Cellularized nerve regeneration graft and method for producing same

Abstract

A cellularized nerve regeneration graft is disclosed, comprising an electrospun biodegradable polymer conduit having an exterior surface and a luminal space, a plurality of fibroblasts seeded on the exterior surface of the conduit, and a system filling the luminal space of the conduit. The system may comprise a hydrogel matrix or a reinforced hydrogel matrix and Schwann cells. The cellularized nerve regeneration graft may be used to repair peripheral nerve injuries. Also disclosed is a method of making the cellularized nerve regeneration graft.

Description

[Technical field]

[0001] The present disclosure relates to cellularized nerve regeneration grafts for use in repairing peripheral nerve injuries and methods for making same. [Background technology]

[0002] Peripheral nerve injury (PNI) represents a direct medical cost of over US$150 billion annually to the global economy. There are also other costs, including lost wages, indirect expenses, and other long-term socio-economic costs due to functional loss PNI, for individuals and their communities, especially since the median age for such injuries is around 35 years. Such injuries may result from physical trauma, cancer, or other neurological pathologies. The associated socio-economic costs have increased significantly worldwide, especially in developing countries where workplace injuries occur frequently in jobs involving manual labor. Despite significant advances in medicine and increasing precision in neurosurgery, nerve injuries still leave patients with little hope of full functional recovery, and in many cases, patients lose sensory and / or motor function. Furthermore, our understanding of neuropathologies in both PNS and CNS disorders is in its infancy, and solutions to degenerative neurological diseases are scarce. Previous experiments have demonstrated the efficacy of generating multi-layered biopolymer scaffolds that guide the growth of supporting cells into a matrix that mimics the human extracellular matrix. See, e.g., Garrison, CM, Singh-Varma, A, Pastino, AK, et al., "A multilayered scaffold for regeneration of smooth muscle and connective tissue layers," J Biomed Mater Res. 2020;109:733-744. Current understanding of tissue development suggests that the ECM, supporting cells, and regulatory molecules such as growth factors play essential roles in tissue growth and remodeling.

[0003] Although surgical techniques to repair nerve damage are improving, there is still no “gold standard” to treat nerve injury. According to Dr. Susan E. MacKinnon, a pioneer in nerve transplant surgery, the best nerve graft, an autograft, is considered merely the “bronze standard.” Current tissue grafts can only partially restore functional and sensory capabilities in most patients after surgery. Furthermore, the cost of managing PNI is also increasing. Currently, nerve injury repair costs approximately $47,000 per patient at the time of repair, and is increasing 9.69% annually. Furthermore, our understanding of regenerating neurons and alleviating debilitating conditions of the nervous system is still in its infancy. Summary of the Invention

[0004] A cellularized nerve regeneration graft is disclosed, comprising an electrospun biodegradable polymer conduit having an exterior surface and a luminal space, a plurality of fibroblasts seeded on the exterior surface of the conduit, and a system for filling the luminal space of the conduit comprising a hydrogel matrix or a reinforced hydrogel matrix and Schwann cells. The reinforced hydrogel matrix comprises a hydrogel selected from the group consisting of RADA16 peptide, collagen, gelatin, alginate, hyaluronic acid, or any combination thereof, combined with growth factors, guidance cues, structural peptides, adhesion factors, other chemical agents, or any combination thereof. The electrospun biodegradable polymer conduit may comprise multiple layers, e.g., two layers, a first layer having aligned biopolymer fibers and a second layer having non-aligned biopolymer fibers. The electrospun biodegradable polymer may be a tyrosine-derived polymer or a tyrosol-derived polymer. The fibroblasts may be present in an amount of about 1.0×10 5 cells / cm 2The Schwann cells may form a continuous cell layer on the outer surface of the conduit at a concentration of greater than about 10 million cells / mL. The Schwann cells may be substantially uniformly distributed throughout the hydrogel matrix or reinforced hydrogel matrix within the luminal space of the conduit, for example at a concentration of greater than about 20 million cells / mL. The cellularized nerve regeneration graft may include a plurality of channels extending the length of the electrospun biodegradable polymer conduit and within the system. A plurality of the channels may be hollow and reinforced with Schwann cells.

[0005] Also disclosed is a method of making the cellularized nerve regeneration graft, comprising the steps of electrospinning a formulation of tyrosine-derived polymer or tyrosol-derived polymer to produce the electrospun biodegradable polymer conduit, culturing fibroblasts, seeding the fibroblasts on the outer surface of the electrospun biodegradable polymer conduit, generating human Schwann cells, embedding the human Schwann cells in the hydrogel matrix or reinforced hydrogel matrix to produce the system, seeding the system into the luminal space of the electrospun biodegradable polymer conduit, and incubating the seeded conduit to produce the cellularized nerve regeneration graft.

[0006] Also disclosed is a method of repairing a damaged peripheral nerve in a patient, comprising implanting a cellularized nerve regeneration graft into the patient.

[0007] Also disclosed is a method of making the cellularized nerve regeneration graft having a plurality of channels extending along the length of the electrospun biodegradable polymer conduit, comprising the steps of electrospinning a formulation of tyrosine-derived polymer or tyrosol-derived polymer to make the electrospun biodegradable polymer conduit, generating human Schwann cells, embedding the human Schwann cells in the hydrogel matrix or reinforced hydrogel matrix to make the system, culturing absorbent fibers with the human Schwann cells, and then loading the cultured absorbent fibers into the electrospun biodegradable polymer conduit in a longitudinal orientation or loading the absorbent fibers into the electrospun biodegradable polymer conduit in a longitudinal orientation. The method includes the steps of loading a spun biodegradable polymer conduit, adding a suspension containing human Schwann cells to the luminal space of the electrospun biodegradable polymer conduit to create a cultured absorbable fiber, inoculating the system into the luminal space of the electrospun biodegradable polymer conduit and between the cultured absorbable fibers, and incubating the inoculated conduit for about 1 week to about 6 weeks, where the absorbable fibers dissolve leaving the cellularized nerve regeneration graft with a plurality of longitudinal hollow channels in the absorbable fiber and, optionally, Schwann cells in the channels. The method further includes culturing fibroblasts and inoculating the outer surface of the electrospun biodegradable polymer conduit with the fibroblasts. The absorbable fibers may be made of polylactic-co-glycolic acid (PLGA), polyglycolic acid (PGA), tyrosine-derived polymers with high concentrations of polyethylene glycol (PEG), or any combination thereof. The spacing between the cultured absorbent fibers may be from about 20 μm to about 100 μm. [Brief description of the drawings]

[0008] 1A-1B are electron micrographs showing aligned and non-aligned fibers in a bi-layer sheet.

[0009] 2A-2B are electron micrographs showing aligned and non-aligned fibers in a bi-layer sheet.

[0010] 3A-3E are an example of an electrospun biodegradable polymer conduit with a reinforced hydrogel matrix and an embedded Schwann cell system enclosed within the luminal space.

[0011] 4A-4F are an example of various 3D printed parts used to assemble the cellularized nerve regeneration graft of the present disclosure.

[0012] FIG. 5 is a schematic diagram showing one example of how Schwann cells can be cultured on absorbable fibers to create longitudinal channels in a polymeric conduit.

[0013] FIG. 6 is a schematic diagram for producing a cellularized nerve regeneration graft of the present disclosure.

[0014] 7A-7D are an example of the construction of a multi-layer biopolymer fixture for initial cell culture.

[0015] 8A-8E are an example of the construction of a tyrosine-based biodegradable polymer conduit and fixture assembly.

[0016] 9A-9C are an example of a method for generating human Schwann cells and a graph of Schwann cell fold expression.

[0017] FIG. 10 shows a second method to generate human Schwann cells using lentivirally delivered transcription factors in combination with growth factors to promote differentiation of either iPSCs or fibroblasts (FBs) into Schwann cells.

[0018] 11A-11C show Schwann cells and fibroblasts cultured on opposite sides of an electrospun biodegradable polymer fiber scaffold.

[0019] FIG. 12 is a scheme for implanting the cellularized nerve regeneration graft of the present disclosure into a mouse peripheral nerve injury model.

[0020] FIG. 13 is an example of a magnified image of rat Schwann cells growing on fibers made of tyrosine-derived polycarbonate.

[0021] FIGS. 14A-C are cross-sectional confocal microscopy images showing rat Schwann cells growing within a collagen hydrogel matrix, showing that tyrosine-derived polymer fibers are dispersed throughout the matrix and maintain longitudinal nerve bundles within the hydrogel matrix. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] A method is disclosed for generating biodegradable polymeric conduits seeded with nerve supporting cells to promote axonal regeneration in PNI patients. The method involves the construction of a cellularized nerve regeneration graft (CNRG) suitable for connecting damaged peripheral nerve tissue. The CNRG is fabricated by combining stem cell biology, biomaterials, and 3D printing.

[0023] The cellularized nerve regeneration grafts disclosed herein utilize preferred multi-layered electrospun biodegradable polymer conduits seeded on the outer surface with fibroblasts (FB) and seeded in the luminal space with Schwann cells (SC). These Schwann cells may be derived via an induced pluripotent stem cell pathway to minimize graft rejection and promote axonal regrowth into a favorable regenerative environment.

