Tissue engineering scaffolds and methods of use thereof

Abstract

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

Description

[0001] TISSUE ENGINEERING SCAFFOLDS AND METHODS OF USE THEREOF

[0002] Jingwei Xie Navatha Shree Sharma

[0003] This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 63 / 662,766, filed June 21, 2024. The foregoing application is incorporated by reference herein.

[0004] This invention was made with government support under Grant No. R01 DE031272 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 extracellular matrix (ECM), methods of synthesizing, and methods of use thereof.

[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] Injury, disease, and chronic disorders can disrupt the coordinated interplay between cells and their microenvironment, leading to harmful conditions such as tissue damage and organ dysfunction or loss. In such scenarios, the tissue damage cannot be fully recovered by the body’s innate regenerative capability and demands a rigorous attempt to restore the lost tissue functions (Eming, et al., Sci. Transl. Med. (2014) 6:265sr6; Lu, et al., J. Orthop. Res. (2005) 23: 1300-7). Despite many advances in grafting techniques, the limited availability of donor tissue for allografting, the high risk of donor site morbidity, and surgical complications make tissue regeneration an unmet clinical challenge. In the past decade, research has been focused on engineering biomaterials that can promote and improve the regenerative process of damaged tissue (Sharma, et al., Front. Mater. (2019) 6: 172; Eldeeb, et al., AAPS PharmSciTech (2022) 23:267). A well-designed biomaterial scaffold can mimic the actual tissue architecture by providing strength, viscoelastic, and anisotropic mechanical cues while withstanding mechanical stress at the targeted site to restore and repair malfunctioned tissues and organs. Advancement in nanotechnology and nanomaterials has led to the development of biomaterials at the nanoscale, which is instrumental in improving regenerative properties of biomaterial scaffold features such as topography and surface free energy (Cai, et al., Nanotechnol. Rev. (2020) 9:971-989; Liu, et al., Mil. Med. Res. (2023) 10: 16; Adhikari, et al., Biomater. Sci. (2023) 11 : 1236-1269). One widely implemented fabrication approach is electrospinning, which can generate fibrous scaffold at the nanometer scale. This approach allows the flexibility of using biodegradable polymers to fabricate scaffolds with precision, reproducibility, and scalability (Xie, et al., J. Mater. Sci. Technol. (2020) 59:243-261; Khorshidi, et al., J. Tissue Eng. Regen. Med. (2016) 10:715-38). Three-dimensional (3D) nanofiber scaffolds show efficient cell adhesion, proliferation, and guidance while retaining the functions of the cells (Chen, et al., Adv. Mater. (2020) 32:e2003754; Chen, et al., Acta Biomater. (2020) 108: 153-167; Owida, et al., Appl. Bionics Biomech. (2022) 2022: 1953861).

[0010] Tissues by nature are comprised of cells and the surrounding extracellular matrix (ECM). ECM is an intricate 3D network composed of structural proteins such as collagen, fibronectin, elastin, glycosaminoglycans (GAGs), and growth factors organized in a tissue-specific manner. It is important for fundamental cellular characteristics such as adhesion, proliferation, migration, polarity, differentiation, and cell death involved in the development, maintenance, and regeneration of tissues (Yue, J. Glaucoma (2014) 23:S20-S23). Hence, the complex nature of ECM in tissue is always a challenge to replicate. 3D electrospun biomaterial scaffolds have been developed to mimic the architecture of the tissue ECM for different tissue engineering applications (Xie, et al., J. Mater. Sci. Technol. (2020) 59:243-261; Owida, et al., Appl. Bionics Biomech. (2022) 2022: 1953861). However, there are challenges associated with the fabrication of biomaterials that can fully simulate ECM. Firstly, there are limited synthetic and natural polymer blends that can be used for fabrication (Su, et al., Small Sci. (2021) 1 :2100003; Nicolas, et al., Biomacromolecules (2020) 21 : 1968-1994). Second, they do not possess adequate functional groups to aid in cellular functions (Gao, et al., ACS Appl. Bio Mater. (2023) 6:2158-2171). Finally, even with modifications to the nanofiber surface with functional groups and peptides, they still fail to incorporate all the required signaling molecules and growth factors involved in tissue remodeling and maintenance. Therefore, improved ECM and 3D scaffolds are needed. SUMMARY OF THE INVENTION

[0011] In accordance with the instant invention, three-dimensional (3D) scaffolds comprising extracellular matrix (ECM) and methods of synthesizing the same are provided. In certain embodiments, the method of synthesis comprises culturing cells (e.g., fibroblasts) within a 3D scaffold (e.g., for sufficient time for the cells to produce extracellular matrix) and then decellularizing the 3D scaffold. In certain embodiments, the 3D scaffold comprises polymer nanofibers. In certain embodiments, the 3D scaffold comprising polymer nanofibers is an expanded electrospun nanofiber mat. In certain embodiments, the nanofibers comprise polycaprolactone (PCL), poly(lactide-co-epsilon- caprolactone) (PLCL), polyglycolic acid (PGA), poly(lactic-co-glycolic) acid (PLGA), or any combination thereof. In certain embodiments, the nanofibers further comprise at least one surfactant (e.g., a pol oxamer). In certain embodiments, the 3D scaffold further comprises a hydrogel coating (e.g., a gelatin coating). In certain embodiments, the nanofibers of the 3D scaffold are radially aligned. In certain embodiments, the nanofibers of the 3D scaffold are laterally aligned. In certain embodiments, the nanofibers of the 3D scaffold are randomly aligned. In certain embodiments, the method further comprises synthesizing the 3D scaffold prior to culturing with cells. In certain embodiments, the 3D scaffold is synthesized by expanding an electrospun nanofiber mat by exposing the electrospun nanofiber mat to gas bubbles (e.g., a gas foaming method). In certain embodiments, the 3D scaffold (e.g., nano- / microfiber hybrid aerogels) is synthesized by the freeze-casting method. In certain embodiments, the 3D scaffold is synthesized by 3D printing. In certain embodiments, the 3D scaffold is synthesized by melt electrospinning. In certain embodiments, the 3D scaffold is synthesized by wet electrospinning. In certain embodiments, the decellularization comprises exposing the 3D scaffold cultured with cells to an ionic detergent (e.g., sodium dodecyl sulfate) or freeze-thaw cycles. In certain embodiments, the decellularization comprises mechanical agitation and / or use of EDTA as chelator. In certain embodiments, the decellularization process further comprises contacting the 3D scaffold with a DNase. Compositions comprising a 3D scaffold of the instant invention and a pharmaceutically acceptable carrier are also provided.

[0012] Methods of using the 3D scaffold of the instant invention are also provided. Methods for treating and / or preventing a disease or disorder in a subject in need thereof are provided. Methods for inducing and / or improving wound healing in a subject in need thereof are also provided. Methods for tailoring the 3D scaffold to the requirements of tissue regeneration in a subject in need thereof are also provided. Methods for inducing and / or improving tissue regeneration in a subject in need thereof are also provided. Generally, the methods comprise administering or delivering a 3D scaffold of the instant to the subject, particularly by direct application or delivery to the site in need of treatment (e.g., a wound).

[0013] BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figures 1 A-1G show the development of decellularized ECM (dECM)-decorated 3D expanded nanofiber scaffolds. Fig. 1 A: Schematic illustrating study design. Top: Fabrication of radially aligned scaffolds (RAS) and laterally expanded scaffolds (LES) by transformation of 2D nanofiber mats into 3D expanded nanofiber scaffolds using a gas-foaming expansion technology. Middle: Cell seeding, growth, ECM deposition, and decellularization to form dECM-decorated RAS and LES. Bottom: In vitro and in vivo evaluation of dECM-decorated RAS and LES through recellularization and subcutaneous implantation. Fig. IB provides a graph of the cell viability in RAS and LES. Fig. 1C provides a graph of the DNA content (ng / scaffold) before and after decellularization by freeze-thawing and SDS methods along with posttreatment with DNAsel. Data is calculated as the mean of three independent experiments with standard error and the significance between groups (p-value) is calculated and is represented in the graph as ns (not significant), * (< 0.05), ** (< 0.01), and ***(< 0.001). Fig. ID provides photographs showing in the top panels from left to right: pristine RAS, RAS with cells and ECM deposition, RAS with cells and ECM deposition decellularized by SDS treatment, and RAS with cells and ECM deposition decellularized by freeze-thawing and in the bottom panels from left to right: pristine LES, LES with cells and ECM deposition, LES with cells and ECM deposition decellularized by SDS treatment, and LES with cells and ECM deposition decellularized by freeze-thawing. Fig. IE provides photographs of RAS (left) and LES (right). Fig. IF provides SEM images of LES (top row) and RAS (bottom row) at different magnifications. Fig. 1G provides a graph of DNA content (ng / scaffold) remaining in RAS and LES after decellularization (by freeze-thawing and SDS methods) before DNase 1 treatment. The cellularized scaffold was used as a control. Data is calculated as the mean of three independent experiments with standard error and the significance between groups is calculated. It is represented in the graph as not significant (ns), * (< 0.05), ** (< 0.01), *** (< 0.001), and **** (< 0.0001).

[0015] Figures 2A-2G show ECM deposition on LES and RAS scaffolds. Figures 2A- 2C provide SEM images at different magnifications showing LES with cells and ECM deposition, LES with cells and ECM deposition after freeze-thawing, and LES with cells and ECM deposition after SDS treatment, respectively. Figures 2D-2F provide SEM images at different magnifications showing RAS with cells and ECM deposition, RAS with cells and ECM deposition after freeze-thawing, and RAS with cells and ECM deposition after SDS treatment, respectively. The panels in the same column share the same scale bar. Fig. 2G provides images of H&E staining of cellularized LES, decellularized LES treated by freeze-thawing, decellularized LES treated with SDS, cellularized RAS, decellularized RAS treated by freeze-thawing, and decellularized RAS treated with SDS.

[0016] Figures 3 A-3F show the collagen 1 A deposition on LES and RAS populated with HDFs. Fig. 3A: Cellularized LES. Fig. 3B: Decellularized LES treated by freezethawing. Fig. 3C: Decellularized LES treated with SDS. Fig. 3D: Cellularized RAS. Fig. 3E: Decellularized RAS treated by freeze-thawing. Fig. 3F: Decellularized RAS treated with SDS. Deposited collagen 1A was stained with anti -Coll A and cell nuclei were stained with Hoechst 33342. Left panels: maximum-intensity projection images of the z-stack. Right panels: the corresponding 3D reconstruction of the series of z-stack images. Left panels in 3A-3F: scale bar = 200 pm. Figure 3G provides quantification of immunofluorescence staining of collagen (left) and fibronectin (right) on cellularized, and dECM-decorated LES and RAS scaffolds treated by freeze-thaw and SDS. Data is calculated as the mean of three independent experiments with standard error and the significance between groups is calculated. It is represented in the graph as not significant (ns), * (<0.05), ** (< 0.01), *** (< 0.001), and **** (< 0.0001).

[0017] Figures 4A-4F show fibronectin deposition on LES and RAS populated with HDFs. Fig. 4A: Cellularized LES. Fig. 4B: Decellularized LES treated by freezethawing. Fig. 4C: Decellularized LES treated with SDS. Fig. 4D: Cellularized RAS. Fig. 4E: Decellularized RAS treated by freeze-thawing. Fig. 4F: Decellularized RAS treated with SDS. Deposited fibronectin was stained with anti -fibronectin and cell nuclei were stained with Hoechst 33342. Left panels: maximum-intensity projection images of the z-stack. Right panels: the corresponding 3D reconstruction of the series of z-stack images. Left panels in Figs. 4A-4F: scale bar = 200 pm.

[0018] Figures 5A-5F provide the quantification of ECM proteins and growth factors present in decellularized LES and RAS after being populated with HDFs for 20, 30, 40, and 50 days, and subsequently decellularized by SDS treatment and freeze-thawing. The ECM content and growth factors including Collagen (Fig. 5A), Fibronectin (Fig. 5B), Elastin (Fig. 5C), GAG (Fig. 5D), VEGF (Fig. 5E), and bFGF (Fig. 5F) in the decellularized scaffolds were measured. Black bars are cellularized scaffold, white bars are decellularized scaffold (SDS), and grey bars are decellularized scaffold (freeze thaw). The data is summarized as the mean of the triplicates with standard error, p-value was calculated and presented as ns (not significant), * (< 0.05), ** (< 0.01), *** (< 0.001) and **** (< 0.0001).

