked to any synthetically feasible position of the two compounds. Exemplary linkers may comprise at least one optionally substituted; saturated or unsaturated; linear, branched or cyclic aliphatic group, an alkyl group, or an optionally substituted aryl group. The linker may be a lower alkyl or aliphatic. The linker may also be a polypeptide (e.g., from about 1 to about 10 amino acids, particularly about 1 to about 5). The linker may be non-degradable and may be a covalent bond or any other chemical structure which cannot be substantially cleaved or cleaved at all under physiological environments or conditions. The term “crosslink” refers to a bond or chain of atoms attached between and linking two different molecules (e.g., polymer chains). The term “crosslinker” refers to a molecule capable of forming a covalent linkage between compounds. A “photocrosslinker” refers to a molecule capable of forming a covalent linkage between compounds after photoinduction (e.g., exposure to electromagnetic radiation in the visible and near-visible range). Crosslinkers are well known in the art (e.g., formaldehyde, paraformaldehyde, acetaldehyde, glutaraldehyde, etc.). The crosslinker may be a bifunctional, tri functional, or multifunctional crosslinking reagent.

[0105] The following example illustrates certain embodiments of the invention. It is not intended to limit the invention in any way.

[0106] EXAMPLE

[0107] Electrospun nanofibers have proven versatile across numerous fields, including environmental, energy, and biomedical applications. Typically, however, electrospun nanofiber materials are fabricated as two-dimensional sheets, membranes, or mats. Here, a straightforward and adaptable foaming method is presented to create three-dimensional microsphere-nanofiber composite structures. This approach involves incorporating expandable microspheres within the nanofiber mats during electrospinning, followed by thermal treatment to achieve the 3D morphology. The expansion ratio and compressive strength increase with higher concentrations of expandable microspheres. In addition, the compressive strength of the 3D composite structures significantly surpasses that of 3D nanofiber scaffolds expanded with subcritical CO2 fluids. This approach presents a pathway for fabricating 3D microsphere-nanofiber composite scaffolds with broad applications.

[0108] Materials and Methods

[0109] Materials

[0110] PVP, CA, and PU were obtained from Sigma-Aldrich (St. Louis, MO). Ethanol, DCM, DMF, and acetone were purchased from Thermo Fisher Scientific (Waltham, MA). Expancel® microspheres were obtained from Nouryon (043-DU-80, Amsterdam, Netherlands), which can be expanded from 94 to 164°C. Preparation of Expandable Microspheres Incorporated Electrospun PVP Nanofiber Mats

[0111] To prepare a 10% PVP solution, 1 g of PVP (Mw -1,3000000 kg mol'1) was dissolved in absolute ethanol. Next, varying amounts of unexpanded Expancel® microspheres (043 -DU-80) were added to the solution to achieve final Expancel® concentrations of 1%, 2%, and 3% before electrospinning. The dispersion of Expancel® in the polymer solution required vigorous shaking using a vortex mixture. The polymer solutions were electrospun at a flow rate of 1 mL h'1under an applied voltage of 10- 12 kV. The distance between the nozzle and collector was set at 10-15 cm, and fibers were collected on a rotating mandrel at -100 rpm. The expandable microspheres tended to settle down during the prolonged electrospinning process. To prevent this, a magnetic stirrer bar was placed inside the syringe and kept stirring continuously throughout the spinning process. For comparison, PVP nanofiber mats without Expancel® microspheres were prepared separately and used to evaluate mechanical properties postexpansion.

[0112] Preparation of Expandable Microspheres Incorporated Electrospun Cellulose Acetate (CA) and Polyurethane (PU) Nanofiber Mats

[0113] Since Expancel® microspheres were unstable in organic solvents such as dichloromethane (DCM), dimethylformamide (DMF), and acetone, a dual electrospinning approach was used. In this setup, CA or PU nanofiber mats and PVP nanofiber mats containing Expancel® microspheres were simultaneously electrospun and deposited on the same rotating mandrel. The electrospinning parameters for PVP containing Expancel® were the same as abovementioned. For CA nanofiber mats, a 20% CA solution was prepared by dissolving CA powder (average Mn -30000) in a 2: 1 (v / v) mixture of acetone:DMF. The solution was electrospun at a flow rate of 1 mL h'1with an applied voltage of 14 kV, and a nozzle-to-collector distance of -10-12 cm. For PU nanofiber mats, PU pellets (Selectophore™) were dissolved in a 1 :2 (v / v) mixture of DCM and DMF to form a 10% PU solution. This solution was electrospun at a flow rate of 1 mL h'1with an accelerating voltage of 12 kV, and a nozzle-to-collector distance of 10-12 cm.