[0024] A cellularized nerve regeneration graft is disclosed that includes an electrospun biodegradable polymer conduit having an exterior surface and a luminal space, a plurality of fibroblasts seeded on the exterior surface of the conduit, and a system that fills the luminal space of the conduit. The system includes a hydrogel matrix or a reinforced hydrogel matrix and Schwann cells. The cellularized nerve regeneration graft may be a solid structure that contains Schwann cells throughout the luminal space and has fibroblasts located on the exterior surface. The Schwann cells may grow three-dimensionally throughout the hydrogel matrix or the reinforced hydrogel matrix.

[0025] The patient is prepared for surgery and a cellularized nerve regeneration graft containing live cells and growth factors is implanted into the patient by suturing or adhering the ends of the graft to the distal and proximal ends of the nerve injury.

[0026] Thus, by developing a pre-established cellular structure prior to implantation, the pre-established tissue, when implanted, promotes cell survival and constitutes a "true in vivo" environment upon implantation. Previous approaches have only injected Schwann cells into the gel, without growing Schwann cells into stable cultures within the hydrogel prior to implantation. Developing a pre-established tissue-like construct reduces the toxicity of apoptotic by-products after implantation and enhances authentic cell-cell interactions present in living tissue. The role of fibroblasts is to establish the basic orientation of Schwann cells and provide growth and adhesion factors to Schwann cells to mimic authentic neural structures.

[0027] The electrospun biodegradable polymer conduit may have a diameter of about 1.0 mm to about 5.0 mm, or about 1.5 mm to about 4.0 mm. The conduit may have a length of about 1.0 cm to about 10.0 cm, or about 1.0 cm to about 5.0 cm. The conduit may have a thickness of about 50 μm to about 500 μm, or about 50 μm to about 300 μm. The conduit may be constructed of tyrosine-derived polymers or tyrosol-derived polymers, which may be referred to as tyrosine polymers or tyrosol-derived polymers, respectively. The tyrosine-derived polymers or tyrosol-derived polymers have non-inflammatory degradable bioproducts. The conduit may be composed of tyrosine-derived polymers, such as desaminotyrosyltyrosine ethyl ester (DTE), desaminotyrosyltyrosine (DT), or combinations thereof. The conduit may be composed of desaminotyrosyltyrosine ethyl ester (DTE), desaminotyrosyltyrosine (DT), and polyethylene glycol (PEG). The molar fraction of free carboxylic acid units and PEG units in the polymers described herein can be adjusted to modify the mechanical properties and degradation rate of NAD made with such polymers. For example, polymers with a lower amount of free carboxylic acid tend to have a longer life in the body. Furthermore, by adjusting the amount of free carboxylic acid in the polymer over a range of preferred molar fractions, the resulting polymer can be adapted for use in various applications requiring different device life. In general, the higher the molar fraction of free carboxylic acid units, the shorter the life of the device in the body, making such devices more suitable for applications in which a shorter life is desired or required.

[0028] The conduit may be comprised of, for example, a tyrosol-derived polymer of U.S. Publication No. 2020 / 0181321 and WO 2021 / 055090, the entireties of which are incorporated herein by reference. The conduit may be comprised of poly(HTy glutarate), poly(HTy suberate), poly(HTy dodecanedioate), poly(HTy phenylenediacetate), or any combination thereof. The conduit may be comprised of poly(HTy glutarate).

[0029] Electrospun biodegradable polymer conduits may be constructed of biodegradable polymers having repeating units of the following structure (Formula I): [ka] wherein a and b are independently 0 or an integer from 1 to 6, c and d are independently 0 or an integer from 1 to 6, each R1 is independently selected from the group consisting of straight and branched chain alkyl groups containing up to 18 carbon atoms, each R2 is independently an alkylene group containing up to 6 carbon atoms, k is from about 20 to about 200, x is in the range of from about 0.02 to about 0.20, z is in the range of from about 0.005 to about 0.10, and x+y+z=1.00.

[0030] In some embodiments, a and b are 2 and 1, respectively. In some embodiments, c and d are 2 and 1, respectively, and R1 is ethyl. In some embodiments, R2 of the polymer is ethylene and k is about 25 to about 50.

[0031] The synthesis of various polycarbonate polymers is generally known in the art, including, for example, the methods disclosed in U.S. Patent Nos. 6,120,491 and 6,475,477, the disclosures of which are incorporated herein by reference. The polyacrylates and other polycarbonates disclosed in U.S. Patent No. 6,120,491 are also incorporated herein by reference for use in constructing the polymeric conduits herein. The polymers having pendant free carboxylic acid groups are preferably prepared with the corresponding benzyl and tert-butyl ester polymers to avoid cross-reaction of the free carboxylic acid groups with comonomers. The benzyl ester polymers may be converted to the corresponding free carboxylic acid polymers by the palladium-catalyzed hydrogenolysis method disclosed in U.S. Patent Application Publication No. 20060034769, also incorporated herein by reference, to selectively remove the tert-butyl groups.

[0032] Polymers may be selected that degrade or resorb within a predetermined time period, and thus, in embodiments, may include polymers having a molar fraction of monomeric repeat units having pendant side chain carboxylic acid groups, such as DT, of from about 2 mol % to about 20 mol %, preferably from about 5 mol % to about 20 mol %.

[0033] Polyalkylene glycol segments such as PEG reduce the surface adhesion of the polymer. By varying the molar fraction of polyalkylene glycol segments in the block copolymers provided by the present invention, the hydrophilic / hydrophobic ratio of the polymer can be altered to tune the ability of the polymer coating to modify cell behavior. Increasing the level of polyalkylene glycol inhibits cell attachment, migration and proliferation. PEG then increases water uptake, thus increasing the rate of degradation of the polymer. Thus, in one embodiment, a polymer is selected in which the amount of polyalkylene glycol is limited to 0.5 mol% to about 10 mol%, preferably about 0.5 mol% to about 5 mol%, more preferably about 0.5 mol% to about 1 mol%. The polyalkylene glycol may have a molecular weight of 1k to 2k.

[0034] Polymers may be selected to have suitable intrinsic physical properties for use in polymeric conduits with suitable mechanical properties including elasticity, stiffness, strength and degradation behavior, including polymers that have a glass transition temperature greater than 37° C. when fully hydrated under physiological conditions if the polymer is amorphous, and a crystalline melting temperature greater than 37° C. when fully hydrated under physiological conditions if the polymer is crystalline.

[0035] It should be understood that other biodegradable and biocompatible polymers can be used to form fibers that provide or enhance certain desired properties of the resulting polymeric conduit. Examples of other polymers that can be used include, but are not limited to, polylactic acid, polyglycolic acid, polylactic-co-glycolic acid, polycaprolactone, various polyamino acids and polyanhydrides. Other natural or non-natural fiber materials, such as collagen, cellulose, chitosan, and derivatives thereof, may alternatively or additionally be utilized to provide or enhance certain desired properties of the resulting polymeric conduit (see, for example, U.S. Pat. No. 8,216,602).

[0036] The electrospun biodegradable polymer conduits may be constructed of any of the polymers disclosed in U.S. Patent Publication No. 2018 / 0280567, the entirety of which is incorporated herein by reference.

[0037] Electrospun biodegradable polymer conduits may be constructed of biocompatible polymers comprising repeating units of Formula XVIII below. [ka] During the ceremony, (a) A is CH2 or CH2CH2, B is a bond, and Y is (CH2)2, (CH2)3, CH2OCH2, (CH2)4, CH2CH=CHCH2, (CH2)5, (CH2)6, and (CH2) 10 is selected from the group consisting of Or, (b) A is CH2CH2, B is selected from the group consisting of -O-CO-CH2CH2, -O-CO-CH2CH2CH2, and -O-CO-CH2OCH2 and is bonded to A through an oxygen, and Y is (CH2)2, (CH2)3, CHOCH2, (CH2)4, CH2CH=CHCH2, (CH2)5, (CH2)6, and (CH2) 10 or other polymers disclosed in U.S. Patent Application Publication No. 2020 / 0181321, which is incorporated herein by reference in its entirety. Electrospun biodegradable polymer conduits may be constructed with PEG block copolymers of the aforementioned polymers.