[0019] Figures 6A-6C show the effect of RAS with and without decoration of dECM on the anti-inflammatory action of M2 macrophages. The macrophages were immunostained with CD68 (pan macrophage marker), CD206 (anti-inflammatory, M2 marker), and Hoechst 33342 (nuclear staining). A merged image was used to visualize co-localization. Pristine RAS (top row), dECM-decorated RAS (freeze-thawing) (middle row), and dECM-decorated RAS (SDS treatment) (bottom row). All images: scale bar = 200 pm. Fig. 6B: Quantification of colocalized CD206 and CD68 positive macrophages. Fig. 6C: IL- 10 secretion by macrophages seeded on pristine RAS and dECM-decorated RAS (freeze-thawing and SDS treatment) at 4, 24, 48, and 72 hours. Black bars are control, white bars are decellularized scaffold (freeze thaw), and grey bars are decellularized scaffold (SDS). The data obtained is represented as the mean of three independent experiments with standard error, p-value calculated and depicted as ns (not significant), * (< 0.05), ** (< 0.01), *** (< 0.001) and **** (< 0.0001). Figures 6D-6F show the effect of RAS with and without decoration of dECM on the pro-inflammatory action of Ml macrophages. The macrophages were immunostained with CD68 (pan macrophage marker), iNOS (pro-inflammatory, Ml marker), and Hoechst 33342 (nuclear staining). A merged image was used to visualize co-localization. Pristine RAS (top row), dECM RAS (freeze-thawing) (middle row), and dECM RAS (SDS-treated) (bottom row). All images: scale bar = 200 pm. Fig. 6E: Quantification of colocalized iNOS and CD68 positive macrophages. Fig. 6F: TNF-a secretion by macrophages seeded on pristine RAS, dECM RAS (freeze-thawing), and dECM RAS (SDS-treated) at 4, 24, 48 and 72 hours. Control: pristine RAS. Black bars are control, white bars are decellularized scaffold (freeze thaw), and grey bars are decellularized scaffold (SDS). The data obtained is represented as the mean of three independent experiments with standard error, p-value calculated and depicted as ns (not significant), * (< 0.05), ** (< 0.01), *** (< 0.001) and **** (< 0.0001). Figures 6G-6I show the effect of LES with and without decoration of dECM on the pro-inflammatory action of Ml macrophages. The macrophages were stained with CD68 (pan macrophage marker), iNOS (pro- inflammatory, Ml marker), and Hoechst 33342 (nuclear staining). A merged image was used to visualize co-localization. Pristine LES (top row), dECM LES (Freeze-thawing) (middle row), and dECM LES (SDS-treated) (bottom row). All images: scale bar = 200 pm. Fig. 6H: Quantification of colocalized iNOS and CD68 positive macrophages. Fig. 61: TNF-a secretion by macrophages seeded on pristine LES, dECM LES (Freezethawing), and dECM LES (SDS-treated) at 4, 24, 48 and 72 hours. Black bars are control, white bars are decellularized scaffold (freeze thaw), and grey bars are decellularized scaffold (SDS). The data obtained is represented as the mean of three independent experiments with standard error, p-value calculated and depicted as ns (not significant), * (< 0.05), ** (< 0.01), *** (< 0.001) and **** (< 0.0001).

[0020] Figures 7A-7C show the effect of LES with and without decoration of dECM on the anti-inflammatory action of M2 macrophages. Control: pristine LES. The macrophages were immunostained with CD68 (pan macrophage marker), CD206 (antiinflammatory, M2 marker), and Hoechst 33342 (nuclear staining). A merged image was used to visualize co-localization. Pristine LES (top row), dECM-decorated LES (freezethawing) (middle row), and dECM-decorated LES (SDS treatment) (bottom row). All images: scale bar = 200 pm. Fig. 7B: Quantification of colocalized CD206 and CD68 positive macrophages. Fig. 7C: IL- 10 secretion by macrophages seeded on pristine LES and dECM-decorated LES (created by freeze-thawing and SDS treatment) at 4, 24, 48, and 72 hours. Black bars are control, white bars are decellularized scaffold (freeze thaw), and grey bars are decellularized scaffold (SDS). The data obtained is represented as the mean of three independent experiments with standard error, p-value represented as ns (not significant), * (< 0.05), ** (< 0.01), *** (< 0.001) and **** (< 0.0001).

[0021] Figures 8A-8E show the repopulation of LES and RAS with and without decoration of dECM with HDFs. Fig. 8 A: Seeding efficiency in pristine scaffolds and dECM decorated scaffolds after 8 hours of incubation. Fig. 8B: Proliferation rate in the pristine scaffolds and dECM decorated scaffolds (LES). Fig. 8C: Proliferation rate in the pristine scaffolds and dECM decorated scaffolds (RAS). Figs. 8D and 8E are representative images of the HDFs stained with Ki67 and the cell nuclei with Hoechst 33342 on day 7. Each image in Figs. 8D and 8E consists of a maximum-intensity projection image of the z-stack on the left and a 3D reconstruction on the right. Fig. 8D: Repopulation of dECM-decorated LES with HDFs decellularized by freeze-thaw and SDS treatment, respectively. Fig. 8E: Repopulation of dECM-decorated RAS of HDFs decellularized by freeze-thaw and SDS treatment, respectively. Images in the left panel: scale bar = 200 pm. p-value calculated and depicted as ns (not significant), * (< 0.05), ** (< 0.01), *** (< 0.001) and **** (< 0.0001). Figures 8F and 8G show the repopulation of dECM-decorated LES and RAS with keratinocytes for 7 days. Top row: dECM-decorated LES (Fig. 8F) and RAS (Fig. 8G) created by freeze-thawing. Bottom row: dECM-decorated LES and RAS created by SDS treatment. The cells were stained with cytokeratin 9 and cell nuclei were stained with Hoechst 33342 on day 7. Each image consists of a maximum-intensity projection image of the z-stack on the left and a 3D reconstruction on the right. The images in the left panel: scale bar = 200 pm.

[0022] Figures 8H and 81 show the mechanical testing of dECM-decorated scaffolds. Compression strength testing was conducted on pristine and dECM scaffolds obtained by SDS and freeze-thaw treatment and the data (mean of n = 6) plotted as line graphs. Fig. 8H: LES. Fig. 81: RAS.

[0023] Figures 9A-9D show the histological assessment of explants of LES and RAS with and without decoration of dECM following subcutaneous implantation in rats. Top row: H & E staining of pristine LES (Fig. 9A) and RAS (Fig. 9B) at 1 and 2 weeks. Middle row: H & E staining of dECM-decorated LES (Fig. 9A) and RAS (Fig. 9B), created via freeze-thawing at 1 and 2 weeks. Bottom row: H & E staining of dECM- decorated LES (Fig. 9A) and RAS (Fig. 9B), created via SDS treatment at 1 and 2 weeks. Fig. 9C: Quantification of cell penetration into the scaffolds. Fig. 9D: Quantification of newly formed blood vessels per mm2of the scaffolds. The statistical significance of the data is notated with p-value as ns (not significant), * (< 0.05), ** (< 0.01), *** (< 0.001), and **** (< 0.0001).

[0024] DETAILED DESCRIPTION OF THE INVENTION

[0025] The use of decellularized extracellular matrix (ECM) products in tissue regeneration is quite alluring yet practically challenging due to the limitations of its availability, harsh processing techniques, and host rejection. Scaffolds obtained by either incorporating ECM material or coating the surface can resolve these challenges to some extent. However, these scaffolds lack the complex three-dimensional (3D) network formed by proteins and growth factors observed in natural ECM. Herein, an approach utilizing 3D nanofiber scaffolds decorated with dECM to enhance cellular responses and promote tissue regeneration is provided. Notably, the dECM can be customized according to specific cellular requirements, offering a tailored environment for enhanced therapeutic outcomes. Two types of 3D expanded scaffolds, namely radially aligned scaffolds (RAS) and laterally expanded scaffolds (LES) fabricated by the gas-foaming expansion were utilized. To demonstrate the proof-of-concept, human dermal fibroblasts (HDFs) were seeded on these scaffolds for up to 8 weeks, resulting in uniform and highly aligned cells deposited ECM on the scaffolds. These cellular components were then removed from the scaffolds through decellularization (e.g., SDS treatment and freeze-thaw cycles). The dECM-decorated 3D expanded nanofiber scaffolds can direct and support cell alignment and proliferation along the underlying fibers upon recellularization. An in vitro inflammation assay indicates that dECM- decorated LES induces a lower immune response than dECM-decorated RAS. Further, subcutaneous implantation of dECM-decorated RAS and LES shows higher cell infiltration and angiogenesis within 7 and 14 days than RAS and LES without dECM decoration. Taken together, dECM-decorated 3D expanded nanofiber scaffolds can be used in tissue regeneration and tissue modeling.

[0026] The decellularized ECM (dECM) products obtained by the decellularization of organs and tissues from human and animal sources have been used as scaffolds in medical interventions (e.g., bone regeneration, skin, nerve, and surgical hernia repair) (Zhang, et al., Bioact. Mater. (2022) 10: 15-31; Mahdian, et al., Front. Neurosci. (2023) 17: 1295563; Golebiowska, et al., Bioact. Mater. (2024) 32:98-123). These products retain their architecture along with all the necessary biological cues (Porzionato, et al., Int. J. Mol. Sci. (2018) 19:4117; Swinehart, et al., Dev. Dyn. (2016) 245:351-60). They can be further processed to serve as starting material for generating electrospun scaffolds. They can be fabricated by either directly electrospinning dECM components along with synthetic or natural polymers or coating the dECM components onto the electrospun scaffolds (Gao, et al., ACS Appl. Bio Mater. (2023) 6:2158-2171; Matthias Santschi, et al., Curr. Opin. Biomed. Eng. (2019) 10: 116-122; Wu, et al., Biomater. Res. (2023) 27:7; Junka, et al., ACS Appl. Bio Mater. (2022) 5:5634-5644). However, during this process, key signaling components important for cell adhesion such as GAGs and growth factors may be degraded (Uhl, et al., Acta Biomater. (2020) 102:231-246). Additionally, animal tissue-derived dECM may trigger immunogenic as well as pathogen transfer complications (Kasravi, et al., Biomater. Res. (2023) 27: 10). ECM has a definite composition that cannot be bioactively customized for a specific application. To overcome these challenges and tailor a functional ECM found in native human tissues, the decellularization of cell-derived ECM has been adopted. This strategy has garnered interest due to the modular composition of cell-derived dECM, which can be customized according to tissue requirements. The cell-derived dECM scaffolds are derived from human cell cultures which are decellularized to remove immunogenic components while preserving the ECM bioactivity. All the cell types used for ECM generation can be prescreened to minimize the risk of disease transmission. It also enables the selection of appropriate ECM-generating cells to layer the desired type of ECM. As it is an in vitro culture, it gives the flexibility to modify the cell type genetically or expose it to specific stimuli to create a distinct ECM that is otherwise unavailable from tissues. It is also flexible in accommodating cocultures or sequential cultures to achieve desired ECM.

[0027] The cell-derived dECM has been successfully used in various applications. For instance, fibroblast-derived dECM can promote the growth and proliferation of re-seeded fibroblasts and show a low immunogenic response in rats (Lu, et al., Biomaterials (2011) 32:9658-9666; Hoshiba, T., J. Mater. Chem. B (2017) 5:4322-4331). dECM deposited by mesenchymal stem cells (MSCs) can enable cell proliferation while enhancing the differentiation of reseeded MSCs into chondrocytes and osteoblasts (Pei, et al., Tissue Eng. Part A (2011) 17:3067-76; Xing, et al., Tissue Eng. Part C Methods (2015) 21 :77- 87; El Ghalbzouri, et al., Biomaterials (2009) 30:71-78). Cell-derived ECM exhibits superior mechanical strength, long-term sustainability, and reproducibility. It can be tailored to meet specific tissue requirements, including applications such as vascular constructs, nerve conduits, bone implants, and various applications (Assuncao, et al., Front. Bioeng. Biotechnol. (2020) 8:602009). However, native tissue represents a well- organized structure that governs many bodily functions and moderates inflammatory responses (Meizlish, et al., Annu. Rev. Immunol. (2021) 39:557-581). Hence, the exploration of an organized 3D nanostructure capable of efficiently replicating various cellular functions remains uncharted in the quest to mimic the complex organization of native tissues. Fibroblasts grown on aligned nano-grated PDMS substrate for 8 weeks showed highly aligned ECM deposition after decellularization (Xing, et al., Adv. Funct. Mater. (2014) 34:3027-3035). However, ECM sheets often lack the 3D architectures and sufficiently high porosity necessary for accommodating tissue defects and facilitating cell infiltration. For example, dECM derived from tissues such as tendons and ligaments are dense, leading to decreased cell infiltration and consequently hindering tissue regeneration (Font Tellado, et al., Adv. Drug Deliv. Rev. (2015) 94: 126-40). It is important to understand the characteristics of each tissue such as pore size, type of ECM modification, and the viscoelastic properties of the tissue which dictate the behavior and fate of the cell. The relationship between scaffold pore size, cellular fate, and function in response to the multivariate mechanical and physical factors presented by ECM is not fully understood. However, the mean pore size is critical when designing scaffolds for tissue engineering applications to ensure permeability and facilitate nutrient transport and cell growth (Murphy, et al., Cell Adh. Migr. (2010) 4:377-81; Han, et al., Front. Bioeng. Biotechnol. (2021) 9:629270). With increasing pore size, there is a reduction in the surface area of cell attachment on the scaffold. Bigger pores offer less surface area while smaller pores restrict cell migration and the transport of nutrients causing cellular patches near the edges of the scaffold. Hence, a balance is needed between mean pore size and the specific surface area to promote cell adhesion, migration, proliferation, and differentiation (Bruzauskaite, et al., Cytotechnology (2016) 68:355-69). In such a scenario, the presence of ECM with its mechanical and biochemical cues can enhance cell attachment and can contribute to surface area. Therefore, it is important to develop 3D ECM constructs with sufficient porosity and highly organized structures to ensure efficient cellular behavior and function for tissue regeneration.

