Serotonin-functionalized and melanin-doped polyhydroxybutyrate nanofibrous scaffolds and a novel hybrid fabrication system for 3D scaffold development as a regenerative modality for neural tissue engineering

Abstract

The disclosure provides fibers comprising a biocompatible polymer and melanin, or fiber assemblies comprising the fibers, a system for producing the fiber assemblies, a method for producing the fiber assemblies, a method for manufacturing fibers, and semiconductors.

Description

[Technical field]

[0001] The present disclosure relates, for example, to a fiber, a fiber assembly, a scaffold, a scaffold material, an implantable material, or an implant. The present disclosure further relates, for example, to a method of implanting a graft material comprising the fibers. The present disclosure further relates, for example, to a system for producing a fiber assembly, a method for producing a fiber assembly, or a method for manufacturing a fiber. [Background technology]

[0002] Neural tissue engineering (NTE) aims to improve regeneration and restoration of normal neural function by combining cells, scaffolds, and biological cues. Electrical stimulation acts as an important physiological signal and helps regulate neural cell proliferation and function (Prabhakaran et al. (Prabhakaran et al., 2011; Shi et al., 2008). In addition to being electrically conductive, a successful NTE scaffold should mimic the fibrous nature of the native extracellular matrix (ECM) (Y. Chen et al. (Y. Chen et al., 2020). Several techniques are available for fabricating nanoscale topographies, including polymer demixing, phase separation, colloidal lithography, chemical etching, and self-assembly (Dalby et al. (Dalby et al., 2002; Qian & Shen, 2005; Wood, 2007). Electrospinning is relatively simple and inexpensive and has been successfully applied to generate nano / micro fibrous scaffolds that mimic the fibrous nature of ECM. These fibrous scaffolds are generally non-conductive and therefore not suitable for propagating electrical stimuli to neural cells seeded on these surfaces (Khorshidi et al., 2016; Vimal et al., 2016). On the other hand, electrically conductive polymers (ECPs) are not easy to electrospin. PHB is a natural polymer and has been used to create implants that promote guided axonal growth (Young et al., 2016). Melanin is also an endogenous pigment that has the ability to donate and accept electrons, interacting with free radicals and other reactive species due to the presence of unpaired electrons, and is therefore naturally conductive (Agrawal et al., 2022; Bettinger et al., 2009; Mostert et al., 2012). Thus, melanin may further act as an antioxidant and minimize toxin-induced tissue destruction and inflammation. We therefore propose to use melanin for the synthesis of nanofiber scaffolds. Interestingly, the presence of melanin is not limited to the skin. A pool of melanin, known as neuromelanin, is present in the brain, mainly in the substantia nigra and locus coeruleus (Gollion et al. (Gollion et al.).Studies have shown that neuromelanin concentrations increase with age, suggesting a role in neuroprotection (neuromelanin can chelate metals and toxic substances) and aging (Haining & Achat-Mendes, 2017). Summary of the Invention

[0003] We verified the potential of the new scaffold as a therapeutic approach in regenerative medicine by investigating its behavior in vitro and in vivo in spinal cord injury (SCI) model mice. Following the success of the preliminary investigation stage in the aforementioned animal models, we further investigated the efficacy of the novel scaffold in patients in collaboration with a medical hospital. Furthermore, based on our experience in scaffold fabrication by electrospinning, photolithography, and 3D printing, we proposed a new electrospinning device that enables efficient and reliable 3D printing. This design provides a new electrospinning device with upgraded features such as computer-aided 3D design, overcoming the limitations of traditional manufacturing methods.

[0004] The present disclosure provides, for example, a fiber, a fiber assembly, a scaffold, a scaffold material, an implantable material, or an implant. The present disclosure further provides, for example, a method of implanting a graft material comprising the fibers. The present disclosure further provides, for example, a system for making a fiber assembly, a method of making a fiber assembly, or a method of manufacturing a fiber.