[0038] Electrospun biodegradable polymer conduits may be constructed of biocompatible polymers that include multiple units of Formula I. [ka] Formula I During the ceremony, A is a bond, C is 1-3 Alkylene, OC 1-3 Alkylene, CH=CH, C 1-3 Alkylene-O-CO-C2-5 Alkylene, C 1-3 Alkylene-O-CO-C 1-2 Alkylene-OC 1-2 is alkylene, B is oxygen or NR a and R a is H, optionally substituted C 1-10 alkyl, or -OE-OCO-, where E is C 1-30 Alkylene, C 2-30 Alkeren, C 1-30 Alkynylene, C 1-30 Heteroalkylene, C 2-30 Heteroalkene, C 1-30 Heteroalkynylene, C 6-30 Arylene, C 7-30 Alkyl arylene, C 8-30 Alkenarylene, C 8-30 Alkynylarylene, and C 2-30 heteroarylene; R 1 H or COOR b and R b H, C containing up to 18 carbon atoms 1-10 Alkyl and C 1-10 alkylaryl; R 2 and R 3 are independently halogen, C 1-4 Alkyl or OC 1-4 is alkyl, In each example, X is [ka] is an amino acid moiety represented by During the ceremony, R c is H or C 1-6 Alkyl, C 1-3 Alkylene-SC 1-4 Alkyl, C 1-3 Alkylene-aryl, C 1-3 Alkylene-heteroaryl, C 1-6 Alkylene-COOH, C 1-6 Alkylene-N(Rx )2, and C 1-6 Alkylene-CON(R x ) 2, each R x are independently H or C 1-6 is alkyl, Y is C 1-10 Alkylene, C 6-10 Arylene, C 1-3 Alkylene-OC 1-3 Alkylene, OC 1-6 Alkylene, C 1-3 Alkylene-SC 1-3 -Alkylene, C 1-3 Alkylene-CH=CH-C 1-3 Alkylene, C 3-8 Cycloalkylene, C 1-3 Alkylene-C 4-8 Cycloalkylene-C 1-3 Alkylene, C 1-3 Alkylene-C 6-10 Arylene-C 1-3 -Alkylene, C 1-6 Alkylene-NR e -, C 1-3 Alkylene -CH(NHBoc)-, and C 1-3 Alkylene-NR e -C 1-3 alkylene; R e , H, C 1-6 Alkyl, C 6-10 Aryl, C 1-3 Alkylene-C 6-10 Aryl and CON(R f ) 2, each R f are independently H or C 1-6 is alkyl, a and b are independently integers ranging from 0 to 4; c is an integer ranging from 1 to 8, d is 0 or 1, and m is an integer ranging from 1 to 3. In some embodiments, A is 1-3 Alkylene, C 1-3 Alkylene-O-CO-C 2-5 Alkylene or C 1-3Alkylene-O-CO-C 1-2 Alkylene-OC 1-2 In some embodiments, A is alkylene. In some embodiments, A is CH2 or CH2CH2. In some embodiments, R 1 In some embodiments, Y is H. In some embodiments, the amino acid moiety is derived from a naturally occurring amino acid. In some embodiments, the amino acid moiety is derived from an essential amino acid selected from the group consisting of phenylalanine, valine, threonine, tryptophan, methionine, leucine, isoleucine, lysine, and histidine. In some embodiments, Y is C 1-5 Alkylene, phenylene, and C 1-2 Alkylene-OC 1-2 In some embodiments, R is selected from the group consisting of alkylene. 2 and R 3 are each independently bromine or iodine. a and b are independently 0, 1, or 2.

[0039] In some embodiments, the biocompatible polymer is represented by formula II-a [ka] In the formula, m' is an integer ranging from 1 to 3.

[0040] In some embodiments, the biocompatible polymer is represented by formula II-b [ka] wherein G is C 2-3 -alkylene, and n is an integer in the range of 4 to 3000.

[0041] In some embodiments, the biocompatible polymer is represented by formula II-c [ka] wherein G is C 2-3 -alkylene, and n' is an integer in the range of 4 to 3,000.

[0042] In some embodiments, the biocompatible polymer further comprises a copolymer unit selected from the group consisting of polyethylene glycol, polycaprolactone-diol, polycaprolactone, poly(trimethylene carbonate), polylactide, polyglycolide, and polylactic-co-glycolic acid.

[0043] In some embodiments, the biocompatible polymer further comprises the structure of formula Ib: [ka] Formula Ib In the formula, w ranges from about 0.001 to 1, x ranges from 0 to 0.999, y ranges from 0 to 0.999, z ranges from 0 to 0.999, and w+x+y+z=1.000. In some embodiments, A is 1-3 Alkylene, C 1-3 Alkylene-O-CO-CH2CH2, C 1-3 Alkylene-O-CO-CH2CH2CH2, and C 1-3 In some embodiments, Y is selected from the group consisting of alkylene-O-CO-CH2OCH2, and B is oxygen. In some embodiments, Y is selected from the group consisting of (CH2)2, (CH2)3, CH2OCH2, (CH2)4, CH2CH=CHCH2, (CH2)5, (CH2)6, and (CH2) 10 In some embodiments, R 1 and R c is H.

[0044] Electrospun biodegradable polymer conduits may be constructed with PEG block polymers of any of the biocompatible polymers mentioned above.

[0045] Also incorporated by reference herein are U.S. Pat. No. 5,099,060, particularly for its disclosures relating to polycarbonate synthesis, U.S. Pat. No. 5,216,115, particularly for its disclosures relating to polyarylate synthesis, U.S. Pat. No. 6,048,521, particularly for its disclosures relating to PEG block copolymerization, and U.S. Patent Application Publication No. 2006 / 0034769, particularly for its disclosures relating to the synthesis of polymers having free acid groups.

[0046] The electrospun biodegradable polymer conduit may be multi-layered. The conduit may include one, two, three, or four layers. Each layer may contain aligned (also called oriented) or non-aligned (also called non-oriented) biopolymer fibers. The electrospun biodegradable polymer conduit may include two layers, with the innermost layer containing aligned biopolymer fibers and the outermost layer containing non-aligned biopolymer fibers. Figures 1A, 1B, 2A, and 2B are electron micrographs of examples of bi-layer sheets with oriented (e.g., Figures 1A and 2A) and non-oriented (e.g., Figures 1B and 2B) layers of biopolymer fibers. The sheet may be rolled up to produce an electrospun biodegradable polymer conduit of the desired diameter.

[0047] Fibroblasts (FB) are cultured at a defined concentration, e.g., approximately 1.0 × 10 5 cells / mL ~ approx. 5.0×10 5 cells / mL, or approximately 2.5 x 10 5 Fibroblasts may be seeded onto the outer surface of the electrospun biodegradable polymer conduit at a density of approximately 1.0×10 cells / mL. Fibroblasts may be allowed to adhere to the outer surface and may reach a density of approximately 1.0×10 cells / mL on the outer surface. 5 cells / cm 2 ~Approx. 1.0×10 6 cells / cm 2 The fibroblasts may be epineural fibroblasts. The fibroblasts may partially (less than about 50% of the surface area), substantially (greater than about 50% of the surface area), or completely (greater than about 80% of the surface area) cover the exterior surface of the polymeric conduit.

[0048] Fibroblasts and Schwann cells may be obtained through derivation of the patient's cells, such as skin cells that can be harvested when the patient is injured. Once obtained from the patient, these skin or other cells are differentiated in vitro into fibroblasts and Schwann cells and then applied to the electrospun biodegradable polymer conduits described herein. The use of autologous cells reduces the likelihood of transplant rejection, as the patient's genetic material is retained in the conduit.

[0049] Using specific differentiation protocols, human Schwann cells (hSCs) may be generated via conversion of human induced pluripotent stem cells (iPSCs) into human Schwann cell progenitor cells (hSCPs).

[0050] A system including a hydrogel matrix or a reinforced hydrogel matrix and Schwann cells promotes regrowth of neurites from damaged neurons and myelination of damaged neurons. A hydrogel matrix may be defined as a cross-linked hydrophilic polymer that is insoluble in water and can absorb large amounts of water or other biological fluids, and may be made of RADA16 peptide, collagen, gelatin, alginate, hyaluronic acid, or any other known hydrogel material. A reinforced hydrogel matrix is ​​a hydrogel matrix that is combined with another biochemical factor, such as a growth factor, guidance cue, structural peptide, adhesion factor, other chemical agent, or combination thereof, via blending, mixing, chemical bonding, or other known methods.

[0051] The reinforced hydrogel matrix may be RADA16 peptide, collagen, gelatin, alginate, or hyaluronic acid hydrogel. The reinforced hydrogel matrix may be functionalized with growth factors that support cell growth by physical mixing with the hydrogel matrix, or by chemical bonding with the hydrogel matrix, or by any combination thereof. The growth factors may be selected from neuregulin 1 (NRG1), EGF, FGF, NGF, PDGF, VEGF, IGF, GMCSF, GCSF, TGF, erythropoietin (EPO), TPO, BMP, HGF, GDF, neurotrophins (e.g., GDNF, CNTF, BDNF, NT3), netrin, MSF, SGF, or any combination thereof. The growth factor may be NRG1.

[0052] The reinforced hydrogel matrix may include one or more additives selected from a basal medium known to be used to support cell growth (e.g., Dulbecco's Modified Eagle's Medium (DMEM)), fetal bovine serum (FBS), antibiotics (e.g., penicillin, streptomycin or combinations thereof), forskolin, and any combination thereof.

[0053] The Schwann cells may be substantially uniformly distributed throughout the hydrogel matrix or reinforced hydrogel matrix within the luminal space of the conduit. The Schwann cells may be distributed throughout the hydrogel matrix or reinforced hydrogel matrix at a concentration of about 5 million cells / mL to about 80 million cells / mL, about 15 million cells / mL to about 75 million cells / mL, or about 20 million cells / mL to about 70 million cells / mL. The Schwann cells may be encapsulated by the hydrogel matrix or reinforced hydrogel matrix within the luminal space of the conduit. Figures 14A-C are cross-sectional confocal microscopy images showing rat Schwann cells growing within a collagen hydrogel matrix. In these figures, tyrosine-derived polymer fibers are shown dispersed throughout the matrix and maintaining longitudinal nerve bundles within the hydrogel matrix. In Figure 14A, the fibers and depth coding of Schwann cells are distributed in three dimensions as shown by the red-blue scale corresponding to the depth within the hydrogel matrix. In Figures 14B and 14C, the tyrosine-derived fibers are shown as large uniform circular structures, and Schwann cell nuclei are shown as small DAPI stained dots scattered within the hydrogel matrix.