[0028] Decellularization is a process aimed at removing immunogenic components from cells without causing damage to the ultrastructure of the ECM and its bioactivity (Neishabouri, et al., Front. Bioeng. Biotechnol. (2022) 10:805299). The effectiveness of decellularization depends largely on the type of tissue, which exhibits varied cellular density and ECM composition. Various conventional decellularization methods for tissues and organs, including thermal shock, detergent treatment, osmatic shock, mechanical disruption, and enzymatic action, are commonly utilized (Neishabouri, et al., Front. Bioeng. Biotechnol. (2022) 10:805299). Among these methods, the use of ionic detergents, such as sodium dodecyl sulfate (SDS), is more effective in removing cellular nuclei but may damage the architecture and mechanical strength of the ECM. On the contrary, thermal shock or freeze-thaw cycling efficiently removes cells without causing a significant reduction in ECM molecules or their mechanical strength. Various decellularization methods on cell sheets have been compared and evaluated for their effect on ECM architecture, mechanical strength, and recellularization (Xing, et al., Tissue Eng. Part C Methods (2015) 21 :77-87). Although this study furnished insights for determining effective decellularization procedures for two-dimensional (2D) cell-ECM mats, it does not evaluate different decellularization methods for a 3D cellularized scaffold. In this context, the size and complexity of the ECM scaffold may vary, especially with differentially organized 3D scaffolds. A fibrocartilage-mimetic scaffold has been developed by combining a poly(e- caprolactone) (PCL) scaffold with dECM from chondrocytes along with calcium phosphate and albumin coating to depict a precursor of woven bone (Junka, et al., ACS Appl. Bio Mater. (2022) 5:5634-5644). This hybrid scaffold promoted better osteoblast attachment, proliferation, differentiation, and mineralization.

[0029] Herein, 3D scaffolds comprising hierarchically assembled nanofibers with controlled alignments were generated by cultivating fibroblasts for 8 weeks, resulting in the deposition of ECM. The scaffolds underwent decellularization through either freezethawing or detergent treatment. The efficiency of the dECM scaffolds was assessed by reseeding fibroblasts and keratinocytes, demonstrating efficient attachment and proliferation on the scaffolds. The immunogenicity of the scaffolds was evaluated by culturing human monocytes and differentiated into anti-inflammatory macrophages. Furthermore, testing these scaffolds in an in vivo rat subcutaneous implantation revealed elevated cell infiltration and neovascularization. These findings collectively demonstrate that a 3D scaffold consisting of hierarchically assembled nanofibers with controlled alignments, when deposited with cell-derived natural ECM and subsequently decellularized, can serve as a powerful grafting material for regenerative applications.

[0030] In accordance with the instant invention, 3D scaffolds (also referred to herein as expanded scaffolds) comprising ECM (e.g., the ECM is contained throughout the 3D scaffold or other scaffold) and methods of synthesizing the 3D scaffolds are provided. 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). In certain embodiments, the 3D scaffolds allow for cell growth and / or infiltration and / or differentiation. The 3D scaffolds of the present invention can be designed to have controlled pore size, a biomimetic surface nanotopography, and / or cell adhesion. The 3D 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 3D scaffolds of the instant invention may also be used as grafts. The 3D 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. For example, the 3D scaffolds of the instant invention may be applied in lung, liver, kidney, and heart tissue engineering. The 3D scaffolds of the instant invention may have a low rejection rate, low toxicity, and / or faster tissue regeneration. The 3D scaffolds of the present invention may also have great regeneration abilities without evoking immune response due to highly aligned nanofibers, deposited ECM, and being devoid of cellular components. The 3D scaffolds of the present invention may also be tailor-made depending on the type of tissue targeted for regeneration or repair. For example, cells consistent with the tissue being regenerated or repaired may be used to produce the ECM in the 3D scaffold. For example, for tissues in general including organ tissue and connective tissue, fibroblasts may be used. For cartilage, chondrocytes may be used. For bone, osteoblasts may be used. In certain embodiments, fibroblasts are used with another cell type. For example, fibroblasts may be seeded with endothelial cells to create vascular ECM or for wound healing, fibroblasts may be seeded with neural progenitor cells and / or Schwann cells to create neural ECM, and fibroblasts may be seeded with bone marrow stem cells to create osteoconductive ECM.

[0031] Methods of synthesizing 3D scaffolds comprising ECM are provided herein. In certain embodiment, 3D scaffolds are expanded nanofiber scaffolds with increased porosity (e.g., compared to non-expanded nanofiber mat). In certain embodiments, the 3D scaffolds comprising ECM are synthesized by A) culturing cells in a 3D scaffold for a time sufficient to allow for deposition of an ECM within the 3D scaffold and B) decellularizing the 3D scaffold to remove the cells. Preferably, the decellularization is performed without significantly impacting the integrity of the ECM. In certain embodiments, the methods of the instant invention further comprise synthesizing the 3D scaffold prior to culturing cells therein. In certain embodiments, the method comprises expanding (e.g., exposure to gas bubbles (e.g., immersion in a solution producing gas)) a 2D nanofiber mat (e.g., electrospun mat) to generate the 3D scaffold. In certain embodiments, the method comprises synthesizing a 2D nanofiber mat (e.g., electrospun mat) and then expanding (e.g., exposure to gas bubbles) the 2D nanofiber mat to generate the 3D scaffold. The expanded nanofiber 3D scaffolds may be fully expanded, partially expanded, or comprising layers wherein some layers may be expanded, and some may not be. In certain embodiments, the 3D scaffolds are fully expanded.

[0032] The 3D scaffolds of the instant invention may be manufactured or synthesized by any method. For example, the 3D scaffolds of the present invention can be manufactured using a variety of methods including, but not limited to: electrospinning, centrifugal spinning, gas-foaming, freeze-casting, self-assembly, weaving, 3D printing, melt electrospinning, and melt electrospinning writing. In certain embodiments, the scaffold is made by 3D printing such as extrusion-based 3D printing. In certain embodiment, the scaffold is made by melt electrospinning writing (e.g., as a type of 3D printing). Notably, 3D printing allows for controlling the shape of the scaffold, controlling the pore sizes within the scaffold, controlling spatial distribution (e.g., microarchitecture), and / or controlling the interconnections of the scaffold.

[0033] The 3D scaffolds of the present invention may comprise any material. In certain embodiments, the nanofibers are made from a variety of inorganic materials including but not limited to bioactive glass, metal oxide, and hydroxyapatite. For example, the 3D scaffolds may comprise plastics, polymers, ceramic, glass, and / or metal or mixtures thereof. 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 3D scaffolds comprise at least one polymer.

[0034] The 3D scaffold of the instant invention may comprise fibers. For example, the 3D scaffold may comprise nanofibers. As used herein, nanofibers are fibers having an average 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 500 nm, about 1000 nm to about 500 nm or about 100 nm to about 400 nm.

[0035] The nanofibers of the instant invention can be fabricated by any method. In certain embodiments, the nanofibers are synthesized by electrospinning. The nanofibers may be aligned fibers (e.g., uniaxially aligned), random fibers, and / or entangled fibers. In certain embodiments, the nanofibers comprise aligned fibers (e.g., uniaxially, radially, laterally, vertically, or horizontally).

[0036] The nanofibers of the instant invention may comprise any polymer. In certain embodiments, the polymer is biocompatible. In certain embodiments, the polymer is 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 hyperbranched (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.

[0037] 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.

[0038] Examples of hydrophilic polymers include, without limitation: polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), poly(ethylene glycol) 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.

[0039] 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. 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 M13 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, collagen / chondroitin sulfate, collagen / chitosan), and composites (e.g, PDLA / HA, PCL / CaCCL, 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.

[0040] 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 isophthalamide, 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).

[0041] 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 comprises 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 comprises poloxamer 188. In certain embodiments, the nanofibers comprises 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).

[0042] In certain embodiments, the polymer solution comprises about 10% polymer (w / v) (e.g., PCL) and about 1.0% poloxamer 407 (w / v) (Pluronic® F127). 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.

[0043] The fibers of the 3D scaffold may be of any orientation. For example, the 3D scaffold may comprise orthogonal fibers, aligned fibers (e.g., uniaxially aligned), partially aligned, random fibers, and / or entangled fibers. In certain embodiments, the 3D scaffold comprises random fibers. In certain embodiments, the 3D scaffold comprises uniform fibers. In certain embodiments, the scaffold comprises aligned fibers (e.g., uniaxially, radially, linearly, vertically, or horizontally). In certain embodiments, the 3D scaffold comprises aligned fibers. In certain embodiments, the 3D scaffold comprises orthogonal fibers. In certain embodiments, the 3D scaffold comprises radially aligned fibers. In certain embodiments, the 3D scaffold comprises laterally aligned fibers.

[0044] In certain embodiments, 3D scaffolds may be composed of layers wherein each layer may have a different thickness, fiber alignment, and / or porosity. 3D scaffolds of the instant invention may be fabricated into any shape, size, or thickness.

[0045] In certain embodiments of the instant invention, the methods further comprise fixing at least one point, edge, end, or side - or a portion thereof - of a nanofiber mat (2D nanofiber mat) and then expanding the nanofiber mat into an expanded nanofiber scaffold (3D scaffold). 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.

[0046] 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.

[0047] 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. To avoid excess fixation and / or damage to the remainder 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).

[0048] In certain embodiments, the nanofiber mat is chemically fixed, 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: dichloromethane (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.

[0049] In certain embodiments, the nanofiber mat is not fixed (e.g., not thermally or chemically fixed).

[0050] The nanofiber mat may be cut, trimmed, or shaped prior to 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). 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).

[0051] While the application generally describes nanofiber mats as the 2D structure prior to expansion, the instant invention also encompasses any nanofibrous structure which can be expanded by the methods provided herein (e.g., structures other than a mat or 3D structures which can be further expanded).

[0052] In certain embodiments, the nanofiber mat is expanded into an expanded nanofiber scaffold by exposing the nanofiber mat to gas bubbles (e.g., immersing the nanofiber mat in a solution producing gas). 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). The bubbles can be generated by chemical reactions or physical manipulations. For example, the nanofiber mat can be submerged or immersed in a bubble / gas producing chemical reaction or physical manipulation. Generally, the longer the exposure to the bubbles, the greater the thickness and 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 gas bubbles) to assist in the formation of a desired shape. The nanofiber mat may be treated with air plasma prior to exposure to gas bubbles (e.g., to increase hydrophilicity). The nanofiber mat may be exposed to gas bubbles more than once (e.g., repeatedly). A negative pressure could be applied to facilitate the expansion.

[0053] After exposure to the bubbles, the expanded nanofiber structure may be washed and / or rinsed in water and / or a desired carrier or buffer (e.g., a pharmaceutically or biologically acceptable carrier). Trapped gas bubbles may be removed by applying a vacuum to the expanded nanofiber structure. For example, the expanded nanofiber structure may be submerged or immersed in a liquid (e.g., water and / or a desired carrier or buffer) and a vacuum may be applied to rapidly remove the gas bubbles. The process may be repeated one or more times. After expansion (e.g., after rinsing and removal of trapped gas), the expanded nanofiber structure may be placed in storage in cold solution or lyophilized and / or freeze-dried.