[0114] Thermally-Induced Expansion of Expancel® Microspheres and Electrospun Fiber Mats with Expancel® Microspheres To perform thermally induced expansion, Expancel® microspheres and electrospun fiber mats containing Expancel® microspheres were incubated in an oven at 110°C for 1 minute. The expansion ratio of the fiber mats was then calculated using the following equation.

[0115] Expansion ratio = (Thickness of fiber mat after expansion) / (Thickness of fiber mat before expansion) / ] 00%

[0116] Compressive Testing for 3D Gas-foamed PVP Nanofiber Scaffolds and Microsphere-Nanofiber Composite Scaffolds

[0117] 3D gas-foamed PVP nanofiber scaffolds were produced by rapidly depressurizing PVP nanofiber mats in subcritical CO2 fluid, following an established protocol (Jiang, et al. (2018) Acta. Biomater., 68:237). For a mechanical test, either a 3D gas-foamed PVP nanofiber scaffold or a microsphere-nanofiber composite scaffold, both shaped into 1 cm3cubes, was placed on a compression plate attached to a load cell with a 10 N measuring capability (Cellscale Univert, Waterloo, Canada). The compression plate applied pressure to achieve 50% displacement over 10 seconds. Each material was tested in quadruplicate to ensure accuracy.

[0118] Statistical Analysis

[0119] All data were expressed as mean ± standard deviation. For the measurement of the expansion ratio and the compressive strength between thermally expanded PVP scaffolds, a minimum of 4 expanded samples for each group was employed. For pairwise comparison, one-way ANOVA with the Tukey test was performed, and significant values were presented as **p < 0.01, ****p < 0.0001, and ns (no significant, p > 0.05). All figures are original, and schematics were created using BioRender.

[0120] Results

[0121] Thermally Induced Expansion of Expancel® Microspheres

[0122] To better understand the behavior and expansion profile of Expancel® microspheres, a small sample was thermally treated at 110°C for 1 minute and subsequently characterized via scanning electron microscopy (SEM) imaging (Figure 2 A). Before expansion, the surface morphology of Expancel® microspheres showed a concave, wrinkle texture, likely due to the presence of blowing agents encapsulated within the core (Figure 2A). The microspheres exhibited a broad diameter range from 5 to 30 pm, with most measuring between 10 and 15 pm (Figure 2B). Upon healing, the thermoplastic shell softened, enabling rapid vaporization of the encapsulated blowing agent and producing a large volume of gas that expanded the microspheres uniformly. Post-expansion, SEM images reveal a smooth surface topography, with diameters ranging from 40 to 100 pm, and the majority between 60 and 70 pm (Figure 2A, 2C).

[0123] Fabrication and Characterization ofExpancel® Microspheres Incorporated PVP Nanofiber Mats

[0124] Various concentrations ofExpancel® microspheres (1%, 2%, and 3%) were then incorporated into electrospun PVP nanofiber mats. The resulting morphology and properties post-electrospinning were examined. Figure 3 A shows that, across all concentrations, the electrospun PVP fibers maintained a bead-free structure with fine diameters, indicating that the inclusion ofExpancel® microspheres in the PVP solution during electrospinning did not disrupt fiber formation. The cross-sectional view of the electrospun mats revealed a dense fiber structure throughout. Notably, the embedded Expancel® microspheres retained a morphology similar to their unexpanded state, as observed in Figure 2A. This similarity indicates that the Expancel® microspheres were unaffected by the ethanol solvent and withstood the high electric field during electrospinning.

[0125] Thermally Induced Expansion ofExpancel® Microspheres Incorporated PVP Nanofiber Mats