[0054] Figures 3A-E show Schwann cells encapsulated in hydrogel within electrospun biodegradable polymer conduits. In Figure 3A, a 96-well optical plate was used to hold and culture electrospun upright biodegradable polymer conduits filled with Schwann cells encapsulated in hydrogel allowing live imaging, and Figure 3B shows GFP (green fluorescent protein)+ rat Schwann cells (SCs) and human neural progenitor cells embedded in a 3D RADA16 peptide hydrogel within an electrospun biodegradable polymer conduit. Figure 3C shows GFP+ rat SCs near the tube end and DIC, with a magnified panel B showing the conduit end and outer well space. Figure 3D is a magnified view of panel B showing only GFP+ SCs. Figure 3E is an example of a 3D reconstruction of a 32 μM Z-stack showing rat SCs (GFP+) embedded in a hydrogel encapsulated by an electrospun biodegradable polymer conduit.

[0055] The system may also contain fibroblasts, which may be substantially uniformly distributed throughout the hydrogel matrix or reinforced hydrogel matrix within the luminal space of the conduit. The fibroblasts may be present at a lower concentration than the Schwann cells, for example, at a ratio of about 2:10 to about 1:20, or about 1:10, of fibroblasts to Schwann cells. The fibroblasts promote Schwann cell function within the cellularized nerve regeneration graft, particularly at the level of the endoneurium, the innermost connective tissue layer found within nerve bundles surrounding myelinated axons. The system may optionally contain, in addition to fibroblasts, other supporting cells, such as, but not limited to, endothelial cells, or other cells that support the graft cellular structure. In one embodiment, the luminal space of the polymer conduit may be filled with a hydrogel matrix or reinforced hydrogel matrix onto which rat SCs grow overall. If present, GFP signal should not be present within the luminal space of the polymer conduit, as GFP+ fibroblasts may be seeded only on the outer surface of the polymer conduit.

[0056] Fibers or hollow tubes having a diameter of about 5 μm to about 50 μm, or about 20 μm, may be added into the hydrogel matrix or reinforced hydrogel matrix as needed to create an endoneurial sheath-like substructure. The fibers or hollow tubes may be made of collagen, other suitable fibers including fast degrading polymer fibers, or water-soluble sacrificial fibers made of materials such as sucrose or other suitable sugars, or may be created by creating channels with metal or polymer wires. Creation of an endoneurial sheath-like substructure may enhance cell-cell signaling during graft development and after implantation.

[0057] Another embodiment is a method of making a cellularized nerve regeneration graft comprising an electrospun biodegradable polymer conduit having an exterior surface and a luminal space, a plurality of fibroblasts seeded on the exterior surface of the conduit, and a system that fills the luminal space of the conduit comprising a hydrogel matrix or a reinforced hydrogel matrix and Schwann cells. The method includes the steps of electrospinning a polymer formulation, such as a tyrosine-derived polymer or a tyrosol-derived polymer, to create an electrospun biodegradable polymer conduit, culturing fibroblasts, inoculating the fibroblasts onto the outer surface of the electrospun biodegradable polymer conduit, generating human Schwann cells, embedding the human Schwann cells in a hydrogel matrix or a reinforced hydrogel matrix to create a system, inoculating the system into the luminal space of the electrospun biodegradable polymer conduit to create a seeded conduit, and incubating the seeded conduit in the system for about 1 week to about 6 weeks or about 2 weeks to about 4 weeks to create a cellularized nerve regeneration graft.

[0058] The fibroblasts may be cultured by any method known in the art. The fibroblasts may be epineurial fibroblasts. The outer surface of the electrospun tyrosine-derived polymeric conduit may be seeded with epineurial fibroblasts. The outer surface of the electrospun tyrosol-derived polymeric conduit may be seeded with epineurial fibroblasts.

[0059] The generation of human Schwann cells may be accomplished by any means known in the art. After fibroblasts are seeded on the outer surface, the generated human Schwann cells are embedded in a hydrogel matrix or a reinforced hydrogel matrix and optionally functionalized with one or more growth factors to support axonal regrowth, Schwann cell proliferation, and myelination of axonal processes. The hydrogel matrix or the reinforced hydrogel matrix at a defined concentration (e.g., about 0.1% to about 0.4%, or about 0.25%) may be mixed with Schwann cells (SCs) at a defined concentration (e.g., about 1 million cells / mL to about 20 million cells / mL, about 2 million cells / mL to about 15 million cells / mL, about 5 million cells / mL to about 10 million cells / mL, or about 10 million cells / mL) to create a system. The system may be injected into the luminal space of an electrospun biodegradable polymer conduit to fill the entire luminal space. The filled electrospun biodegradable polymer conduit may be incubated in the system for about 2 to about 4 weeks, or about 3 weeks, to allow both epineurial fibroblasts and Schwann cells to grow in the conduit. A medium solution may be added during incubation and replaced periodically. The medium solution may be Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS), antibiotics (e.g., penicillin, streptomycin, or combinations thereof), and any combinations thereof. The medium solution may be DMEM / 10% FBS (fetal bovine serum) / 1% penicillin / streptomycin, and may be supplemented with forskolin and / or growth factors at a specified concentration, as needed. Forskolin may be added at a concentration of about 1 μM to about 5 μM, or about 2 μM. The growth factor may be neuregulin-1, and may be added at a concentration of about 5 ng / ml to about 15 ng / ml, or about 10 ng / ml. The media solution may be 1.2 mL of DMEM / 10% FBS / penicillin / streptomycin.

[0060] During incubation, the fibroblasts and Schwann cells were allowed to grow to confluency in the culture medium, such that at the end of this period, the fibroblasts had deposited approximately 1.0×10 cells on the outer surface of the conduit. 5 cells / cm 2 ~about 1.0x10 6 cells / cm 2 While constituting an outer continuous cell layer at a defined concentration of about 5 million cells / mL to about 80 million cells / mL, about 15 million cells / mL to about 75 million cells / mL, or about 20 million cells / mL to about 70 million cells / mL, within the luminal space, there may be a confluent, 3D, substantially uniform distribution of Schwann cells throughout the hydrogel matrix or reinforced hydrogel matrix such that the Schwann cells occupy the entire luminal space of the conduit at a defined concentration of about 5 million cells / mL to about 80 million cells / mL, about 15 million cells / mL to about 75 million cells / mL, or about 20 million cells / mL to about 70 million cells / mL.

[0061] To seed the exterior surface of the electrospun biodegradable polymer conduit with fibroblast cells, a microdevice may be created and assembled by 3D printing. The 3D printed microdevice may include multiple parts, including one or more rods and one or more gears, that are constructed as separate pieces but may be assembled or reversibly or irreversibly linked to create the microdevice for seeding with fibroblast cells.

[0062] Alternatively, the 3D printed microdevice may be used to seed the system within the luminal space of an electrospun biodegradable polymer conduit. The 3D printed microdevice may be the same microdevice used to seed the fibroblast cells.

[0063] For example, as shown in FIG. 4, a microdevice may be assembled from a rod (FIG. 4A), a hollow gear (FIG. 4B), a solid gear (FIG. 4C), and a hollow tube with a cap (FIG. 4E). To assemble the microdevice, the rod may be inserted through the hollow gear. The rod may have a diameter of about 1.0 mm to about 4.0 mm and a total length of about 5.0 mm to about 5.0 cm or more depending on the application. Each of the hollow and solid gears may have a diameter of about 8.0 mm to about 2.0 cm and a thickness of about 1.0 mm to about 2.0 mm. Each of the parts of the 3D printed microdevice may be constructed of polylactic acid (PLA), polycaprolactone (PCL), or any other material known to be used in the art. The electrospun biodegradable polymer conduit is then placed on (or around) the rod and capped with the solid gear, resulting in an assembly with the electrospun biodegradable polymer conduit shown in transparent shading as shown in FIG. 4D. The assembly is then placed into a well plate (shown in FIG. 8E below) and incubated at a predetermined concentration, e.g., approximately 1.0×10 5 cells / mL ~ approx. 5.0×10 5 cells / mL, or approximately 2.5 x 10 5 The fibroblast cell suspension may be inoculated at 1000 cells / mL. The assembly may be immersed in the medium in the well plate and then left to rest for about 2 minutes to about 4 minutes to allow the fibroblast cells to attach to the scaffold, after which the assembly may be rotated 90 degrees about its longitudinal axis while remaining in the well. This process may be repeated three times until four arc lengths of the conduit are inoculated with the fibroblast cell suspension. If necessary, the assembly may be immersed in the medium solution by pipetting fresh medium into the bottom of the well until the assembly is completely covered with medium.