[0054] The gas bubbles of the instant invention can be made by any method known in the art. The bubbles may be generated, for example, by chemical reactions or by physical approaches. Electrospun nanofiber mats can be expanded three dimensionally using a gas-foaming based expansion method (example methods may be found in WO 2016 / 053988; WO 2019 / 060393; WO 2020 / 124072; US Patent No. 11,033,659; US Patent No. 11,813,377; US Patent No. 11,427,936; US Patent No. 11,946,164; and Jiang et al. (2018) Acta Biomater., 68:237-248, each incorporated herein by reference). Electrospun nanofiber mats can be expanded in the third dimension with ordered structures using gas bubbles generated by chemical reactions in an aqueous solution (see, e.g., WO 2016 / 053988; WO 2019 / 060393; US Patent No. 11,033,659; US Patent No. 11,813,377; US Patent No. 11,427,936; US Patent No. 11,946,164; Jiang et al. (2018) Acta Biomater., 68:237-248; Jiang, et al. (2015) ACS Biomater. Sci. Eng., 1 :991-1001; Jiang, et al. (2016) Adv. Healthcare Mater., 5:2993-3003; Joshi, et al. (2015) Chem. Eng. J., 275:79-88; each of the foregoing incorporated by reference herein). In certain embodiments, the chemical reaction or physical manipulation does not damage or alter or does not substantially damage or alter the nanofibers (e.g., the nanofibers are inert within the chemical reaction and not chemically modified). As explained hereinabove, the nanofiber mat may be submerged or immersed in a liquid comprising the reagents of the bubble-generating chemical reaction. Examples of chemical reactions that generate bubbles include, without limitation:

[0055] NaBH4+ 2H2O = NaBO2+ 4H2

[0056] NaBH4+ 4H2O = 4H2(g) + H3BO3 + NaOH HCO3- + H+= CO2+ H2O NH4+ NO2- = N2+ 2H2O H2CC>3=H2O + co22H++ S2’ = H2S 2H2O2= O2+ 2H2O 3HNO2= 2NO + HNO3 + H2O HO2CCH2COCH2CO2H = 2CO2+ CH3COCH3 2H2O2= 2H2+ O2CaC2+H2O = C2H2Zn+ 2HC1 =H2+ ZnCl22KMnO4+ 16HC1 = 2KCl+2MnCl2+H2O+5Cl2

[0057] In certain embodiments, the chemical reaction is the hydrolysis of NaBH4(e.g., NaBH4+ 2H2O = NaBO2+ 4H2). In certain embodiments, CO2gas bubbles (generated chemically or physically) are used (e.g., for hydrophilic polymers).

[0058] Examples of physical approaches for generating bubbles of the instant invention include, without limitation: 1) create high pressure (fill gas) / heat in a sealed chamber and suddenly reduce pressure; 2) dissolve gas in liquid / water in high pressure and reduce pressure to release gas bubbles; 3) use supercritical fluids (reduce pressure) like supercritical CO2; 4) use subcritical gas liquid (then reduce pressure) (e.g., liquid CO2, liquid propane and isobutane); 5) fluid flow; 6) apply acoustic energy or ultrasound to liquid / water; 7) apply a laser (e.g., to a liquid or water); 8) boiling; 9) reduce pressure boiling (e.g., with ethanol); and 10) apply radiation (e.g., ionizing radiation on liquid or water). The nanofiber mat may be submerged or immersed in a liquid of the bubblegenerating physical manipulation. In certain embodiments, the nanofiber mats are expanded using a subcritical or supercritical fluid or liquid (e.g., CO2, N2, N2O, hydrocarbons, and fluorocarbons). In certain embodiments, the nanofiber mats are expanded by exposure to depressurized CO2. In certain embodiments, liquid CO2 is utilized. For example, nanofiber mats may be expanded by exposing to, contacting with or being placed into (e.g., submerged or immersed) a subcritical liquid / fluid (e.g., subcritical CO2) and then depressurized. The cycle of placing the nanofibrous structures into subcritical CO2 and depressurizing may be performed one or more times. Generally, the more times the expansion method is used the thickness and porosity of the nanofibrous (or microfibrous) structure increases. For examples, the cycle of exposure to subcritical CO2 and then depressurization may be performed one, two, three, four, five, six, seven, eight, nine, ten, or more times, particularly 1-10 times, 1-5 times, or 1-3 times. In certain embodiments, the cycle of exposure to subcritical CO2 and then depressurization is performed at least 2 times (e.g., 2-10 times, 2-5 times, 2-4 times, or 2-3 times). In certain embodiments, the method comprises placing the nanofibrous mat and dry ice (solid CO2) in a sealed container, allowing the dry ice to turn into liquid CO2, and then unsealing the container to allow depressurization.

[0059] The nanofiber mat and subcritical fluid (e.g., subcritical CO2; or solid form of subcritical fluid (e.g., dry ice)) may be contained in any suitable container (e.g., one which can withstand high pressures). For example, the subcritical fluids and the nanofiber mat may be contained within, but not limited to: chambers, vessels, reactors, and tubes. In certain embodiments, the equipment or container used during the methods of the present invention will have a feature or component that allows control of the depressurization rate of the subcritical fluid. Depressurization of the subcritical fluid can be done using a variety of methods including but not limited to manually opening the container to decrease pressure or by using some type of equipment that can regulate the rate of depressurization of the reaction vessel.

[0060] The 3D scaffolds of the instant invention may be coated with a coating material such as gelatin. The methods of the instant invention may further comprise coating the 3D scaffold 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, starch-acrylonitrile 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.

[0061] Prior to coating, the scaffold 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.

[0062] 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).

[0063] 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 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 scaffold by immersing or soaking the scaffold in a solution or suspension comprising the coating material.

[0064] 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.

[0065] The 3D 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 3D 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).

[0066] The 3D scaffolds of the instant invention may also be sterilized. Sterilization may occur after expansion and / or after coating (if present). For example, the 3D scaffolds can be sterilized using various methods (e.g., by treating with ethylene oxide gas, gamma irradiation, or 70% ethanol). In certain embodiments, the 3D scaffolds are sterilized by treating with ethylene oxide.

[0067] As stated hereinabove, the methods of the instant invention further comprise culturing cells on and / or within the 3D scaffold. In certain embodiments, the cells are seeded on and / or in the 3D 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 3D scaffold of the instant invention. Different cells or combinations of cells may be seeded on the 3D 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. In certain embodiments, a co-culture of at least two or more different types of cells on the 3D 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. The cells may be cultures in the 3D scaffold as long as desired. In certain embodiments, the cells are cultured at least long enough to produce ECM throughout the 3D scaffold. 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 3D 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 3D 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 3D scaffolds in differentiation media. In certain embodiments, the cells or tissue may be cultured within the 3D scaffolds in the presence of different growth factors (e.g., TGF-beta) which can modulate secretion of ECM.

[0068] As stated hereinabove, the methods of the instant invention comprise decellularization of the 3D scaffold after the culturing of the cells has produced the desired ECM. In certain embodiments, the decellularization results in the elimination of viable cells from the 3D scaffold. 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 3D scaffold with DNase (e.g., DNase I).

[0069] 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.

[0070] 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.

[0071] The 3D 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 3D scaffolds may also be stored in a cold solution, lyophilized, frozen in a vacuum, and / or freeze-dried after decellularization.

[0072] The 3D scaffolds of the instant invention may also be sterilized after decellularization. For example, the 3D scaffolds can be sterilized using various methods (e.g., by treating with ethylene oxide gas, gamma irradiation, or 70% ethanol). In certain embodiments, the 3D scaffolds are sterilized by treating with ethylene oxide.

[0073] The 3D scaffolds comprising ECM 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 3D scaffolds comprising ECM. 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.

[0074] 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

[0075] T1 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).

[0076] 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.

[0077] In certain embodiments, the 3D scaffold comprises and / or encapsulates cells or tissue (after establishment of the ECM and decellularization). In certain embodiments, the cells are autologous to the subject to be treated with the 3D scaffold. The 3D scaffold may comprise and / or encapsulate any cell type. Cell types 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, the cells comprise stem cells. In certain embodiments, the cells comprise fibroblasts (e.g., dermal fibroblasts). In certain embodiments, the cells comprise bone marrow mesenchymal stem cells (BMSCs). In certain embodiments, the cells comprise endothelial cells (e.g., human umbilical vein endothelial cells (HUVECs)). In certain embodiments, the cells comprise BMSCs and endothelial cells (e.g., human umbilical vein endothelial cells (HUVECs)). In certain embodiments, the 3D scaffold comprises and / or encapsulates cell spheroids. In certain embodiments, the 3D scaffold comprises and / or encapsulates tissue samples (e.g., minced tissue), such as skin tissue samples or bone samples. The cells or tissue may be cultured within the nanofiber, porous nanofibrous microsphere, and / or nanofibrous microsphere hydrogel composite (e.g., the cells or tissue may be cultured for sufficient time to allow for growth within and / or infiltration into the nanofiber, porous nanofibrous microsphere, and / or nanofibrous microsphere hydrogel composite). For example, the cells or tissue may be cultured with the 3D scaffold for 1 day, 2 days, 3 days, 4 days, 5 days, or more. 3D scaffolds synthesized by the methods of the instant invention are also encompassed herein. Compositions comprising the 3D scaffolds of the instant invention and a carrier (e.g., a pharmaceutically acceptable carrier) are also encompassed herein.

[0078] The 3D 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. 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.

[0079] The three-dimensional 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 three-dimensional scaffolds may be used ex vivo to generate tissue or tissue constructs / models. The three-dimensional 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 three- dimensional scaffold stimulates the growth of existing tissue and / or repair of a wound or defect when applied in vivo. The 3D scaffolds can be used for engineering, growing, and / or regeneration of a variety of tissues including but not limited to skin, bone, cartilage, muscle, nervous tissue, and organs (or portions thereof).

[0080] In accordance with the instant invention, the 3D scaffolds may be used in inducing and / or improving / enhancing wound healing and inducing and / or improving / enhancing tissue regeneration. The 3D 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 3D scaffold, optionally comprising an agent, as described herein. 3D scaffolds of the instant invention can be loaded with different agents as necessary for regeneration of various tissues. In a particular embodiment, the 3D scaffold comprises blood clotting factors (e.g., for accelerating blood clot formation and / or preventing blood loss). For example, the 3D 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 3D 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 3D scaffolds also may be delivered directly into a cavity (such as the peritoneal cavity) (e.g., using a pressurized cannula).

[0081] In accordance with the instant invention, the 3D scaffolds of the present invention can be used in medicine. In certain embodiments, the 3D 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 3D scaffolds of the instant invention to the subject (e.g., at or in a wound). In a particular embodiment, the method comprises administering 3D scaffolds comprising an agent and / or cell as described herein. 3D scaffolds of the instant invention can be loaded with agents as necessary for regeneration of various tissues. In a particular embodiment, the 3D 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 3D scaffolds to the subject and an agent as described herein (i.e., the agent is not contained within the 3D scaffold). When administered separately, the 3D scaffold may be administered simultaneously and / or sequentially with the agent. The methods may comprise the administration of one or more 3D scaffold. When more than one 3D scaffold is administered, the 3D scaffolds may be administered simultaneously and / or sequentially. In certain embodiments, the 3D scaffold is used for wound healing. In certain embodiments, the ECM is from seeding / culturing fibroblasts alone or with endothelial cells. In certain embodiments, the 3D scaffold comprises PGA and / or PLGA.

[0082] In certain embodiments, the 3D scaffold is used for nerve repair (e.g., peripheral nerve repair). In certain embodiments, the ECM is from seeding / culturing fibroblasts alone or with neural progenitor cells and / or Schwann cells. In certain embodiments, the 3D scaffold comprises PGA, PLGA, and / or PLCL. In certain embodiments, the 3D scaffold and / or nanofibers comprise or are coated with laminin.

[0083] In certain embodiments, the 3D scaffold is used for bone regeneration. In certain embodiments, the ECM is from seeding / culturing bone marrow derived stem cells or mesenchymal stem cells, optionally with fibroblasts. In certain embodiments, the 3D scaffold comprises PCL or PLCL.

[0084] In certain embodiments, the 3D scaffold is used for regenerating blood vessels. In certain embodiment, different cells may be seeded / cultured in different layers to create scaffold more like native blood vessels (endothelial cells on the inside and smooth muscle cells and fibroblasts on the outside). The present invention may use 3D expanded material or tubular aerogel that may result in highly porous scaffolds for better cellular infiltration. PGA or variations of PLGA scaffolds may be used.