[0126] To further examine the expansion capability of the incorporated Expancel® microspheres, the prepared PVP fiber mats were cut into small sections (1 cmx 1 cm) and subjected them to thermal treatment at 110°C for Iminute. No significant change was observed for PVP nanofiber mats after thermal treatment for 1 minute (Figure 3B). As shown in Figure 4, the microspheres retained their expansion potential, which led to a rapid increase in the thickness of the initially 2D fiber mats, transforming them into 3D composite scaffolds (Figure 4A). Cross-sectional SEM images (Figure 4B) of the expanded 3D scaffolds reveal expanded microspheres embedded within the nanofiber matrix, with a higher concentration of microspheres visible in the cross-sections as loading increased. The expansion ratio of the 2D fiber mats correlated positively with the concentration ofExpancel® microspheres (Figure 4C). Mats loaded with 1% Expancel® microspheres expanded by nearly four-fold, while those with 2% and 3% Expancel® microspheres expanded approximately six- and eight-fold, respectively. This embedding of Expancel® microspheres enabled substantial expansion and likely reinforced the mechanical stability of the resulting 3D structures. To validate this mechanical enhancement, the compressive strength of these 3D expanded PVP microsphere-nanofiber composite scaffolds were compared with that of a control sample: pure PVP nanofiber mats expanded into 3D structures using subcritical CO2 gas-foaming (Jiang, et al. (2018) Acta. Biomater., 68:237). Figure 4D shows the compressive strengths of various samples. The 3D gas-foamed PVP fiber scaffold had the lowest compressive strength, ~2 kPa. In contrast, the 3D PVP microsphere-nanofiber composite scaffolds demonstrated a significant increase in compressive strength due to the expanded microspheres embedded within the structure. Specifically, the 3D scaffold with 1% Expancel® achieved a compressive strength of ~50 kPa, a 25-fold improvement over the gas-foamed scaffold, while scaffolds with 2% and 3% Expancel® reached compressive strengths of ~88 and 102 kPa, respectively.

[0127] Thermally Induced Expansion of Expancel® Microspheres Incorporated CA and PU Nanofiber Mats

[0128] To explore the feasibility of this expansion approach for other fiber mats, cellulose acetate (CA), a naturally derived polymer, and polyurethane (PU), a synthetic polymer, both widely used in biomedical applications, were chosen to fabricate expandable 3D microsphere-nanofiber composite scaffolds (Bifari, et al. (2016) Curr. Pharm. Res., 22:3007; Azarmgin, et al. (2024) ACS Biomater. Sci. Eng., 10:6828). Unlike PVP, which can be dissolved in ethanol, the preparation of CA and PU polymer solutions requires organic solvents such as dichloromethane (DCM), dimethylformamide (DMF), or acetone. These solvents, however, can dissolve the thermoplastic shell of Expancel® microspheres. To overcome this, dual electrospinning of a CA solution (or a PU solution) alongside a PVP solution containing Expancel® microspheres was used (Baker, et al. (2012) Proc. Natl. Acad. Sci., 109: 14176). As expected, both electrospun CA and PU nanofiber mats exhibited the same expansion behavior upon thermal treatment, transforming into 3D structures through the expansion of incorporated Expancel® microspheres at 110°C for 1 minute (Figure 5 A, 5B). SEM images show top and cross-sectional views of the 3D CA / PVP microsphere-nanofiber nanofiber mats, both before and after thermal treatment (Figure 5 A). Similarly, Figure 5B presents top and cross-sectional views of 3D PU / PVP microsphere-nanofiber mats, highlighting unexpanded and expanded Expancel® microspheres embedded within the nanofiber matrix.

[0129] Herein, Expancel® microspheres were used as a foaming agent to prepare 3D microsphere-nanofiber composite scaffolds. The incorporation of these expandable microspheres into nanofiber mats was demonstrated through direct electrospinning or dual electrospinning. Additionally, microspheres can be introduced into the mats via electrospraying or gas-spraying deposition alongside electrospinning (Park, et al. (2023) Nat. Commun., 14:4896; Henao, et al. (2017) Acta Mater., 125:327). Expancel microspheres require relatively high temperatures (94-164°C) to expand, which exceed the glass transition temperatures of many polymers commonly used in the biomedical field such as poly(e-caprolactone), poly(lactide-co-glycolide), and poly(lactic acid) (Fernandez-Tena, et al. (2023) Macromolecules 56:4602; Baker, et al. (2008) Polym. Rev., 48:64; Park, et al. (2020) Mol. Pharmaceutics 18: 18). To address this, microspheres with custom size and composition could be developed to expand at lower temperatures using fabrication methods like solvent evaporation, coaxial-electrospray, or microfluidics (Galogahi, et al. (2020) J. Sci. Adv. Mater. Dev., 5:417; U.S. Patent Application Publication No. 2022 / 0363859). While this study produced fiber mats with random nanofiber orientations, mats with aligned fibers could be generated by increasing the mandrel’s rotation speed (Robinson, et al. (2021) Matter 4:821). This adjustment could extend the approach to create 3D microsphere-nanofiber composite scaffolds with controlled fiber alignment and tailored compositions. Furthermore, by utilizing principles of solids-of-revolution and confined expansion, various 3D composite shapes - including spheres, cones, cylinders, hollow spheres, and hollow cylinders - could be fabricated, broadening the scope of possible scaffold geometries (Chen, et al. (2019) Nano Lett., 19:2059; Chen, et al. (2020) Appl. Phys. Rev., 7:021406).