[0064] After seeding the outer surface with fibroblasts, which may take about 1 hour to about 24 hours, the assembly may be removed from the well and placed upright with the hollow gear resting on the capped hollow tube in the adjacent well. The solid gear may be removed from the assembly and discarded or reused after sterilization, if desired. As shown in FIG. 4F, pressure may be applied from above to the rod to push the rod through the hollow gear and into the capped hollow tube, thereby exposing the interior of the biopolymer tube (or conduit). The device shown in FIG. 4F may then be placed in any known culture tube, such as a polystyrene culture tube, and the system (e.g., a mixture of hydrogel matrix or reinforced hydrogel matrix and Schwann cells) may be inserted into the luminal space of the electrospun biodegradable polymer conduit seeded with fibroblasts on the outer surface. If desired, a medium solution may be added to the culture tube to immerse the device. The culture tube may be capped and incubated, for example, at about 33°C to 40°C and about 3% to 7% CO2, or at about 37°C and about 5% CO2, for about 15 to about 30 minutes or about 20 minutes. After the initial incubation period, at least a portion of the medium solution may be replaced, and this process may be repeated 2 to 4 times every about 15 to about 30 minutes, or may be replaced every about 20 minutes. The culture tube may then be incubated for about 3 weeks, with at least a portion of the medium solution in the culture tube being replaced every about 2 to 3 days.

[0065] When fibroblasts and Schwann cells are grown to confluence, the filled and seeded electrospun biopolymer conduit is a solid graft-like structure containing Schwann cells throughout the lumen and fibroblasts located on the outside. Structurally, fibroblasts may form the outer epineural sheath, a layer of connective tissue that surrounds peripheral nerve bundles. Fibroblasts accomplish the goal of directing the apical / basal orientation of Schwann cells while secreting growth factors that support Schwann cell survival in vitro and in vivo.

[0066] The terms used in connection with this embodiment (i.e., the method of making) have the same meanings and definitions as discussed above.

[0067] A method of repairing an injured peripheral nerve is disclosed, comprising the step of implanting a cellularized nerve regeneration graft into a patient by adhering the cellularized nerve regeneration graft to the distal and proximal ends of the injured peripheral nerve. The cellularized nerve regeneration graft comprises an electrospun biodegradable polymer conduit having an outer surface and an inner luminal space, a plurality of fibroblasts seeded on the outer surface of the conduit, and a system filling the inner luminal space of the conduit, comprising a hydrogel matrix or a reinforced hydrogel matrix and Schwann cells. The Schwann cells occupying the interior of the cellularized nerve regeneration graft support ingrowing axons by secreting signaling molecules and growth factors, such as ciliary neurotrophic factor (CNTF), and when the graft is implanted into a patient, they achieve the goal of inducing axonal growth through the graft and myelination of the regenerated axons.

[0068] A method is disclosed for forming multiple channels in a hydrogel matrix or a reinforced hydrogel matrix. In this method, Schwann cells are cultured on absorbent (also called dissolvable) fibers (which may be about 10 μm to about 50 μm in diameter) to create multiple channels in the hydrogel matrix or the reinforced hydrogel matrix, and optionally in a biodegradable polymer conduit. The absorbent fibers may be made of polylactic-co-glycolic acid (PLGA), polyglycolic acid (PGA), tyrosine-derived polymers or tyrosol-derived polymers containing high concentrations of polyethylene glycol (PEG), or any combination thereof. One or more coating materials may be applied to the surface of the absorbent fibers. The coating materials may be selected from poly-D-lysine or other charged molecules and collagen hydrogel, hyaluronic acid, or other polymeric coatings that may mimic components of the extracellular matrix, and any combination thereof. Cells in suspension may be applied to the absorbent fibers and evenly coated on the fibers by capillary action. FIG. 13 is an example of a 10x magnification image of rat Schwann cells growing on approximately 50 μm E1001k fibers.

[0069] The absorbent fibers cultured with Schwann cells may be loaded into the biodegradable polymer conduit in a longitudinal arrangement with an inter-fiber spacing of about 20 μm to about 100 μm (alternatively, the fibers may be present in the biodegradable polymer conduit at the time of cell seeding). The remaining luminal space may be filled with a hydrogel matrix or reinforced hydrogel matrix such that the interior is composed of parallel oriented fibers surrounded by Schwann cells, or filled with Schwann cells directly attached to the absorbent fibers, and the remaining portion of the luminal space may be occupied by a hydrogel matrix or reinforced hydrogel matrix, or a hydrogel matrix or reinforced hydrogel matrix mixed with cells, such as (but not limited to) Schwann cells, fibroblasts and / or other cells. When mixed with cells (for example, this may be a cell suspension with a medium having a density of about 5 million cells / mL to 80 million cells / mL), the remaining luminal space may be filled with a hydrogel matrix or reinforced hydrogel matrix and cell mixture with a volume ratio of about 3:2 (hydrogel:cells) as needed.

[0070] After a period of time, for example, about one week to about six months, the absorbable fibers dissolve, leaving Schwann cells in channels extending longitudinally within the hydrogel matrix or reinforced hydrogel matrix. The absorbable fibers may comprise a composition in which an inner rapidly dissolving material is coated with an absorbable material, such as, but not limited to, sucrose, polyvinyl alcohol (PVA), polyethylene glycol (PEG), or combinations thereof, such as, but not limited to, PLGA, PGA, tyrosine-derived polymers or tyrosol-derived polymers with large amounts of PEG, or combinations thereof that dissolve over time such that the arrangement results in hollow channels with attached / surrounding cells. These channels act as a template endoneurium for ingrowing axons during nerve regeneration, and Schwann cells line the nerve bundles to myelinate the ingrowing axons. The hollow channels may contain Schwann cells within the longitudinal space. Using this method, channels can be created without the use of Schwann cells, allowing nutrients and media to diffuse across the length of the conduit and throughout the hydrogel matrix or reinforced hydrogel matrix.

[0071] FIG. 5 is an example of a method for forming multiple Schwann cell-reinforced channels in a hydrogel matrix or reinforced hydrogel matrix. As shown in FIG. 5, a biodegradable polymer conduit 1, also referred to as a scaffold tube, is shown to be filled with absorbable fibers 2. A suspension 3 containing Schwann cells is then seeded onto the absorbable fibers, and the cells are attached to the fibers to create SC-coated fibers 4. After about 4 hours to about 5 days, the remaining luminal space within the biodegradable polymer conduit is filled with a suspension of hydrogel or reinforced hydrogel matrix containing Schwann cells 5, as desired. Upon addition, the biodegradable polymer conduit 1 remains filled with hydrogel-encapsulated Schwann cells 6 surrounding the Schwann cell-coated fibers 4. After about 3 weeks, the absorbable fibers degrade leaving Schwann cell-reinforced channels 7 that extend longitudinally and are used for ingrowing axons during nerve regeneration. As shown in FIG. 5, the biodegradable polymer conduit 1 includes an outer non-oriented electrospun layer 10 and an inner oriented electrospun layer 11 .

[0072] The features and advantages of the present disclosure will be more fully demonstrated by the following examples, which are provided for illustrative purposes and should not be construed as limiting the invention in any way. EXAMPLES

[0073] [Example 1]

[0074] Preparation of tyrosine-derived polymer electrospun scaffolds Scaffolds were prepared from a random block copolymer poly(DTE-co-10%DT-co-1%PEG carbonate) composed of desaminotyrosyltyrosine ethyl ester (DTE), desaminotyrosyltyrosine (DT), and polyethylene glycol (PEG), designated E1001(1k), where 10 and 01 are the mole percentages of DT and PEG, respectively, and 1k is the molecular weight of PEG (1000 Da). 5,6The three components of the polymer serve different purposes. The backbone of the DTE fragment segment aids in the processing of the polymer, has the necessary mechanical properties, and provides chemical stability during processing and use. Increasing the percentage of DT units increases the degradation rate from days at 25 mol% DT to hours at 50 mol% DT. The degradation rate with 10 mol% DT used herein is approximately one year. Both DTE and DT are very hydrophobic, so PEG is incorporated to increase the water content and allow degradation. PEG(1k) remains biocompatible after degradation and does not crystallize within the scaffold, unlike PEG(2k). The polymers were dissolved in hexafluoropropylene to prepare a 16% solution. The electrospinning apparatus consisted of a syringe pump (kd Scientific, Model 780100, Holliston, MA), a high voltage DC power supply with an 18G needle (Gamma High Voltage Research, Model ES30P / 5W / DAM, Ormand Beach, FL), and a rotating mandrel. The syringe was positioned 10 cm away from the mandrel.

[0075] Culture of rat Schwann cells Primary rat Schwann cells (SCs) were cultured on Matrigel (Corning)-coated plates in DMEM (Dulbecco's modified Eagle's medium) supplemented with 10% FBS, 1% penicillin / streptomycin, 2 μM forskolin, and 10 ng / ml neuregulin-1 (NRG1). Rat SCs were routinely passaged using Accutase (StemCell Technologies).

[0076] Generation of induced Schwann cells from human induced pluripotent stem cells (iPSCs) and / or human fibroblasts To generate human induced Schwann cells, human iPSCs were passaged with Accutase (StemCell Technologies) and plated onto growth factor-reduced Matrigel (Corning) plates in induction medium containing 1:1 DMEM / F12 (Hyclone) and Neurobasal Medium (Gibco) supplemented with 1X B27 (Gibco), 3 μM CHIR99021 (StemCell Technologies), 20 μM SB431542 (StemCell Technologies), and 50 ng / ml neuregulin-1 (Peprotech) for 18 days with medium changes every other day. On day 18, the medium was changed to 1:1 DMEM / F12 and Neurobasal medium supplemented with 1X B27, 200ng / ml Neuregulin-1, 4μM Forskolin (Sigma), 10ng / ml PDFG-BB (Peprotech), and 100nm All-Trans Retinoic Acid (Sigma) for 3 days. After 3 days, the cells were fed the same medium, minus the All-Trans Retinoic Acid and Forskolin, and cultured for another 3 days. The induced Schwann cells were then maintained in 1:1 DMEM / F12 and Neurobasal medium supplemented with 1X B27 and 200ng / ml Neuregulin-1, and fed every 3-4 days until ready for experiments. Human fibroblasts may be directly induced into human Schwann cells by ectopic expression of pro-Schwann cell developmental transcription factors, such as SOX10, KROX20, or other transcription factors.