[0005] We have fabricated a novel biomaterial that can be used to fabricate implants / conduits for neural tissue engineering. By mixing melanin and poly(3-hydroxybutyrate) (PHB) with 1.5 mM 5-hydroxytryptamine (5-HT) in a ratio of 2:3, we took advantage of electrospinning to develop a biodegradable and conductive composite polymer nanofibrous scaffold. The surface morphology, physicochemical properties, and electrical conductivity of the obtained fibrous scaffold were evaluated. As a result, the surface of the 5-HT / melanin / PHB composite fibers was rough, with an average diameter of 290 nm. Furthermore, these fibers exhibited a melting temperature of 179.05 °C, a Young's modulus of 10-90 MPa, a hydrophilic index of 61.8 ± 3.4°, and an electrical conductivity of 1.3 × 10 -3 Scm 1 showed high thermal and mechanical stability. To prove its biocompatibility with neural tissues, human motor neurons and mouse sensory neurons were successfully cultured on the composite fibers. Furthermore, DRG culture on the aligned fibers promoted the vectorial growth of axons along the fibers. These results suggest that the 5-HT / melanin / PHB composite fibrous scaffold is a biodegradable, conductive, biocompatible material suitable for neural tissue engineering applications. Furthermore, we also propose a novel grid-based designed electrospinning device that allows printing computer-aided designs of 3D scaffolds and hydrogels and generating implants with controlled 3D shapes and well-defined parameters precisely tailored to fit the injury site. This device is considered to be highly advantageous for the development of cost-effective and personalized regenerative treatments for various tissues. [Brief description of the drawings]

[0006] [Figure 1](A) Typical electrospinning setup for fiber production. (Ai) Cylindrical target for random fiber collection. (Aii) Blade drum target for aligned fiber collection. (B) SEM images of random and aligned fibers showing the ultrastructure of PVA scaffold, (C) melanin and PHB mixed fibers, (D) 5-HT-melanin and PHB mixed fibers, and (E) PHB fibers. (F) Bar graph shows fiber diameter. Data are shown as mean ± sem. Asterisks indicate statistically significant differences (One-way ANOVA with Tukey's post hoc test; ****p < 0.0001).

[0007] [Diagram 2] FTIR UV-Vis spectra of (A) PHB scaffold, (B) melanin + PHB scaffold, (C) 5-HT-melanin + PHB scaffold, (D) PVA scaffold with characteristic peaks of functional groups. XPS spectra for elemental analysis of (E) C1S, (F) O1S, (G) N1S. (Gi) indicates the atomic percentage of C1S, O1S, and N1S in the scaffold.

[0008] [Diagram 3] Thermal and mechanical properties of scaffolds. Differential scanning calorimetry (DSC) curves of (A) blank sample, (B) 5-HT-melanin and PHB composite fibers, (C) melanin and PHB composite fibers, (D) PHB fibers, and (E) PVA fibers. Heating cycles are shown in red and cooling cycles in blue. Tm indicates the peak melting temperature. Representative AFM images of (F) 5-HT-melanin·PHB composite fibers, (G) melanin·PHB composite fibers, (H) PHB fibers, and (I) PVA fibers showing the anisotropic distribution of Young's modulus (DMT modulus) calculated from the height sensor measurements. Scale bar of height sensor: 5 μm. Color-coded scale bars indicate scaffold height (left panel) and DMT modulus (right panel).

[0009] [Figure 4]Conductivity and hydrophilicity measurements. (A) Four-probe resistivity and conductivity measurements of 5-HT-melanin and PHB composite fibers in the semiconducting region. (B-Bi) Contact angle measurements for wettability / hydrophilicity analysis of PHB, melanin + PHB, 5-HT-melanin + PHB, and PVA scaffolds. Data are shown as mean ± sem. Asterisks indicate statistically significant differences (One-way ANOVA with Tukey's post hoc test; ****p < 0.0001).

[0010] [Diagram 5] Culture of mouse sensory neurons and hMNs. (A-Aii) Confocal images of DRG neuron cultures on (A) glass, (Ai) random and (Aii) aligned 5-HT-melanin-PHB scaffold surfaces. (B-Bi) hMNs growing on 2D control glass and (Bi) mixed 5-HT-melanin and PHB fibrillar surfaces. Neurons were stained with anti-βIII-tubulin antibody (red): (A-Bi) Scale bar: 50 μm.