[0130] A new foaming approach has been demonstrated for converting 2D nanofiber mats into 3D microsphere-nanofiber composite scaffolds. By adjusting the loading of expandable microspheres, the expansion ratio can be precisely controlled, with higher loadings yielding greater expansion. Additionally, the expanded nanofiber scaffolds containing expandable microspheres exhibited significantly higher compressive strength compared to gas-foamed 3D nanofiber scaffolds, with mechanical strength positively correlated with the microsphere concentration. This approach may allow for the fabrication of microsphere-nanofiber composites with tailored composition and fiber alignment, unlocking potential applications of electrospun nanofiber materials across environmental, energy, and biomedical fields. While certain of the preferred embodiments of the present invention have been described and specifically exemplified above, it is not intended that the invention be limited to such embodiments. Various modifications may be made thereto without departing from the scope and spirit of the present invention, as set forth in the following claims.

Claims

What is claimed is:

1. A method of synthesizing a nanofiber-microsphere composite scaffold, said method comprising: a) electrospinning a nanofiber mat with embedded expandable microspheres, and b) thermally treating the electrospun mat from step a), wherein the thermal treatment causes expansion of the expandable microspheres, thereby resulting in the synthesis of said nanofiber-microsphere composite scaffold.

2. The method of claim 1, wherein the nanofiber comprises at least one polymer.

3. The method of claim 2, wherein said polymer comprises a synthetic polymer, naturally derived polymer, inorganic material, or a combination of thereof.

4. The method of claim 2, wherein said polymer comprises cellulose, cellulose acetate (CA), polyvinylpyrrolidone (PVP), polyurethane (PU), polycaprolactone (PCL), poly(lactide-co-epsilon-caprolactone) (PLCL), polyglycolic acid (PGA), polylactide (PLA), polydioxanone (PDO), poly(lactic-co-glycolic) acid (PLGA), or a combination thereof.

5. The method of claim 1, wherein the electrospun nanofibers of the nanofiber mat comprises uniformly aligned fibers or randomly aligned fibers.

6. The method of claim 1, wherein the electrospun nanofibers of the nanofiber mat are aligned radially or laterally.

7. The method of claim 1, wherein the glass transition temperature of the nanofibers of the nanofiber mat is greater than the temperature of the thermal treatment in step b).

8. The method of claim 1, wherein said nanofiber mat of step a) is fixed or fused on one side or a portion thereof prior to thermal treatment in step b).

9. The method of claim 1, wherein step a) comprises electrospinning a solution comprising a polymer and the expandable microspheres.

10. The method of claim 1, wherein step a) comprises electrospinning a first solution comprising a polymer and a second solution comprising the expandable microspheres.

11. The method of claim 1, wherein step a) comprises electrospinning a solution comprising a polymer and electrospraying or gas spraying the expandable microspheres onto the forming electrospun nanofiber mat.

12. The method of claim 1, wherein said expandable microspheres increase in volume by at least 5 times after the thermal treatment of step b).

13. The method of claim 1, wherein said thermal treatment of step b) is at least 80°C.

14. The method of claim 1, further comprising coating the nanofiber-microsphere composite scaffold with a hydrogel or gelatin.

15. The method of claim 1, further comprising adding cells and / or extracellular matrix to the nanofiber-microsphere composite scaffold.

16. The method of claim 1, further comprising adding at least one agent to the nanofiber-microsphere composite scaffold.

17. A nanofiber-microsphere composite scaffold synthesized by a method of any one of claims 1-16.

18. A nanofiber-microsphere composite scaffold comprising electrospun nanofibers and microspheres, wherein said microspheres are interwoven among the electrospun nanofibers.

19. A composition comprising the nanofiber-microsphere composite scaffold of claim 17 or claim 18 and a pharmaceutically acceptable carrier.

20. A method for treating and / or preventing a disease or disorder in a subject in need thereof, said method comprising administering to said subject the nanofiber-microsphere composite scaffold of claim 17 or claim 18.

21. The method of claim 20, wherein the disease or disorder is selected from the group consisting of wounds, ulcers, infections, hemorrhage, tissue injury, tissue defects, tissue damage, bone fractures, and bone degeneration.

22. The method of claim 20, wherein the administration is injection into the area in need of treatment.

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