[0085] Generally, the polymer and orientation of the fibers may be selected to suit the intended purpose. For example, for uses that require biomaterials with slower degradation, higher mechanical strength and tunable alignment (e.g., bone or cartilage), PCL or PLCL may be used. In another embodiment, skin tissue regeneration may be faster with moderate mechanical properties and alignment. As such, PGA or PLGA may be utilized in such tissues. With regard to fiber orientation, RAS may be preferred for hard tissues while LES may be preferred for soft tissues.

[0086] Further, the 3D scaffolds of the present invention may also have various pore sizes (e.g., by altering length of time of expansion or the number of times expanding the nanofiber mat). Different pore sizes may be beneficial depending on the type of tissue desired for repair or regeneration. Smaller pore sizes may be more beneficial for regenerating skin, blood vessels, and cartilage. Larger pore sizes may be more beneficial for bone tissue and peripheral nerve regeneration.

[0087] The 3D 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 3D 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 3D scaffolds comprise autologous cells).

[0088] The 3D 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, 3D 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 3D scaffolds when together with chondrocytes (Liu, et al., Nat. Mater. (2011) 10:398- 406).

[0089] The 3D scaffolds of the present invention may be administered by any method. The 3D scaffolds described herein may be administered to a subject or a patient as a pharmaceutical composition. The compositions of the instant invention comprise a 3D 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.

[0090] 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.

[0091] 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 3D scaffolds dispersed in a medium that is compatible with the parenteral injection. 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.

[0092] 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 3D scaffold to the wound or site of bleeding. In a particular embodiment, the 3D scaffolds comprise a blood clotting factor such as thrombin and / or fibrinogen.

[0093] 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 3D 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 3D scaffolds are mineralized. In a particular embodiment, the 3D 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).

[0094] In accordance with the instant invention, antimicrobial (e.g., antibiotic)-loaded 3D scaffolds are provided. In a particular embodiment, the antimicrobial (e.g., antibiotic)-loaded 3D scaffold is in the form of a wound dressing. The antimicrobial (e.g., antibiotic)-loaded 3D 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 3D scaffold is in a wound dressing.

[0095] Definitions

[0096] The singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. 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 yarns, 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.

[0097] “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.

[0098] 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.

[0099] 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.

[0100] “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. 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.

[0101] 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.

[0102] The term “antimicrobials” as used herein indicates a substance that kills or inhibits the growth of microorganisms such as bacteria, fungi, viruses, or protozoans.

[0103] 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.

[0104] 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.

[0105] 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. 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).

[0106] 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.

[0107] As used herein, the term “subject” refers to an animal, particularly a mammal, particularly a human.

[0108] 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.

[0109] 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.

[0110] 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.

[0111] 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.

[0112] 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, trifunctional, or multifunctional crosslinking reagent.

[0113] The following example illustrates certain embodiments of the invention. It is are not intended to limit the invention in any way.

[0114] EXAMPLE

[0115] Materials and Methods

[0116] Materials

[0117] PCL (MW= 80 kDa), Pluronic®-F-127, gelatin, sodium borohydride, osmium tetroxide, paraformaldehyde, Sodium Cacodylate Buffer, phenylmethyl sulfonyl fluoride (PMSF), Ethylenediaminetetraacetic acid (EDTA), sodium phosphate buffer, Urea, hexamethyldisilazane (HMDS), Papain, and DNase 1 were procured from Sigma-Aldrich (St. Louis, MO). Dichloromethane (DCM) and N, V-di methyl form am ide (DMF) were purchased from BDH Chemicals (Dawsonville, GA). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), Dulbecco’s phosphate buffer saline (DPBS), RPMI 1640 (RPMI), and penicillin-streptomycin were obtained from Invitrogen (Carlsbad, CA). Human growth factors such as Macrophage-colony stimulating factor (M-CSF), Tumor necrosis factor alpha (TNF-a), Interleukin 4 (IL-4), IL-13, and Interferon-gamma (IFN-y) were procured from Peprotech. Human adult dermal fibroblasts and the standard media components were obtained from ATCC. Primary antibodies such as Collagenla, fibronectin, Ki67, cytokeratin 9, CD206, CD68, iNOS, and Alexa Fluor™ 546 phalloidin were purchased from Abeam (Cambridge, MA). Secondary antibodies, Alexa 488, 647, and Hoechst 33342 were obtained from ThermoFisher Scientific. ECM quantification kits, and cell counting kit-8 were purchased from Sigma-Aldrich (hydroxyproline), R & D systems (fibronectin, Vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF)), Abeam (TNF- a, IL-10), and Biocolor (elastin, GAG).

[0118] Fabrication of radially aligned (RAS) and laterally expanded (LES) scaffolds

[0119] The RAS and LES were prepared by the expansion of 1-mm thick 2D PCL nanofiber mats fabricated based on established protocols (Chen, et al., Adv. Mater. (2020) 32:e2003754; Chen, et al., Acta Biomater. (2020) 108: 153-167; Chen, et al., Sci. Adv. (2021) 7:eabg3089; McCarthy, et al., Nano Select (2021) 2: 1566-1579). The mats were prepared by utilizing an electrospinning machine (Fluidnatek® LE-100) following a reported method (Fig. 1 A) (Chen, et al., Adv. Mater. (2020) 32:e2003754; Chen, et al., Sci. Adv. (2021) 7:eabg3089). Briefly, PCL (10% w / v) and Pluronic® F127 (1% w / v) were completely dissolved in a mixture of DCM / DMF at a ratio of 4: 1 (v / v). The prepared solution was loaded into a 20-emitter array equipped with 21 -gauge needles and electrospun onto a high-speed rotating drum collector at an applied voltage of 30 kV. The polymer flow rate was fixed at 16 mL / hour. The 2D PCL nanofiber mats were collected when the 1-mm thickness was achieved. The aligned fiber mats were further processed to obtain RAS and LES. For RAS, the aligned fiber mats were cut into rectangular shapes in liquid nitrogen (7 mm x 20 mm). The long side of rectangular mats was thermally fixed at 85°C for 3 seconds and transferred into freshly made sodium borohydrate (NaBEL, IM) solution for expansion. The RAS was then collected and washed several times with distilled water followed by freezing in a vacuum before being freeze-dried. To further maintain the morphology and enhance the mechanical property, the RAS was coated with 0.1% gelatin (type A) and freeze-dried again. Gelatin-coated RAS was further sliced into smaller pieces with an approximate height of 1-1.5 mm and circularly punched by an 8-mm punch (Chen, et al., Sci. Adv. (2021) 7:eabg3089). The LES was prepared by cutting the aligned fiber mat into small strips of 1.5 mm x 40 mm dimensions such that the length of these strips aligned with the direction of the fibers on the mat. The strips were then placed in freshly made NaBEL (IM) solution for expansion. After expansion, the LES was washed several times with DI water to remove residual NaBEL and coated with 0.1% gelatin (type A). The LES were frozen in a vacuum and then freeze-dried. LES were collected and circularly punched by an 8-mm punch. RAS and LES were sterilized using ethylene oxide gas before use in in vitro and in vivo studies.

[0120] ECM deposition by fibroblasts.

[0121] Human adult dermal fibroblasts (HDF) were cultured in media composition as per ATCC protocol. Fibroblasts in passages 2-5 were utilized for the ECM deposition. 2 x 105cells in 100 pl were plated on each scaffold placed in a 48-well low attachment plate and incubated for 30 minutes for proper distribution. After the incubation time was completed, 250 pl of the complete media was added to each well -containing scaffold and placed in a cell culture incubator maintained at 37°C and 5% CO2. To ensure better attachment and less disturbance from media changes, 250 pl media was added to scaffolds on day 2 of culture. From day 4 onwards, media is replaced every two days until the end of the experiment. Samples were collected at different time points for further analysis. The viability and proliferation of the fibroblasts on the scaffolds were assessed at various time points during the experiment using cell counting kit-8 as per the manufacturer’s instructions.

[0122] Decellularization of fibroblasts

[0123] Two decellularization methods were used to remove cells from the scaffolds including SDS treatment and freeze-thawing. For SDS treatment, the cellularized scaffolds were placed in 0.1% wt. SDS solution containing 10 mM Tris and 25 mM EDTA at room temperature with gentle shaking for 8 hours. The scaffolds were then washed thrice with 1 x DPBS to remove any traces of SDS and EDTA. For the freezethawing method, the scaffolds containing cells were frozen at -80°C for 30 minutes and thawed at 37°C with warm 1 x DPBS for 2 minutes. The DPBS was removed, and the scaffolds were placed at -80°C for another 30 minutes. The freeze-thawing cycle was repeated 8 times to ensure efficient decellularization. The scaffolds were washed once with l x DPBS. The decellularized scaffolds obtained by SDS and freeze-thaw treatments were then treated with 50 U / ml of DNase 1 for 4 hours. The scaffolds were washed with 1 x DPBS and freeze-dried for in vitro and in vivo evaluation.

[0124] Quantification of DNA content

[0125] The amount of DNA present in the cellularized and decellularized scaffolds was quantified by Quant-IT™ PicoGreen™ dsDNA assay kit. The scaffolds were treated with a digestion buffer comprised of papain (125 mg / ml), L-cysteine (2 mM), and EDTA (0.4 M) in DPBS at 65°C overnight. One hundred pl of each sample was mixed with 100 pl PicoGreen™ diluted in 1 xTris-EDTA buffer and placed in a 96-well plate in triplicates. The mixture was incubated for 5 minutes at room temperature in the dark and the fluorescent intensity was measured using a plate reader with an excitation wavelength of 480 nm and an emission wavelength of 520 nm. The readings were recorded, and the total DNA content was calculated and represented as a graph.

[0126] Scanning electron microscopy (SEM) For SEM imaging, the scaffolds were washed with 1 *DPBS twice and fixed in fixative comprising of 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M Sorenson phosphate buffer (or Sodium Cacodylate Buffer) overnight at room temperature. The scaffolds were washed twice in 0.1 M Sorenson phosphate buffer and fixed for 30 minutes in 1% osmium tetroxide. Following the second fixation, the scaffolds were washed with 0.1 M Sorenson phosphate buffer twice. They were dehydrated by a series of ethanol dilution treatments (30%, 50%, 70%, 2x95%, and 2x 100%, 5 minutes each). The samples were then chemically dried by leaving them in 100% HDMS overnight. On the next day, the scaffolds were mounted, sputter coated, and imaged using FEI Nova NanoSEM™ 450.

[0127] Quantification of ECM content

[0128] ECM proteins such as collagen, fibronectin, elastin, and GAG as well as growth factors such as VEGF and bFGF were quantified.

[0129] 1. Collagen

[0130] Collagen was measured using a hydroxyproline assay kit as per the manufacturer's protocol. Duplicates of cellularized and dECM-decorated scaffolds were homogenized with deionized water and an equal volume of 12 M HC1 was added to hydrolyze at 120°C for 4 hours. The mixture obtained from the reaction was then centrifuged at 10,000xg for 5 minutes. Fifteen pl of the supernatant was transferred into a 96-well plate. Then, 100 pl of Chloramine T / Oxidation Buffer was added and incubated at room temperature for 5 minutes. One hundred pl of diluted DMAB mixture (1: 1 v / v of p-Dimethylaminobenzaldehyde (DMAB) with Perchloric Acid / Isopropanol solution) was added to each well, mixed, and incubated for 90 minutes at 60°C. The plate was read at an absorbance of 560 nm using a plate reader. Using the standard hydroxyproline curve and the reading obtained, the total hydroxyproline content in the scaffolds was determined. The total collagen content was calculated using a 10: 1 w / w ratio of collagen to hydroxyproline and plotted as a graph.

[0131] 2. Fibronectin

[0132] For fibronectin quantification, fibronectin was extracted from the scaffolds by homogenization with 50 Mm sodium phosphate buffer (pH 6.0) containing 2 mM PMSF, 2 M urea, and 10 mg / ml heparin for 4 hours with constant stirring at room temperature. The mixture was centrifuged at 8000*g for 20 minutes. The supernatant was used to measure fibronectin content using a fibronectin Quantikine™ ELISA kit. The supernatant was diluted 10,000-fold with calibrator diluent prior to use in the assay. The assay was performed as per the protocol provided by the manufacturer. The optical density was measured at 450 nm with correction set at 540 or 570 nm. The concentration of fibronectin in the samples was calculated using the standard curve and by multiplying the dilution factor. The values thus obtained were represented as a graph.