[0011] [Figure 6A] (Hybrid Grid-Based Fabrication System) Detailed mechanical design and incorporated technology of the hybrid fabrication system. [Figure 6B] (Hybrid grid-based fabrication system) Our proposed fabrication system can also be configured with a conventional ES system with a rotating cylindrical target. [Figure 6C] (Hybrid Grid-Based Fabrication System) The grid patterned samples can be used as collectors to fabricate various 3D scaffolds. [Figure 6D] (Hybrid grid-based fabrication system) A typical structure of a nanogrid (2 cm × 2 cm) pattern fabricated on a glass surface by deposition of a 4 nm titanium layer and a 20 nm gold layer.

[0012] [Figure 7]FTIR UV-Vis spectra of (A) PHB (B) melanin (C) 5-HT powders mixed with KBr showing characteristic peaks of functional groups. (D-E) SEM-EDX traces of 9% PHB fibers (D and Di) confirming the presence of C (73.5%) and O (26%). 5-HT-melanin-PHB fibers (E and Ei) are characterized by the presence of C (73.3%), O (25.1%) and N (0.7%). The presence of N only in the mixed fibers confirms the well-mixed 5-HT and melanin in the fibers and documents their distribution. Scale bar: 25 μm.

[0013] [Figure 8] Swelling and degradation analysis of PHB and 5-HT-Melanin-PHB scaffolds. Weight increase due to swelling of fibers in PBS after 12 hours for A) PHB scaffold and (Ai) 5-HT-Melanin-PHB scaffold. (B) Comparison of percent (%) swelling capacity or swelling degree of PHB and melanin-PHB scaffolds after 12 hours. mi = initial weight of dry scaffold, ms = weight after swelling, mx = constant remaining weight after drying. (A-Ai) AFM images of dried and swollen PHB fibers and 5-HT-Melanin-PHB mixed fibers after 12 hours. Scale bar: 2 μm, height color-coded scale bar, scale bar: 8 μm. (C-E) Mass degradation of (C) PHB scaffold and (G) 5-HT-Melanin-PHB scaffold in PBS after 10 days (D10). (H) Comparison of percent degradation of PHB and 5-HT-Melanin-PHB scaffolds at D10. Data are shown as mean ± sem; asterisks indicate statistically significant differences (n = 12 scaffolds for each condition, Student t-test; ***p <= 0.01 and ***p <= 0.0001). Detailed Description of the Invention

[0014] As used herein, the term "fiber" refers to a material having a thread-like structure. As used herein, the term "fiber aggregate" refers to a material formed of multiple fibers. As used herein, the term "nanofiber" refers to fibers having a diameter of submicrometers or less than 1000 nm.

[0015] As used herein, the term "biocompatible" means having no deleterious effects on animals, such as mammals, preferably humans.

[0016] The present disclosure provides a fiber or fiber assembly. In an embodiment, the fiber can be a biocompatible fiber, a nanofiber, or a biocompatible nanofiber. In a preferred embodiment, the fiber or biocompatible fiber can be a fiber of a biodegradable polymer, more preferably a fiber of a natural biodegradable polymer. In a preferred embodiment, the fiber has electrical conductivity. The electrical conductivity is preferably sufficient for neural cells to adhere and / or grow on the fiber. The fiber assembly includes a plurality of fibers, including one or more fibers as described above. In a preferred embodiment, the fiber assembly includes fibers as described above.

[0017] In a preferred embodiment, examples of biocompatible polymers include, but are not limited to, polyhydroxyalkanes (PHAs), preferably polyhydroxybutyrate (PHB), more preferably poly(3-hydroxybutyrate).

[0018] In preferred embodiments, the fibers may further comprise melanin. In preferred embodiments, the fibers may further comprise serotonin (5-HT). In more preferred embodiments, the fibers may further comprise serotonin and melanin. In embodiments, the biodegradability of the fibers may preferably be increased. In these embodiments, the fibers are made of a polymer, preferably a biocompatible polymer, and melanin and / or 5-HT. In embodiments, the biocompatible polymer may be a non-conductive polymer, since melanin and 5-TH may render the fibers conductive in a dose-dependent manner.