[0077] Encapsulation of Schwann cells in hydrogels To encapsulate Schwann cells in RADA16 peptide hydrogels, either rat Schwann cells or human induced Schwann cells were dissociated with Accutase, resuspended in 10% sucrose water, and counted with a hemocytometer to ensure appropriate loading density of ~1 million cells / ml to ~20 million cells / ml. RADA16 peptide hydrogel and 20% sucrose water were mixed 1:1. The cell suspension and hydrogel mixture were then combined 1:1, gently mixed, and loaded into electrospun upright biodegradable polymer conduits. The final concentration of hydrogel is 0.25%. Immediately after loading the hydrogel-cell mixture, medium consisting of DMEM, 10% FBS, 1% penicillin / streptomycin, 2 μM forskolin, and 10 ng / ml neuregulin-1 was added to the wells to initiate hardening of the hydrogel. The medium was replaced after the first 20 min to alleviate acute acidity caused by the hydrogel. The electrospun biodegradable polymer conduits containing hydrogel-encapsulated Schwann cells were then cultured in an incubator at 37 °C and 5% CO. The medium was changed every 1-2 days, which may be accomplished using a continuous flow reactor loop.

[0078] result

[0079] Overall design of cellularized nerve regeneration graft (CNRG)

[0080] The goal is to construct a cellularized nerve regeneration graft to be used for reconstruction and repair of damaged peripheral nerves. The overall design of the graft is shown in Figure 6. Briefly, 1) construct a biopolymer conduit (tube) using electrospun tyrosine-derived polymer as a substrate, 2) culture epineural fibroblasts (FBs) and inoculate the fibroblasts on the outer surface of the conduit using the developed microdevice (see Figures 7C-7E and 4A-4F), 3) generate Schwann cells (SCs), which are myelinating cells of peripheral nerves, and coat the Schwann cells on absorbable fibers and / or mix them with functionalized hydrogels (containing growth factors), and 4) inoculate the hydrogel / SC mixture into the inner space of the biodegradable polymer conduit using the microdevice shown in Figures 7C-7E.

[0081] To help promote axonal regeneration, myelination, and function, the cellularized nerve regeneration grafts were cultured in culture dishes for approximately 1 to 6 weeks before implantation at the lesion site.

[0082] Fabrication of biopolymers and assemblies for cellularized nerve regeneration grafts

[0083] A large diameter (5 cm) mandrel, vibrated laterally, was used to produce flat sheets, resulting in a 13 x 21 cm mat. The mandrel speed was controlled by a DC power supply (Model 1627A, BK Precision, Yorba Linda, Calif.). The linear speed was set at 30 meters per minute (mpm) for the non-aligned layers and 650 mpm for the aligned layers.

[0084] Multilayer scaffolds were prepared in three steps: a 16% polymer solution was spun at 2 mL / h for 30 min to form a non-aligned layer, followed by a 10% solution spun at 1 mL / h for 30 min to form a further non-aligned layer, and finally a 10% solution spun at 1 mL / h for 30 min to form an aligned layer.

[0085] These multilayer scaffolds were initially electrospun as flat sheets, slowly dried at RT (room temperature), and refrigerated at 4 °C until needed. Just prior to culture experiments, the polymers were cut into 8 mm circular sections (as shown in Figure 7F) using an 8 mm diameter steel hollow punch, if needed. After all parts were sterilized under UV light for 30 min, the cut scaffolds were fixed into snap-fit ​​fixtures shown in Figures 7C-E.

[0086] Following the demonstration of cytocompatibility with flat biopolymer sheets, hollow biopolymer conduits were prepared using mandrels of smaller diameters (1.5 mm, 2 mm, and 4 mm). To facilitate the release of the conduits after electrospinning, the mandrels were coated with PEG gel. These mandrels were mounted on a chuck (IKA, model R20DS1) and spun at approximately 200 rpm for 10 min to 2 h to obtain tubes of different wall thicknesses and tube diameters. After electrospinning, the mandrels were removed from the chuck, slightly wetted with deionized water, and the polymer conduits were carefully removed by sliding them off the mandrels. The conduits were slowly dried and then refrigerated at 4 °C until required to prevent degradation. For culture experiments, immediately prior to cell seeding, the conduits were cut into sections 5 mm in length using stainless steel surgical scissors and sterilized under UV light for 30 min. The thickness of the scaffolds was measured using a micrometer. Fiber morphology was evaluated using a scanning electron microscope (SEM) (Phenom ProX, Nanoscience Instruments, Phoenix, AZ).

[0087] An SEM image at 500x magnification of the non-aligned E1001(k) biopolymer fibers forming an electrospun flat sheet is shown in Figure 7A. An SEM image at 5000x magnification of the same electrospun sheet as in Figure 7A is shown in Figure 7B. Figure 7C shows the bottom half of a 3D printed snap fixture to hold the flat biopolymer sheet. An 8mm diameter piece of electrospun E1001(k) scaffold may be placed into the round insert shown in the device. Figure 7D shows the top half of a 3D printed snap fixture. Once the electrospun biodegradable polymer sheet is placed into the bottom half piece, the top half piece snaps together, locking the electrospun biopolymer sheet into place. This fully assembled configuration is shown in Figure 7E. Once the device is fully assembled, a cell suspension can be pipetted into the wells created on either side of the snap fixture. This allows cells to adhere to only one side of the scaffold fibers, preventing cell migration to the other side. The exterior width of the device (tab to tab) is approximately 12 mm, allowing the entire device to fit into the well of a standard 24-well culture dish and rotate inward. Holes through the sidewalls allow media to flow through the device. Indentations in the tabs allow the device to be handled using forceps. Figure 7F is a sample 8 mm circular DTE biopolymer sheet.

[0088] To seed cells on the outside and inside of the electrospun biodegradable polymer conduit, also referred to herein as electrospun biopolymer tube or electrospun biodegradable polymer conduit, the device was assembled such that a rod (FIG. 4A) was threaded through a hollow gear (FIG. 4B). An electrospun biopolymer tube, shown herein in transparent shading, was then placed over the rod and capped with a solid gear (FIG. 4C), resulting in the assembly shown in FIG. 4D. The assembly was then placed longitudinally in a 24-well plate (shown in FIG. 8E below) and seeded with 2.5×10 5A fibroblast cell suspension of 40 μL at a density of 100 cells / mL was inoculated by pipetting the volume over the entire outer length of the exposed electrospun biopolymer tube. After allowing the cells to attach to the scaffold for 2 min, the device was rotated 90 degrees around its longitudinal axis by applying force to the tabs of one of the gears with a pair of forceps while the device remained in the well. This process was repeated three times until all four arc lengths of the tube were inoculated with the fibroblast cell suspension. The assembly was then immersed in 1.2 mL of DMEM / 10% FBS / 1% P / S by pipetting fresh medium to the bottom of the well until the assembly was completely covered with medium. After allowing the fibroblasts to attach overnight, the assembly was removed intact from the well with a pair of forceps and the assembly was placed upright with the hollow gear (shown in Figure 4B) resting on a capped hollow tube (shown in Figure 4E) in an adjacent well of a 24-well plate. The solid gear was then removed from the assembly using a pair of forceps and discarded. Forceps were used to apply pressure directly from above on the rod, forcing the rod through the hollow gear and into the capped hollow tube, exposing the interior of the electrospun biopolymer tube as shown in Figure 4F. The entire device was then carefully placed into a polystyrene culture tube, and the hydrogel and Schwann cell mixture was pipetted into the lumen of the electrospun biopolymer tube. Immediately after dispensing the cell mixture, 1.5 mL of medium was added to the polystyrene tube to immerse the entire device. The tube was then capped and incubated at 37°C and 5% CO2 for 20 min. After 20 min, 1.0 mL of medium was replaced, and the process was repeated after 40 min. The medium in the tube was replaced in the same manner every 2-3 days for 3 weeks.

[0089] Construction of tyrosine-based biopolymer tubes and fixtures for seeding cells into the inner and outer layers

[0090] Using the above method, a tyrosine-based biopolymer tube was constructed as shown in FIG. 8. FIG. 8A is an SEM image of the outer surface of a biopolymer conduit at 500x magnification. FIG. 8B is an SEM image of the outer surface of the same biopolymer conduit as FIG. 8A at 5000x magnification. FIG. 8C is a 10mm long tyrosine-based polymer conduit. FIG. 8D is a photograph of the gear and rod assembly shown in FIG. 4D (shown here without the biopolymer conduit). FIG. 8E is a photograph of the gear and rod assembly with a biopolymer conduit seeded with fibroblasts immersed in media in a standard 24-well plate. A 40μL volume of fibroblast cell suspension was applied at 2.5x10 cells across the length of the exposed polymer conduit. 5 The cells were pipetted at a density of 1000 cells / mL.