[0133] 3. Elastin

[0134] A Fastin™ dye assay kit was used to quantify elastin content in the scaffolds. Elastin was extracted from the scaffolds by adding 500 pl of 0.25 M oxalic acid and incubating it on a hot plate maintained at 100°C for 1 hour. The undigested scaffolds were collected into fresh tubes and the extraction step with oxalic acid was repeated four times to ensure efficient recovery of elastin from the scaffolds. The elastin extract from all three steps was pooled and equal volumes of elastin precipitating reagent were added and vortexed. The mixture was allowed to stand for 15 minutes at room temperature and centrifuged at 13,000xg for 10 minutes. The supernatant was discarded and 1 ml of Fastin™ dye was added to the pellet and mixed for 90 minutes. The unbound dye was removed by centrifugation at 13,000*g for 10 minutes and pat drying. The bound dye was then dissociated from the elastin pellet with 250 pl of the dye dissociation reagent with constant mixing for 10 minutes. 100 pl of the dye from each sample was transferred to a 96-well plate and absorbance was measured using a plate reader set at 513 nm. The elastin content was calculated using the elastin standard curve and presented on a graph.

[0135] 4. Glycosaminoglycan (GAG)

[0136] For GAG quantification, the GAG content in the ECM-containing scaffolds was determined using the Blyscan™ sulfated glycosaminoglycan assay kit. The scaffolds were digested with freshly prepared papain extraction reagent (containing papain, sodium acetate, EDTA, and cysteine dissolved in sodium phosphate buffer) for 4 hours at 65 °C with occasional mixing. After removing the scaffold, the digested material was centrifuged at 10,000*g for 10 minutes and the supernatant was discarded. To the pellet, 1 ml of Blyscan™ was added and mixed by shaking every 5 minutes for 30 minutes. The mixture was centrifuged at 13,000*g for 10 minutes and pat dried to remove unbound dye. 0.5 ml of the dye dissociation reagent was added, mixed for 10 min, and centrifuged at 13,000*g for 5 minutes. Two hundred pl of the dye from each sample was transferred to a 96-well plate and absorbance was read at 656 nm. The total sulfated GAG content was calculated from the standard curve.

[0137] 5. Growth factors

[0138] VEGF and bFGF were measured with DuoSet™ ELISA assay. The ECM from the scaffolds was extracted by incubating them in an extraction buffer containing 2 M urea and 10 mg / mL heparin in 50 mM Tris (pH 7.4) at 4°C for 24 hours. The extract was centrifuged at 13,000*g for 10 min in a cold centrifuge at 4°C. The supernatant was collected and analyzed as per the manufacturer’s procedure. The absorbance was measured at 450 nm with correction set at 540 or 570 nm. The concentrations of VEGF and bFGF in the samples were calculated using the standard curve. Similarly, TNF-a and IL- 10 were quantified by ELISA assay kits per the protocol provided by the manufacturer.

[0139] Immunogenicity testing by macrophages

[0140] Human monocytes isolated from the peripheral blood of healthy donors were obtained from the elutriation core facility located at UNMC. 2 * 106monocytes per 10 cm2dish were cultured in RPMI supplemented with 10% FBS and 1% Pen-Strep along with 20 ng / ml of M-CSF. The media was replaced with fresh M-CSF every two days until 7 days to differentiate into naive macrophages (M0). These macrophages were then polarized into Ml and M2 macrophages using a cocktail of growth factors for another 3 days. Ml macrophages were induced with 100 ng / ml TNF-a and 100 ng / ml IFNy along with 20 ng / ml M-CSF. M2 macrophages were induced with 40 ng / ml IL4, 20 ng / ml IL- 13, and 20 ng / ml M-CSF. The respective factors were added to the media every two days. The Ml and M2 macrophages were then gently scraped and centrifugated at 1000xg for 5 minutes to obtain cell pellets. The pellets were then resuspended in the media and counted. 0.5 x io6cells were added to the pristine and decellularized scaffolds (obtained from freeze-thawing and SDS treatment) and allowed the cells to attach to the scaffolds for 20 minutes in a minimal volume of media. Then media containing Ml and M2 inducing factors were added in respective sets and cultured for 3 days to test the immunogenic response of the decellularized scaffolds. The pristine and decellularized scaffolds were collected and processed using the immunostaining protocol described next for Ml and M2 markers after 3 days. The levels of TNF-a and IL-10 were measured at 4, 24, 48, and 72 hours in respective conditions by ELISA assay kits.

[0141] Recellularization

[0142] Recellularization of dECM-decorated scaffolds was tested with human adult dermal fibroblasts and keratinocytes (HaCaT). HaCaT cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin. 2 x 105cells / scaffold were cultured on the dECM-decorated scaffolds placed in a 48-well low attachment plate. The media was changed on alternative days until 7 days. Cell seeding efficiency was measured by collecting the remaining cells that were present in the media after 6 hours by CCK-8 assay (as per assay protocol). The scaffolds were assessed for cell proliferation by CCK-8 assay at 0, 1, 3, 5, and 7 days and stained with Ki67, smooth muscle actin (fibroblasts), and cytokeratin 8 (keratinocytes) to visualize the recellularization of the dECM-decorated scaffolds on day 7.

[0143] Mechanical testing

[0144] The mechanical properties of the pristine, cellularized, and dECM-decorated scaffolds (SDS and freeze-thaw) were assessed by compression testing using a mechanical tester (Univert Model UV-200-01, Cell Scale Biomaterials Testing), with a 10-N load cell and a displacement rate of 1.2 mm / minute. For each condition, scaffolds with 8 mm diameter and 2 mm thickness were used. The experimental data was collected and processed using the UniVert software. Data was obtained for each scaffold, and the corrected force and displacement were plotted to depict the compression ability of the scaffolds.

[0145] Immunofluorescence studies

[0146] The cellularized and dECM-decorated scaffolds were washed with 1 xDPBS thrice and fixed with 4% buffered formaldehyde for 15 minutes at room temperature. The scaffolds were then washed twice with 1 xDPBS to remove remnants of fixative. The scaffolds were blocked with a blocking buffer containing 0.3% Triton™ X-100 and 1% BSA in 1 xDPBS for 1 hour. Primary antibodies (Collagen 1A1, fibronectin, Cytokeratin 9, Ki67, and alpha-smooth muscle actin), diluted 1 : 1000 in blocking buffer, were added to the scaffolds and incubated at 4°C overnight. For macrophage staining, an antibody combination of CD68 (common macrophage marker) and iNOS (pro- inflammatory, Ml) or CD206 (anti-inflammatory, M2) (1 : 1000) were added to the scaffolds. On the following day, the primary antibody was removed, and the scaffolds were washed thrice with 1 *DPBS. Then, secondary antibodies tagged with a fluorophore (diluted 1 :10000 in blocking buffer) were added to the scaffolds and incubated in the dark at 4°C for 1 hour. The secondary antibody was washed with 1 *DPBS twice and Hoechst 33342 was added to counterstain the nucleus and DNA present in the scaffold for 5 min. The scaffolds were washed and observed using Zeiss LSM 800 with an Airyscan microscope. The data were acquired and processed with Zeiss ZEN 2010 software and presented as images. The collagen, fibronectin, and colocalization of the markers were quantified using ImageJ from images obtained from three independent experiments.

[0147] Subcutaneous implantation

[0148] Sprague-Dawley rats (8-10 weeks old) were purchased from Charles River laboratories for the experiment. All animal procedures were performed under the approved UNMC IACUC protocol No. 17-103-11-Fc. All scaffolds were sterilized using ethylene oxide gas for 24 hours before implantation. For this study, 6 different scaffolds- RAS, dECM-decorated RAS (freeze-thawing), dECM-decorated RAS (SDS treatment), LES, dECM-decorated LES (freeze-thawing) and dECM-decorated LES (SDS treatment) - were tested. A total of 12 scaffolds of each type were used for each time point to be assessed. The animals were anesthetized using 2% isoflurane gas and kept on a heating pad to maintain body temperature at 37°C. Sterile eye ointment was applied to the anesthetized animal to protect and lubricate the eyes. The dorsal side of the animals was shaved and cleaned thrice with povidone-iodine solution and 70% ethanol. Four subcutaneous pockets were made through 1.5 cm incisions at the supraspinal area on the dorsum. Each sample was placed into a subcutaneous pocket by a tweezer, and the pocket was closed with a stapler. Each animal received four different scaffolds maintained in a predetermined sequence. The animals were euthanized at 7- and 14 days post-implantation. Tissues with the scaffolds were collected and placed in formalin for use in histological evaluation.

[0149] Histological analysis

[0150] All samples were fixed using buffered formalin for 2 days. They were washed in PBS and processed for paraffin embedding. The samples were dehydrated in a graded ethanol series (70%, 90%, 95%, and 100%), embedded in paraffin, and then 5-pm-thick paraffin slides were prepared for histological analysis. H&E and Masson trichrome staining were performed using standard protocols. The slides were scanned, and the tissue images were visualized by Aperio imagescope (Leica biosystems, IL). Cell infiltration (%), collagen content, and neovascularization were quantified using ImageJ.

[0151] Statistical analysis

[0152] All in vitro experiments and histology evaluations were repeated at least three times, and the data was represented as mean ± standard error. All statistical analysis was performed using GraphPad Prism 10. One-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test was conducted to compare samples. Statistical significance was expressed as ns (not significant, p < 0.05), * (p < 0.05), ** (p < 0.01), *** (p < 0.001) and **** (p < 0.0001).

[0153] Results

[0154] Deposition of ECM on 3D scaffolds consisting of hierarchically assembled nanofibers with controlled alignments

[0155] Fig. 1 A shows a schematic illustrating the design of this study. Fig. 1 A shows the fabrication procedures following established protocols (Chen, et al., Adv. Mater. (2020) 32:e2003754; Chen, et al., Acta Biomater. (2020) 108: 153-167; Chen, et al., Sci. Adv. (2021) 7:eabg3089). Briefly, PCL nanofiber mats were fabricated using electrospinning. Then, the nanofiber mats were cut in liquid nitrogen. The nanofiber mats, with predetermined geometric dimensions achieved through cutting, were expanded in a NaBEL solution to achieve 3D laterally expanded nanofiber scaffolds (LES). Alternatively, one side of the cut nanofiber mat was fixed through thermal treatment and then it was expanded to form 3D radially aligned nanofiber scaffolds (RAS). These two types of scaffolds were then utilized to evaluate and compare the rate of deposition of ECM. Fig. 1A also shows the procedures of the generation of dECM- decorated 3D expanded nanofiber scaffolds. Fibroblasts were seeded to the RAS and LES and cultured for 50 days to deposit ECM. Subsequently, dECM-decorated 3D expanded nanofiber scaffolds were achieved by decellularization through either freezethawing or SDS treatment. Fig. 1 A also illustrates the further in vitro and in vivo studies. dECM-decorated 3D RAS and LES were either repopulated with cells or subcutaneously implanted in rats to examine cell proliferation, immune response, cell infiltration, and angiogenesis. Fig. IE show photographs of RAS and LES after fabrication. SEM images indicate that the LES has layered structures and aligned fibers present along the length of the scaffolds (Fig. IF). SEM images of RAS revealed that it consists of numerous layered nanofiber membranes and aligned nanofibers along the radial direction (Fig. IF).