[0019] In preferred embodiments, the fibers satisfy at least one, two, three, four, five, or all of the following: (i) diameter 100 nm to 2 μm (e.g., 250 nm to 1.5 μm); (ii) has a crystallization temperature of 160°C to 190°C; (iii) having a Young's modulus between 10 MPa and 90 MPa; (iv) exhibits a contact angle of less than 90° (e.g., between 55° and 80°); (v) 1×10 -2 S / cm~1×10 -5 S / cm, 1×10 -2 S / cm~1×10 -4 S / cm, or 1×10 -2 S / cm~1×10 -3 S / cm; and (vi) It exhibits higher biodegradability than fibers that do not contain melanin or serotonin.

[0020] In preferred embodiments, the fibers have a diameter of 100 nm to 10 μm, 100 nm to 8 μm, 100 nm to 5 μm, 100 nm to 4 μm, 100 nm to 3 μm, or 100 nm to 2 μm (e.g., 250 nm to 1.5 μm, 250 nm to 1 μm, or 500 nm to 1.5 μm).

[0021] In preferred embodiments, the fibers have a crystallization temperature of 120°C to 200°C, 120°C to 150°C, 160°C to 190°C, or 140°C to 180°C.

[0022] In preferred embodiments, the fibers have a Young's modulus of 10 MPa to 90 MPa, 10 MPa to 50 MPa, 40 MPa to 90 MPa, 20 MPa to 80 MPa, or 30 MPa to 80 MPa.

[0023] In a preferred embodiment, the fibers have sufficient electrical conductivity for neural cells to grow on the fibers. Melanin and 5-HT can impart such electrical conductivity to non-conductive fibers such as polyhydroxyalkanes (PHAs) in a dose-dependent manner. Thus, the fibers may preferably contain sufficient amounts of melanin and / or 5-HT. In a preferred embodiment, the fibers have a conductivity of 1×10 -2 S / cm~1×10 -5 S / cm, 1×10 -2 S / cm~1×10 -4 S / cm, or 1×10-2 S / cm~1×10 -3 It has a conductivity of S / cm.

[0024] In a preferred embodiment, the fibers contain melanin and are 1×10 -2 S / cm~1×10 -5 S / cm, 1×10 -2 S / cm~1×10 -4 S / cm, or 1×10 -2 S / cm~1×10 -3 In a preferred embodiment, the fibers contain melanin and 5-HT and have a conductivity of 1×10 -2 S / cm~1×10 -5 S / cm, 1×10 -2 S / cm~1×10 -4 S / cm, or 1×10 -2 S / cm~1×10 -3 It has a conductivity of 1.0 S / cm.

[0025] In a preferred embodiment, the fibers have a hydrophilic surface. In an embodiment, the fibers have a hydrophilic surface exhibiting an angle of 90 degrees or less, 80 degrees or less, 70 degrees or less, 60 degrees or less, 50 degrees or less, 40 degrees or less, 30 degrees or less, 20 degrees or less, or 10 degrees or less. In an embodiment, the fibers have a hydrophilic surface exhibiting an angle of 10 degrees or more, 20 degrees or more, 30 degrees or more, 40 degrees or more, 50 degrees or more, 60 degrees or more, 70 degrees or more, or 80 degrees or more. In one embodiment, the fibers have a hydrophilic surface exhibiting an angle of 10 degrees to 90 degrees, 10 degrees to 50 degrees, 50 degrees to 90 degrees, 55 degrees to 80 degrees, or 20 degrees to 50 degrees.

[0026] In a preferred embodiment, the fibers are coated with an extracellular matrix, such as collagen, fibronectin, gelatin, laminin, etc. Extracellular matrices are known to support the attachment of animal cells to surfaces coated with the extracellular matrix. In a preferred embodiment, the fibers are coated with poly-L-lysine. In a preferred embodiment, the fibers are coated with laminin and poly-L-lysine. This coating allows animal cells, such as nerve cells, to grow on the fibers or fiber aggregates. In these embodiments, the fibers or fiber-containing aggregates are suitable for culturing cells on their surface.