[0091] Generation of human Schwann cells

[0092] To generate human Schwann cells for incorporation into cellularized nerve regeneration grafts, Schwann cells were differentiated from human induced pluripotent stem cells (iPSCs) using a previously published method shown in Figure 9A (see Kim, HS, et al., "Directly induced human Schwann cell precursors as a valuable source of Schwann cells," Stem Cell Res Ther 11, 257 (2020) and https: / / doi.org / 10.1186 / s13287-020-01772-x), in which small molecules and growth factors are used to differentiate Schwann cells from patient iPSCs. Briefly, iPSCs were seeded on growth factor-reduced Matrigel plates in a cocktail of 20 μM SB431542 and 50 ng / ml neuregulin-1 for 18 days. On day 18, the cocktail was changed to 200ng / ml Neuregulin-1, 4μM Forskolin, 10ng / ml PDFG-BB, and 100nm All-Trans Retinoic Acid for 3 days. After 3 days, the same medium was given, minus All-Trans Retinoic Acid and Forskolin, and cultured for another 3 days. The induced Schwann cells were then maintained in 50ng / ml Neuregulin-1 until ready for experimentation. Figure 9B shows brightfield images of cells at various stages of the differentiation process. These are representative cell culture images of Schwann cell induction at day 0 (human stem cells), day 3 (Schwann cell precursors (hSCPs)), and day 16 (hSCs) using this method.

[0093] Cell-specific biomolecular markers are used to identify the conversion of iPSCs to human Schwann cells. Myelin protein zero (MPZ) (a myelin protein expressed by Schwann cells), SOX10 (a transcription factor important for neural crest cell progression and Schwann cell differentiation) and GAP43 (expressed by Schwann cell precursors and non-myelinating Schwann cells) of each cell at days 0, 3 and 16 were analyzed and graphed as shown in Figure 9C. As is evident from the data, the expression of cellular markers as indicators of the presence of Schwann cells increased over time.

[0094] We also tested a second published method of Schwann cell induction (see Mazzara, P, et al., "Two factor-based reprogramming of rodent and human fibroblasts into Schwann cells," Nat Commun 8,14088 (2017), https: / / doi.org / 10.1038 / ncomms14088) using transcription factors to promote the induction of human Schwann cells. In method 2, we use transcription factors delivered by lentivirus in combination with growth factors to promote the differentiation of either iPSCs or fibroblasts (FBs) into Schwann cells. As shown in Figure 10, SOX10 and KROX20 transcription factors are delivered into either human iPSCs or fibroblasts, followed by induction for 2 weeks by the addition of doxycycline. Additionally, we add neuregulin-1 and forskolin during this process to promote the differentiation of human Schwann cells.

[0095] Viability and insulating properties testing of electrospun biopolymers

[0096] The requirement was to demonstrate that tyrosine ester-derived electrospun biodegradable polymer scaffolds could support the growth of human fibroblasts and rat Schwann cells in vitro. To demonstrate the polymer scaffold and cytocompatibility, human fibroblasts were grown on one side of a multi-layered E1001(k) polymer sheet. Fibroblasts were grown at 2.5x10 5 Fibroblasts in 100 µL of cell suspension in DMEM / 10% FBS / 1% P / S were seeded at an initial concentration of 1.0 × 10 cells / mL. 5 cells / cm 2 were pipetted into the wells of the device shown in Figure 8E.

[0097] 11A-11C show Schwann cells and fibroblasts cultured on opposite sides of an electrospun biodegradable polymer scaffold. In FIG. 11A, one cell type (herein Schwann cells) is first cultured on the top layer of the fibers. Once these cells have attached to the fibers, the scaffold is inverted and another cell type (herein fibroblasts) is cultured on the bottom layer of the scaffold. The intervening scaffold localizes the cells to each side while preventing either cell from migrating to the other side. In FIG. 11B, human fibroblasts are grown on the non-aligned fibers of the biopolymer scaffold. Note the multidirectional projects of the actin cytoskeleton shown with Texas Red phalloidin staining. FIG. 11C shows rat Schwann cells (nuclei in blue and actin cytoskeleton in red) growing in linear alignment along the biopolymer fibers shown in green. Such alignment of various cell types demonstrates the ability to localize and control cell growth and proliferation using the biopolymer scaffolds presented herein.

[0098] Construction of cellularized nerve regeneration grafts

[0099] Having demonstrated that both SCs and FBs can be grown on biodegradable polymers, the next goal is to construct an implantable cellularized nerve regeneration graft in a 3D format. As illustrated in the overall scheme (Figure 6), FBs of the nerve sheath are cultured on the outer surface of a living biopolymer tube, SCs are cultured within a functionalized hydrogel and hydrogel seeded within the tube to form a 3D matrix within the tube, the biopolymer wall, and then a solid structure with SCs cultured within the FBs of the nerve sheath on the outer biopolymer wall.

[0100] Transplantation of cellularized nerve regeneration grafts into animal models of peripheral nerve injury

[0101] To prove the principle use of CNRG, further tests are being performed to confirm that CNRG can help repair peripheral nerve injury in an animal model. Specifically, B6 / C57 mice are deeply anesthetized with isoflurane, the left sciatic nerve is exposed, and a 5 mm lesion gap is created in the nerve. The removed nerve gap is replaced with a similarly sized CNRG, and the biopolymer layer is sutured together with the epineural connective tissue membrane of the sciatic nerve. The distal end of the CNRG hydrogel contains nerve growth factors (10 ng / ml brain-derived neurotrophic factor (BDNF), 10 ng / ml neurotrophin-3 (NT3), and 10 ng / ml glial-derived neurotrophic factor (GDNF)). Post-operative care, including antibiotics (amoxicillin) and analgesics (buprenorphine), is provided to these animals. The condition of the animals is monitored every 6 hours, the animals' gait is monitored, and sensation is monitored in the left hind paw using von Frey filaments. Recovery of injured animals is compared to a) that of sham-operated animals (mice in which the skin is incised but the nerves are not severed) and b) that of animals with a 5 mm lesion gap repaired with autograft.

[0102] Figure 12 shows a general scheme for the implantation of CNRGs into a mouse peripheral nerve injury model. Figure 12 shows in vitro modeling of axonal growth through CNRGs. Axonal processes are expected to grow through the hydrogel and be myelinated by Schwann cells. Fibroblasts and non-myelinating Schwann cells secrete ECM (extracellular matrix) components and growth factors into the different layers of the CNRG. Once the cells have grown to confluence within the CNRG, they are surgically inserted into a mouse peripheral nerve injury model. Recovery of motor and sensory abilities is evaluated in CNRG-treated, autograft-treated and sham animals.

[0103] In conclusion, the goal of the study is to construct a CNRG suitable for reconstruction and repair of damaged peripheral nerves. Through this initial investigation, the following objectives have been achieved: 1) To prove the compatibility of fibroblasts and Schwann cells with electrospun tyrosine-derived biopolymer E1001(k), cells have been successfully cultured on flat sheets of electrospun biopolymer; 2) To prove the distinct localization and organization of cells, fibroblasts and Schwann cells were cultured on opposite sides of a flat biopolymer sheet, where fibroblasts and Schwann cells use separate layers without crossing the biopolymer fiber layer and use a specific alignment based on the alignment of the polymer fibers; 3) To prove the compatibility of Schwann cells with hydrogel, Schwann cells were successfully cultured on hydrogel matrix and live imaged to demonstrate the survival and proliferation of Schwann cells in the three-dimensional hydrogel matrix; 4) To prove the compatibility of Schwann cells with hydrogel, Schwann cells were successfully cultured on hydrogel matrix and live imaged to demonstrate the survival and proliferation of Schwann cells in the three-dimensional hydrogel matrix; To prove the ability to grow cells inside the lumen of the tube, Schwann cells were mixed with hydrogel matrix or reinforced hydrogel matrix and injected into the inside of electrospun tubes with a diameter of 3.5 mm and a length of 5.0 mm, and the survival and proliferation of Schwann cells were confirmed by confocal fluorescence microscopy and differential interference contrast (DIC) microscopy; 5) To prove the ability, Schwann cells were cultured on the biodegradable fibers; 6) To prove the final structure of CNRG, fibroblasts were first seeded on the outer surface of the biopolymer tube, and then after filling it with hydrogel-encapsulated Schwann cells as described in point "4"), the survival of both fibroblasts and Schwann cells was confirmed on the outer surface and throughout the lumen of the tube by imaging and cryosectioning.

[0104] While various aspects of the disclosure and what are presently believed to be certain desired embodiments have been described, those skilled in the art will recognize that changes and modifications can be made without departing from the spirit of the disclosure, and it is intended to include all such changes and modifications that fall within the true scope of the disclosure.

[0105] Although the invention has been described in detail with reference to certain preferred embodiments, variations and modifications exist within the scope and spirit of the invention as described and defined in the following claims.

Claims

1. a. an electrospun biodegradable polymer conduit having an exterior surface and an interior lumen space; b. a plurality of fibroblasts inoculated onto the outer surface of the conduit; c) a system for filling the luminal space of said conduit, said system comprising a hydrogel matrix or a reinforced hydrogel matrix and Schwann cells.

2. The cellularized nerve regeneration graft of claim 1 , wherein the electrospun biodegradable polymer conduit comprises multiple layers.

3. The cellularized nerve regeneration graft of claim 2, wherein the electrospun biodegradable polymer conduit has two layers, a first layer comprising aligned biopolymer fibers and a second layer comprising non-aligned biopolymer fibers.