[0156] The growth of fibroblasts was first examined on the two types of scaffolds, and the cell proliferation was assessed at different time points by cell counting kit-8 (Fig. IB). HDFs on LES exhibited higher cell viability than those grown on RAS on day 1. With increasing time, the cells proliferated at an exponential rate in both types of scaffolds. Cell proliferation on LES was higher than in RAS at all time points. One possible explanation for this observation is that the uniform porous architecture of LES allows for the seeding and infiltration of a higher number of cells during the initial seeding within the scaffolds. Once the cellularization was confirmed, the cellularized RAS and LES were then decellularized at day 50 to obtain dECM scaffolds through either freeze-thaw cycles or SDS treatment. To evaluate the efficiency of decellularization, the DNA content present before and after decellularization was measured. When compared to the cellularized scaffolds, the DNA content in SDS and DNase 1 -treated scaffolds was reduced to 17 ng (RAS) and 10 ng (LES), while freeze drying along with DNase 1 treatment decreased the DNA content to 23 ng and 12 ng (LES) (Fig. 1C). DNA content present in the decellularized scaffolds was higher with SDS treatment or freeze-thawing alone, as illustrated in Fig. 1H. dECM scaffolds produced by SDS treatment alone exhibited approximately 310 ng (RAS) and 415 ng (LES) of remaining DNA, while those generated by freeze-thawing showed around 398 ng (RAS) and 541 ng (LES). Due to the additional treatment of dECM scaffolds with DNase 1 after decellularization, the DNA content was greatly reduced in the decellularization conditions. Owing to its uniform porous structure, LES may be more amendable to SDS treatment and freeze-thaw cycles. In contrast, RAS comprises of radially aligned nanofibers with a dense center, which can impede access to decellularizing agents. Consequently, both decellularization methods could more effectively reduce DNA levels in LES compared to RAS with the inclusion of DNase- 1 treatment (Fig. 1C). Therefore, rigorous decellularization via SDS treatment proved to be more effective than freeze-thaw cycles in RAS. However, these two decellularization methods showed no evident morphological differences in RAS and LES (Fig. ID). On the contrary, decellularization by SDS is more disruptive than the freeze-thaw method in the 2D scaffold (Xing, et al., Tissue Eng. Part C Methods (2015) 21 :77-87). Minimal ECM loss was observed during decellularization in SDS and freeze-thaw cycles along with additional DNase treatment steps. It was theorized that the DNase treatment removed cellular DNA remnants and ensured intact ECM architecture by reducing excessive SDS and freeze-thaw treatments. Hence, the work indicates that the architecture of the scaffold and the decellularization method dictate the decellularization efficacy. dECM morphology on RAS and LES

[0157] To examine the morphology of deposited ECM, scanning electron microscopy (SEM) imaging of cell-seeded scaffolds before and after decellularization was performed (Fig. 2). The left panels in Fig. 2A and D show the distribution of cells as well as the ECM on LES and RAS. At higher magnification, ECM nanofiber bundles bound with proteins and adhesive molecules were observed surrounding the cells in the cellularized scaffolds (middle and right panels in Fig. 2 A and D). ECM deposition was also observed along the PCL nanofibers. In LES, decellularization by SDS and freezethawing caused the effective removal of cells as well as some of the ECM material (Fig. 2B and 2C). However, in RAS, the freeze-thawing method eliminated cells while retaining most of the ECM along with a few cell debris on the scaffold (Fig. 2E). SDS treatment in RAS removed the cells efficiently but caused the removal of adhesion proteins found in the ECM, leaving the ECM network alone (Fig. 2F). These observations from SEM images indicate that freeze-thawing can effectively retain ECM and its related proteins but can leave cell debris in a dense nanofiber scaffold such as RAS than in LES. On the contrary, SDS treatment showed efficient decellularization with the loss of some ECM proteins. H&E staining data showed the cellular distribution in the LES and RAS (Fig. 2G). The efficiency of decellularization by SDS and freezethawing was also observed as cells were absent in dECM decorated LES and RAS (Fig. 2G).

[0158] Characterization of ECM content

[0159] To visualize the distribution of cells and ECM content before and after decellularization, immunostaining for abundant ECM proteins such as collagen and fibronectin was performed. The distribution of fibroblasts in the LES and RAS was observed by staining the nucleus with Hoechst 33342 in blue. Cells were growing on both scaffolds, as evidenced by the presence of the cell nuclear staining (Fig. 3 A and D). In the LES, cells layered the collagen in an intricate network throughout the scaffold (Fig. 3 A), while in the RAS, they grew along the aligned nanofibers and deposited collagen (Fig. 3D). Decellularization by freeze-thawing effectively removed cells (evidenced by the absence of nuclear staining) and retained collagen on the scaffold in both RAS and LES (Fig. 3B and E). However, treatment with SDS induced some structural damage to the collagen deposited in LES (Fig. 3C), while it remained intact in RAS (Fig. 3F). The mean collagen staining showed a similar trend as discussed in LES and RAS (Fig. 3G).

[0160] Immunofluorescence staining of fibronectin on cellularized scaffolds revealed its localization in discrete locations interspersed among fibroblasts in LES (Fig. 4A). In contrast, in RAS, it is presented as fibrous structures deposited adjacent to the cells along the nanofibers (Fig. 4D). Freeze-thawing exhibited no influence on the localization of fibronectin on LES and RAS (Fig. 4B and E). However, SDS treatment severely affected the architecture of fibronectin in both LES and RAS (Fig. 4C and 4F, Fig. 3G). The immunostaining results of collagen and fibronectin indicate that fibroblasts grow along the aligned nanofiber in both RAS and LES. However, they deposit ECM proteins in a unique fashion. In RAS, ECM is deposited along the nanofiber orientation, while in LES, it is layered in a branched fashion, providing higher coverage to the cells.

[0161] ECM retention in dECM-decorated scaffolds

[0162] To assess the retention of ECM, the ECM protein levels before and after decellularization were quantified. As depicted in Fig. 5, the content of all ECM proteins in the cellularized scaffolds increased over time, with measurements taken at 20, 30, 40, and 50 days. However, SDS treatment during decellularization resulted in a decrease in ECM content, whereas freeze-thawing either decreased or showed a similar amount of ECM proteins compared to cellularized scaffolds. Notably, LES exhibited a higher promotion of ECM deposition than RAS. In Fig. 5A-D, quantification of cellularized RAS after 50 days showed -150 pg of collagen, -125 pg of fibronectin, -115 pg elastin, and -55 pg of GAG proteins. However, SDS treatment caused a significant decrease in ECM content in RAS, with -80 pg of collagen, -60 pg of fibronectin, -75 pg of elastin, and -35 pg of GAG remaining in the scaffold. The freeze-thawing method did not lead to a significant loss of ECM, with RAS retaining -115 pg, 109 pg, 103 pg, and 35 pg of collagen, fibronectin, elastin, and GAG proteins, respectively. In LES, the average content of ECM proteins was consistently higher than in RAS. Even as early as 20 days, cellularized LES exhibited a greater amount of ECM compared to RAS (Fig. 5A-5D). However, no significant difference in these proteins was observed between LES and RAS after 50 days of cellularization (Fig. 5A-5D). Surprisingly, decellularization by SDS and freeze-thawing did not significantly affect the ECM content, except for a reduction in collagen. At day 50, cellularized LES promoted the deposition of -190 pg collagen, -125 pg fibronectin, -135 pg elastin, and -55 pg GAG along its nanofibers (Fig. 5A-5D). After decellularization by SDS and freezethawing, collagen significantly reduced to -140 pg and -160 pg, respectively (Fig. 5A). However, the content of all other proteins after both SDS and freeze-thawing was not statistically different from the cellularized LES (Fig. 5B-5D).

[0163] In addition to assessing various matrix proteins, the levels of growth factors abundantly bound to the ECM were measured. Specifically, the concentrations of VEGF and bFGF in the scaffolds were evaluated. Similar to the ECM proteins, growth factors exhibited an increase in their levels over time in both LES and RAS (Fig. 5E and 5F). Cellularized RAS and LES demonstrated the highest levels of these growth factors, with no apparent differences between them. For VEGF, cellularized RAS contained -1500 pg per scaffold, while LES had -1550 pg per scaffold after 50 days of culture (Fig. 5E). However, decellularization by SDS treatment significantly decreased VEGF levels in RAS to -800 pg per scaffold (Fig. 5E), whereas freeze-thawing preserved VEGF levels in RAS. Contrastingly, both decellularization methods showed no significant changes in VEGF levels in LES (Fig. 5E). The bFGF levels in cellularized RAS and LES after 50 days were -1500 and -1600 pg per scaffold, respectively (Fig. 5F). However, SDS- treated and freeze-thawed RAS showed decreased concentrations to -1100 and -1400 pg per scaffold, respectively (Fig. 5F). No change was observed in bFGF levels in LES after decellularization. These findings collectively indicate that LES promotes more ECM deposition than RAS due to its architecture. Decellularization by SDS treatment and freeze-thawing methods significantly affects ECM content in RAS more than in LES. Furthermore, the loss of matrix proteins and growth factors is higher in SDS- treated RAS. The retention of ECM proteins and growth factors is greater in LES after decellularization. Together, the intricate ECM deposition observed in Fig. 3A and 4A, combined with higher ECM content retention in LES, indicates that LES can withstand harsh SDS treatment and freeze-thaw cycles while preserving ECM. Interaction between macrophages and dECM-decorated scaffolds

[0164] Human fibroblasts possess immunosuppressive functions and the ECM deposited by these cells shares similar immunomodulatory properties (Davidson, et al., Nat. Rev. Immunol. (2021) 21 :704-717; Cavagnero, et al., Front. Immunol. (2022) 13: 1058862; Gauthier, et al., Front. Immunol. (2023) 14: 1137659). However, the understanding of dECM materials is currently limited, hindering their application in regenerative medicine. Assessing the inflammatory properties of dECM materials involves examining the activation of macrophages, specifically Ml and M2 types. Earlier studies on dECM materials often overlooked the pro- and anti-inflammatory functions of macrophages (Junka, et al., ACS Appl. Bio Mater. (2022) 5:5634-5644; Xing, et al., Tissue Eng. Part C Methods (2015) 21 :77-87; Xing, et al., Adv. Funct. Mater. (2014) 24:3027-3035). Macrophages can be classified into Ml and M2. Ml macrophages become activated in response to remnant DNA after ineffective decellularization, releasing pro-inflammatory factors such as TNF-a, IL-6, and IL-ip, which may lead to inflammation and graft rejection (Kasravi, et al., Biomater. Res. (2023) 27: 10). Conversely, M2 macrophages are stimulated by the presence of ECM proteins like elastin, hyaluronic acid, and growth factors (VEGF, FGF, and TGF-P). They promote the release of anti-inflammatory cytokines, such as IL- 10, which plays a crucial role in immune regulation, ECM deposition, and tissue remodeling (Sell, et al., Polym. Int. (2007) 56: 1349-1360; Franz, et al., Acta Biomater. (2013) 9:5621-5629; Reing, et al., Biomaterials (2010) 31 :8626-8633.). Understanding the interplay between dECM materials and macrophage activation can provide valuable insights for utilizing these materials in regenerative applications.

[0165] The pro- and anti-inflammatory responses of the decellularized scaffolds were investigated by seeding Ml and M2 macrophages on pristine and decellularized scaffolds. Immunofluorescence staining of Ml (iNOS) and M2 (CD206), along with naive macrophages (CD68) was performed, along with the quantification of TNF-a and IL-10 secretion at different time points (Fig. 6). iNOS Immunostaining revealed that pristine scaffolds exhibited less attachment of Ml polarized macrophages on both RAS and LES (Figs. 6A and 6D). On the contrary, freeze-thawed dECM-decorated RAS showed higher Ml polarization than SDS-treated RAS (Figs. 6D). Quantitation of the staining showed 7%, 22%, and 14% colocalization of iNOS with CD68 in pristine RAS and dECM-decorated RAS produced by freeze-thawing and SDS treatment, respectively (Fig. 6E). Next, the inflammatory response of dECM was evaluated by measuring TNF- a secretion from Ml macrophages cultured on the RAS at 4, 24, 48, and 72 hours postseeding. In general, all the scaffolds showed high levels of TNF-a at 4 hours (Fig. 6F). These levels decreased with time, as observed at 24, 48, and 72 hours. Of the three scaffolds, freeze-thawed dECM-decorated RAS showed higher secretion of TNF-a followed by SDS-treated dECM RAS at 4 hours post-seeding. Pristine RAS showed comparatively lower levels of TNF-a than the dECM-decorated RAS. At 72 hours, TNF-a levels decreased, and no significant difference was observed between the dECM- decorated scaffolds (Fig. 6F). iNOS staining was higher in freeze-thawed dECM- decorated LES followed by SDS-treated dECM-decorated LES, and the least was observed in pristine LES (Fig. 6G). Nearly 5%, 15 %, and 11% colocalization of iNOS with CD68 was observed in pristine RAS and dECM-decorated RAS produced by freeze-thawing and SDS treatment, respectively (Fig. 6H). Further analysis of TNF-a levels revealed significant differences in cytokine secretion between the pristine and dECM-decorated LES at early 4 and 24 h after plating (Fig. 61). No change was observed between the dECM-decorated LES samples generated by freeze-thawing and SDS treatment.