[0027] In one embodiment, the present disclosure provides a scaffold or scaffolding material, implantable material or implant comprising the fibers or fiber aggregates of the present disclosure. In an embodiment, the present disclosure provides a nonwoven fabric or web comprising the fibers or fiber aggregates of the present disclosure. The term "nonwoven fabric" or "nonwoven web" refers to an article or sheet having a structure of individual fibers, which are, for example, overlapping in a mesh-like manner, but not in a distinguishable manner. The scaffold or scaffolding material can be used to culture organs or tissues in vitro or in vivo. The scaffold or scaffolding material can be suitable and / or used for culturing neurons or nerves. The scaffolding material of the present invention can be suitable and / or used for inducing nerve regeneration in vitro or in vivo to obtain graft material.

[0028] The present disclosure provides a method of implanting a graft material into a subject (e.g., a human subject) in need thereof. The method may include implanting the graft material into a subject. In one embodiment, the graft material includes a fiber or fiber assembly, a scaffold or scaffold material, a transplantable material, or an implant, comprising a plurality of fibers of the present disclosure; wherein the plurality of fibers may form a nonwoven fabric or be arranged in a mesh in the graft material; and wherein the graft material may further include nerve cells for nerve regeneration on the surface of the graft, thereby inducing nerve regeneration in the subject. In one embodiment, the fiber may include melanin and 5-HT, and may be coated with an extracellular matrix, preferably laminin, and poly-L-lysine. In a preferred embodiment, the fiber is PHB with melanin and 5-HT to make it conductive.

[0029] In one embodiment, the fibers or aggregates can be dried, for example, by air drying or preferably by lyophilization. Thus, the present disclosure provides a dried form of the fibers or aggregates, preferably a lyophilized form of the fibers or aggregates. In one embodiment, the fibers or aggregates are suitable for attachment of cells such as, for example, neural stem cells, neurons such as sensory neurons, interneurons, motor neurons, ganglion neurons such as dorsal root ganglion neurons, trigeminal ganglion neurons, and glial cells such as astrocytes, oligodendrocytes, microglia, ependymal cells, Schwann cells, satellite cells, etc. In a preferred embodiment, the attached cells may be motor neurons and ganglion neurons, more preferably dorsal root ganglion neurons.

[0030] The present disclosure further provides a system for producing a fiber aggregate. FIG. 6A shows the overall configuration of a system according to one embodiment. In one embodiment, as shown in FIG. 6A, the system 20 has a nozzle 21 to which a spinning solution is supplied, and a collector 22 disposed at a position away from the nozzle 21. In a preferred embodiment, examples of the spinning solution include, but are not limited to, a biocompatible polymer (preferably a biodegradable biocompatible polymer, more preferably a natural biodegradable biocompatible polymer selected from the group consisting of, for example, PHA, PHB, or poly(3-hydroxybutyrate)), chitosan, pectin, gelatin, agar, polycaprolactone, and collagen, and the polymer may further carry a drug, a neurohormone or neuropeptide such as serotonin (5-HT), and / or another hormone or peptide such as melanin {for example, the spinning solution as described above can be supplied to the nozzle 21}.

[0031] The nozzle 21 and the collector 22 are disposed in a spinning chamber 27. The chamber 27 may be provided with at least one of a humidity controller 27a for measuring the humidity in the chamber 27 and a temperature sensor 27b for measuring the temperature in the chamber 27, so that the humidity and / or temperature in the chamber 27 can be monitored when the spinning solution is discharged from the nozzle 21. The chamber 27 may also be provided with an exhaust unit 27c (e.g., a fan) for exhausting the air from the chamber 27.