4. The cellularized nerve regeneration graft according to any one of claims 1 to 3, wherein the electrospun biodegradable polymer is a tyrosine-derived polymer or a tyrosol-derived polymer.

5. The fibroblasts were approximately 1.0 x 10 ５ cells / cm ２ ~Approx. 1.0×10 ６ cells / cm ２ The cellularized nerve regeneration graft of claim 1, which forms a continuous cell layer on the outer surface of the conduit at a concentration of

6. The cellularized nerve regeneration graft of claim 1 , wherein the Schwann cells are substantially uniformly distributed throughout the hydrogel matrix or reinforced hydrogel matrix within the luminal space of the conduit.

7. The cellularized nerve regeneration graft of claim 6, wherein the Schwann cells are distributed throughout the hydrogel matrix or reinforced hydrogel matrix at a concentration of about 5 million cells / mL to about 80 million cells / mL.

8. The cellularized nerve regeneration graft of claim 1 , wherein the reinforced hydrogel matrix comprises fibers, hollow tubes, or a combination thereof having a small diameter.

9. The cellularized nerve regeneration graft of claim 1, wherein the reinforced hydrogel matrix comprises a hydrogel selected from the group consisting of RADA16 peptide, collagen, gelatin, alginate, hyaluronic acid, or any combination thereof, in combination with growth factors, guidance cues, structural peptides, adhesion factors, other chemical agents, or any combination thereof.

10. The cellularized nerve regeneration graft of claim 1 , wherein the system further comprises fibroblasts, other support cells, or any combination thereof.

11. The cellularized nerve regeneration graft of claim 10, wherein the concentration of the fibroblasts and Schwann cells in the system is approximately 1:

10.

12. A method for producing the cellularized nerve regeneration graft according to claim 1, comprising: a. electrospinning a formulation of tyrosine-derived polymer or tyrosol-derived polymer to produce said electrospun biodegradable polymer conduit; b. Culturing fibroblasts; c) seeding the fibroblasts onto the exterior surface of the electrospun biodegradable polymer conduit; d. generating human Schwann cells; e. embedding the human Schwann cells into the hydrogel matrix or reinforced hydrogel matrix to form the system; f. inoculating the luminal space of the electrospun biodegradable polymer conduit with the system; g. incubating the seeded conduit for about 2 weeks to about 4 weeks to produce the cellularized nerve regeneration graft.

13. 13. The method of claim 12, wherein a 3D printed microdevice is used to facilitate seeding of the fibroblasts.

14. 14. The method of claim 13, wherein the 3D printed microdevice is used to facilitate seeding of the system into the lumen space of the electrospun biodegradable polymer conduit.

15. 14. The method of claim 13, wherein the 3D printed microdevices include rods, hollow gears, solid gears, and hollow tubes with caps.

16. The step of inoculating the fibroblasts comprises: The electrospun biodegradable polymer conduit was then coated with approximately 1.0×10 ５ cells / mL ~ approx. 5.0 x 10 ５ subjecting fibroblasts in suspension at a concentration of 1000 cells / mL; rotating the electrospun biodegradable polymer conduit in the fibroblast suspension; immersing the electrospun biodegradable polymer conduit in a culture medium solution; and maintaining the electrospun biodegradable polymer conduit in an immersion state for about 10 hours to about 24 hours until fibroblasts are seeded on the outer surface of the electrospun biodegradable polymer conduit.

17. Inoculating the luminal space of the electrospun biodegradable polymer conduit with the system comprises: a. capping one end of the electrospun biodegradable polymer conduit; b. inserting the system into the lumen space of the capped conduit to create a filled conduit; c) immersing the filled conduit in a medium solution; 13. The method of claim 12, comprising the steps of: a.) incubating the filled conduit to seed the system and form the cellularized nerve regeneration graft.

18. The incubation step may be carried out at about 33°C to about 40°C and about 3% to about 7% CO ２ 18. The method of claim 17, wherein the incubation is carried out at about 30 minutes to about 60 minutes, with at least a portion of the medium solution being replaced every about 15 minutes to about 30 minutes.

19. 20. The method of claim 18, wherein the incubating step is maintained for an additional period of about 3 weeks with at least a portion of the medium solution being replaced every about 1 to about 2 days.

20. A method for repairing a damaged peripheral nerve in a patient, comprising the step of implanting the cellularized nerve regeneration graft of claim 1 into the patient.

21. 21. The method of claim 20, further comprising adhering the cellularized nerve regeneration graft to the distal and proximal ends of the injured peripheral nerve.

22. a. an electrospun biodegradable polymer conduit having an exterior surface and an interior lumen space; b. a system for filling the luminal space of said conduit, said system comprising a hydrogel matrix or a reinforced hydrogel matrix and Schwann cells; c) a plurality of channels extending longitudinally of the electrospun biodegradable polymer conduit and within the system;

23. 23. The cellularized nerve regeneration graft of claim 22, wherein the electrospun biodegradable polymer conduit comprises multiple layers.

24. 24. The cellularized nerve regeneration graft of claim 23, wherein the electrospun biodegradable polymer conduit has two layers, a first layer comprising aligned biopolymer fibers and a second layer comprising non-aligned biopolymer fibers.

25. The cellularized nerve regeneration graft according to any one of claims 22 to 24, wherein the electrospun biodegradable polymer is a tyrosine-derived polymer or a tyrosol-derived polymer.

26. and a plurality of fibroblasts inoculated onto the exterior surface of the conduit, optionally comprising about 1.0 x 10 ５ cells / cm ２ ~Approx. 1.0×10 ６ cells / cm ２ The cellularized nerve regeneration graft of claim 22, which forms a continuous cell layer on the outer surface of the conduit at a concentration of

27. 23. The cellularized nerve regeneration graft of claim 22, wherein the Schwann cells are substantially uniformly distributed throughout the hydrogel matrix or reinforced hydrogel matrix within the luminal space of the conduit.

28. 28. The cellularized nerve regeneration graft of claim 27, wherein the Schwann cells are distributed throughout the hydrogel matrix or reinforced hydrogel matrix at a concentration of about 5 million cells / mL to about 80 million cells / mL.

29. 23. The cellularized nerve regeneration graft of claim 22, wherein the reinforced hydrogel matrix comprises fibers, hollow tubes, or a combination thereof having a small diameter.

30. 23. The cellularized nerve regeneration graft of claim 22, wherein the system further comprises fibroblasts, other support cells, or any combination thereof.

31. The cellularized nerve regeneration graft of claim 30, wherein the concentration of the fibroblasts and Schwann cells in the system is approximately 1:

10.

32. 23. The cellularized nerve regeneration graft of claim 22, wherein a plurality of said channels are hollow and enriched with Schwann cells.

33. 23. A method for producing a cellularized nerve regeneration graft according to claim 22, comprising: a. electrospinning a formulation of tyrosine-derived polymer or tyrosol-derived polymer to produce said electrospun biodegradable polymer conduit; b. generating human Schwann cells; c) embedding the human Schwann cells into the hydrogel matrix or reinforced hydrogel matrix to form the system; d. culturing absorbable fibers using the human Schwann cells; e. loading the cultured absorbable fibers into the lumen space of the electrospun biodegradable polymer conduit so that the fibers are longitudinally arranged and spaced apart; f. seeding the system into the luminal space of the electrospun biodegradable polymer conduit and between the cultured absorbable fibers; g. incubating the seeded conduit for about 1 week to about 6 weeks, wherein the absorbable fibers dissolve, leaving behind the cellularized nerve regeneration graft having a plurality of longitudinal hollow channels therein.

34. 23. A method for producing a cellularized nerve regeneration graft according to claim 22, comprising: a. electrospinning a formulation of tyrosine-derived polymer or tyrosol-derived polymer to produce said electrospun biodegradable polymer conduit; b. generating human Schwann cells; c) embedding the human Schwann cells into the hydrogel matrix or reinforced hydrogel matrix to form the system; d. Loading absorbable fibers into the lumen space of the electrospun biodegradable polymer conduit so that the fibers are longitudinally arranged and spaced apart; e. adding a suspension containing the human Schwann cells to the luminal space of the electrospun biodegradable polymer conduit to create a cultured absorbable fiber; f. seeding the system into the luminal space of the electrospun biodegradable polymer conduit and between the cultured absorbable fibers; g. incubating the seeded conduit for about 1 week to about 6 weeks, wherein the absorbable fibers dissolve, leaving behind the cellularized nerve regeneration graft having a plurality of longitudinal hollow channels therein.

35. A method as described in claim 33 or 34, comprising, before step d, a step of culturing fibroblasts and a step of inoculating the fibroblasts onto the outer surface of the electrospun biodegradable polymer conduit.

36. 34. The method of claim 33, wherein the absorbent fibers have a diameter of about 10 μm to about 50 μm and / or comprise polylactic-co-glycolic acid (PLGA), polyglycolic acid (PGA), tyrosine-derived polymers or tyrosol-derived polymers with high concentrations of polyethylene glycol (PEG), or any combination thereof.

37. 34. The method of claim 33, wherein the spacing between the cultured absorbent fibers is from about 20 μm to about 100 μm.

38. 34. The method of claim 33, wherein the cellularized nerve regeneration graft comprises Schwann cells within the hollow channels.

Images