[0166] Concurrently, the anti-inflammatory function of dECM-decorated scaffolds was studied by staining them with CD206 (M2 macrophages). After 3 days of culture, CD206-positive macrophages significantly increased on pristine and dECM-decorated scaffolds (Figs. 6A and 7A). CD206 colocalization with CD68 was higher in all the samples, and no evident differences were observed between these samples (Figs 6B and 7B). The IL- 10 levels released into the media by the M2 macrophages was also assessed at different time points. In RAS, an increase in IL-10 over time was observed. However, a significant difference between pristine and dECM-decorated RAS was only observed at 4 hours (Fig. 6C). When comparing pristine LES with the dECM-decorated ones, an increase in IL-10 was observed at 4 and 24 h time points (Fig. 7C). From the in vitro inflammation evaluation, a higher pro-inflammatory profile in RAS was observed than in LES in both freeze-thawed and SDS-treated dECM samples. Furthermore, the SDS- treated samples showed a lower inflammatory response than freeze-thawed conditions in RAS, while both treatment conditions showed a similar response in LES. TNF-a levels were higher in both dECM conditions in RAS than in LES until 48 hours. On the contrary, anti-inflammation was high in both the treatment conditions in dECM- decorated LES and RAS, with no significant difference in IL- 10 levels. High DNA content can trigger inflammation, which was observed in freeze-thawed and SDS treatments in RAS and LES. However, RAS showed a higher inflammatory response than LES, as it contains a dense core where freeze-thawing is less efficient than SDS treatment in removing the DNA. Additionally, the presence of ECM proteins promotes anti -inflammation, validating the increase in IL- 10 levels and CD206 staining observed in dECM-decorated scaffolds. dECM-decorated RAS and LES have been shown to negate pro-inflammation while promoting anti-inflammatory properties. Interestingly, along with DNA, other components of the cell such as the membrane and mitochondria can elicit an immune response.

[0167] Recellularization of dECM-decorated scaffolds with fibroblasts and keratinocytes

[0168] To evaluate the capability of the dECM-decorated scaffolds to assist in cell growth, viability, and function, human fibroblasts were seeded on the scaffolds and cultured for 7 days. The seeding efficiency of these cells was calculated by quantifying DNA content after collecting unattached cells from the scaffolds at 8 hours post-seeding. In RAS, the fibroblasts seeding efficiencies were 48.8± 3.8%, 72.9± 1.2%, and 71.8± 0.9% for pristine scaffolds and dECM-decorated scaffolds produced by freeze-thawing and SDS treatment (Fig. 8A). In LES, the efficiencies were 61.2.8± 1.1%, 84.6± 2.3% and 84± 0.8% for the three groups (Fig. 8 A). The cell attachment efficiency was higher in all the dECM-decorated scaffolds compared to the pristine ones (Fig. 8A). No significant difference was observed between dECM-decorated LES and RAS generated by freeze-thawing and SDS treatment. The proliferation of fibroblasts on these scaffolds was analyzed by CCK8 at different time points. It was observed that the cells populated the scaffolds by more than 91% in all the dECM samples by day 7 in both types of scaffolds (Fig. 8B and 8C). The proliferation rate was higher in dECM layered scaffolds than in pristine scaffolds and tended to increase with time until reaching confluency at day 7 (Fig. 8B and 8C). No apparent difference was found between the freeze-thawed and SDS-treated samples in both scaffolds. Immunostaining of fibroblasts with Ki67 showed cells populating the scaffolds on day 7 in RAS and LES (Fig. 8D-8E). Similarly, HaCaT cells stained with cytokeratin 9 showed a similar trend in the proliferation of the cells (Fig. 8F-8G). All the dECM-decorated scaffolds irrespective of RAS or LES showed no variance in their growth. Within the two decellularization methods, no difference in the repopulation of the cells was observed but were higher than the pristine scaffold. This indicates that the ECM on scaffolds provides the mechanical and signaling cues necessary for accelerated cell acclimatization, attachment, and survival which is lacking in pristine scaffolds. The data cumulatively indicates that cell-derived ECM layered scaffolds could promote cellular properties by providing a conducive environment in a 3D architecture.

[0169] Mechanical load performance of dECM-de corated scaffolds

[0170] ECM is crucial to the mechanical stress and strain of tissues. It was examined whether ECM decoration by the cells provides any mechanical properties to the scaffolds and the effect of decellularization on them. A compression strength test was conducted to compare the pristine scaffold separately with cellularized and dECM scaffolds obtained by SDS and freeze-thaw methods (Fig. 8H). In LES, the pristine scaffold showed the least force for compression, nearly 0.3 ± 0.02 N. However, deposition of ECM increased the force to 2.07 ± 0.13 N in cellularized, 1.54 ± 0.34 N, and 1.61 ± 0.48 N in SDS and freeze-thaw treated LES, respectively. No significant difference was observed between the SDS-treated and freeze-thaw methods. Compared to LES, RAS required a higher force about 13.05 ± 4.45 N to compress (Fig. 8H and 81). This change can be attributed to the presence of a dense core in RAS. Cellularized RAS required 20.21 ± 8.01 N to compress. Compression forces of decellularized RAS by SDS and freeze-thawing were 14.71 ± 5.11 N and 19.49 ± 7.83 N, respectively (Fig. 81). These data imply that the presence of ECM on the scaffold irrespective of orientation can improve their mechanical strength. With the modified decellularization protocols, minimal loss in mechanical attributes was observed. Based on the mechanical properties, dECM RAS can be more suitable for load-bearing tissues such as bone while dECM LES for soft tissue regeneration.

[0171] In Vivo Evaluation of dECM-decorated scaffolds

[0172] To examine the influence of layered dECM on cell infiltration and angiogenesis in 3D expanded nanofiber scaffolds, dECM-decorated LES and RAS, along with pristine scaffolds, were subcutaneously implanted into rats for 1 week and 2 weeks. H&E staining revealed that, compared to pristine scaffolds, dECM-decorated scaffolds exhibited better cell penetration in both LES and RAS (Fig. 9A- 9B). Due to the porous nature of RAS and LES, coupled with dECM deposition, 70-80% of the scaffold was populated by cells in dECM samples after 14 days in rats (Fig. 9C). Pristine RAS and LES showed 40-60% cell coverage after 14 days (Fig. 9C). Both of the decellularization methods exhibited similar cellular infiltration, significantly surpassing those of pristine scaffolds. Additionally, LES decorated with dECM showed elevated cell infiltration after 7 days of implantation compared to RAS (Fig. 9A- 9B). However, no significant difference was observed between dECM-decorated LES and RAS after 14 days. Furthermore, quantitation of neo-angiogenesis showed a substantial increase in the number of newly formed blood vessels in all dECM-layered samples compared to pristine RAS and LES, with no difference in the decellularization methods (Fig. 9D). Blood vessel formation was higher in all the samples at 14 days than at 7 days (Fig. 9D). Trichrome staining of the tissues showed an increase in the collagen deposition (in blue) as early as 7 days in all dECM-decorated scaffolds compared to pristine scaffolds (Fig. 10). The deposited collagen further increased from day 7 to day 14 in dECM-layered RAS and LES (Fig. 10). Collagen quantitation from the tri chrome staining confirmed these findings (Fig. 10). Altogether, the in vivo data indicate that dECM-decorated, 3D expanded nanofiber scaffolds exhibit greater cell infiltration, higher angiogenic function, and better regenerative properties than their counterparts (Zhang, et al., Bioact. Mater. (2022) 10: 15-31; Meizlish, et al., Annu. Rev. Immunol. (2021) 39:557-581). The cell- derived dECM scaffolds obtained herein has many attributes that make it distinctive from other scaffolds reported. The dECM scaffolds are porous to enable better cell infiltration and 3D structures enable the cells to deposit ECM in a natural ECM-like manner, thereby mimicking native tissues. The porosity of the scaffold can be controlled to allow ECM deposition similar to dense tissues. Cell-derived ECM scaffolds can withstand the harsh environment of decellularization and retain maximum ECM both in architecture and content with the modified decelluarization protocols. These nanofibrous scaffolds can be employed to test different aspects of ECM either by genetically modifying cells or inducing cells with potent inducers to modify the expression of proteins present in the ECM (Lamande, et al., Anat. Rec. (2020) 303: 1527-1542; Naba, A., Mol. Cell. Proteomics (2023) 22: 100528). Specifically, dECM LES scaffolds can be used in wound healing, deep tissue, cardiovascular, and neural regeneration applications while dECM RAS can be employed in hard tissues such as bone. These dECM scaffolds can also be employed to address various aspects of cancer biology where treatment remains palliative. Such cell-derived ECM scaffolds can promote basic research and therapeutic approaches.

[0173] Expanded 3D scaffolds comprising hierarchically assembled nanofibers with controlled alignments were fabricated. Cell-derived ECM was deposited by culturing cells on these scaffolds, which were then decellularized using either freeze-thawing or SDS treatment to create dECM-decorated scaffolds. SDS detergent-based decellularization was more effective in decreasing DNA content compared to the freezethawing method in RAS. No difference was observed between the decellularization methods in LES. However, the addition of DNase treatment enhanced the decellularization efficiency. Deposition of ECM was guided by the orientation of the nanofibers in both RAS and LES. Retention of ECM proteins and growth factors was higher in dECM-decorated LES than in dECM-decorated RAS. These scaffolds exhibited higher immunosuppressive capabilities, decreased pro-inflammatory, and elevated anti-inflammatory profile. The dECM-decorated scaffolds showed efficient recellularization and directional cell alignment due to their 3D architecture. The dECM- decorated scaffolds also showed promising cell infiltration and neo-angiogenesis as early as day 7 after subcutaneous implantation. The cell-derived ECM scaffold acquired in this study stands out from other scaffolds owing to its numerous distinctive features. The dECM-adomed scaffolds boast porosity, facilitating superior cell infiltration, while their 3D configuration enables cells to deposit ECM in an ECM reminiscent of native tissues. The scaffold’s porosity can be regulated to facilitate ECM deposition akin to that found in dense tissues. dECM-decorated scaffolds exhibit resilience in the demanding conditions of decellularization, maintaining optimal ECM integrity in both structure and composition. Lastly, dECM-decorated scaffolds can be tailored to suit all types of tissues. Therefore, the development of expanded 3D scaffolds, featuring hierarchically assembled nanofibers with controlled alignments, combined with cell-based ECM deposition and effective decellularization, are a versatile biomaterial for tissue regeneration applications.

[0174] 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 for synthesizing a three-dimensional scaffold comprising extracellular matrix, wherein said method comprises:A) culturing cells within a three-dimensional scaffold comprising polymer nanofibers; andB) decellularizing the three-dimensional scaffold, thereby synthesizing said three- dimensional scaffold comprising extracellular matrix.

2. The method of claim 1, wherein said three-dimensional scaffold comprising polymer nanofibers is an expanded electrospun nanofiber mat.

3. The method of claim 1, wherein the polymers comprise poly caprolactone (PCL), poly(lactide-co-epsilon-caprolactone) (PLCL), polyglycolic acid (PGA), poly(lactic-co- glycolic) acid (PLGA), or any combination thereof.

4. The method of claim 1, wherein said nanofibers further comprise at least one surfactant.

5. The method of claim 1, wherein said three-dimensional scaffold further comprises a gelatin coating.

6. The method of claim 1, further comprising synthesizing the three-dimensional scaffold comprising polymer nanofibers prior to step A).

7. The method of claim 6, wherein the three-dimensional scaffold comprising polymer nanofibers is synthesized by expanding an electrospun nanofiber mat by exposing the electrospun nanofiber mat to gas bubbles.

8. The method of claim 1, wherein said decellularization of step B) comprises exposing the three-dimensional scaffold comprising polymer nanofibers cultured with cells in step A) to an ionic detergent or to freeze-thaw cycles.

9. The method of claim 8, wherein said ionic detergent is sodium dodecyl sulfate (SDS).

10. The method of claim 1, wherein said decellularization of step B) comprises adding DNase to the three-dimensional scaffold comprising polymer nanofibers cultured with cells in step A).

11. The method of claim 1, wherein the nanofibers of the three-dimensional scaffold are radially aligned.

12. The method of claim 1, wherein the nanofibers of the three-dimensional scaffold are laterally aligned.

13. The method of claim 1, wherein said cells comprise fibroblasts.

14. A three-dimensional scaffold comprising extracellular matrix synthesized by a method of any one of claims 1-13.

15. A three-dimensional scaffold comprising extracellular matrix, wherein said three- dimensional scaffold is an expanded electrospun nanofiber mat, and wherein said extracellular matrix is deposited within said three-dimensional scaffold by cells.

16. A composition comprising the three-dimensional scaffold of claim 14 or claim 15 and a pharmaceutically acceptable carrier.

17. 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 three-dimensional scaffold comprising of claim 14 or claim 15.

18. The method of claim 17, 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.

19. The method of claim 17, wherein the administration is injection into the area in need of treatment.

Images