[0032] In one embodiment, as shown in FIG. 6A, the collector 22 includes a non-conductive base 22a and a conductive target electrode 22b having a pattern disposed on the base 22a. In the embodiment shown in FIG. 6A, the base 22a has a flat plate shape. In a preferred embodiment, an insulating material such as quartz glass can be used as the material of the base 22a. The dimensions of the base 22a may be, for example, but are not limited to, 20 mm×20 mm×1 mm.

[0033] In the embodiment shown in FIG. 6A and FIG. 6C(A), the target electrode 22b has a grid pattern. In a preferred embodiment, a conductive material such as gold may be used as the material of the target electrode 22b. The target electrode 22b may be formed on the substrate 22a by known techniques such as imprinting (nanoimprinting), printing, and etching. The line width of the grid lines of the target electrode 22b may be, for example, 20 μm or less, 10 μm or less, or 8 μm or less, or 5 μm or less. In addition, the interval between two adjacent grid lines may be, for example, 1 μm or more and 40 μm or less, 1 μm or more and 20 μm or less, or 1 μm or more and 10 μm or less.

[0034] In one embodiment, as shown in Fig. 6D, the target electrode 22b may be provided with a voltage input point 22c. The target electrode 22b is electrically connected to a high voltage power supply 28 via the voltage input point 22c, and a high voltage of 5 kV or more, for example, 10 kV to 30 kV, can be applied between the nozzle 21 and the target electrode 22b.

[0035] In alternative embodiments, the target electrode 22b may have a concentric rectangular pattern (see FIG. 6C(B)), a concentric circular pattern (see FIG. 6C(C)), or a continuous planar shape (see FIG. 6C(D)).

[0036] In one embodiment, the collector 22 is a replaceable or removable collector. The collector 22 is replaceably or removably attached to and supported by a stage or mount 26 disposed within the chamber 27. The collector 22 may be removably secured to the stage or mount 26, for example, but not limited to, by clips 26a. By replacing the collector 22 and changing the pattern of the target electrodes 22b, the arrangement, diameter, orientation and fiber spacing of the fibers in the fiber assembly deposited on the collector 22 may be controlled. In an alternative embodiment, the collector 22 may be a non-replaceable or non-removable collector. The stage or mount 26 may be electrically grounded.

[0037] The nozzle 21 is made of a conductive material such as metal, and is disposed above the collector 22 at a distance from the collector 22. The tip of the nozzle 21 is directed vertically downward and is covered with an insulator. A metallic syringe 21a for storing the spinning solution is fixed to the main body of the nozzle 21. A syringe pump 21b for controlling the flow rate of the spinning solution supplied to the nozzle 21 is connected to the syringe 21a. The flow rate of the spinning solution supplied to the nozzle 21 may be, for example, 0.01 to 1 ml / h. The nozzle 21 is electrically connected to a high-voltage power supply 28 via the syringe 21a, and a high voltage of 5 kV or more, for example, 10 kV to 30 kV, can be applied between the nozzle 21 and the target electrode 22b.

[0038] When a high voltage is applied between the nozzle 21 and the target electrode 22b while the spinning solution is being supplied to the nozzle 21, the spinning solution becomes charged and is discharged in a charged state toward the target electrode 22b. The discharged spinning solution is stretched by the repulsive force of the electric charge, and the solvent in the spinning solution volatilizes to form fibers (e.g., nanofibers), and the formed fibers are deposited on the collector 22 as a fiber aggregate.

[0039] In a preferred embodiment, the stage or mount 26 may be provided with a movement mechanism 25 for moving the collector 22 within the chamber 27. The movement mechanism 25 may be configured to move the collector 22 on the stage or mount 26 in X and Y directions (horizontal directions) perpendicular to the ejection direction of the nozzle 21, or in a Z direction (vertical direction) parallel to the ejection direction of the nozzle 21. The movement mechanism 25 may have a piezoelectric motor and be capable of controlling the amount of movement of the collector 22 with an accuracy of 200 nm or less.

[0040] In an alternative embodiment, both the substrate 22a and the target electrode 22b may have a cylindrical shape (see FIG. 6B and FIG. 6C(E)). In this case, one axial end and the other axial end of the cylindrical collector 22 may be rotatably and replaceably or removably attached and supported by a pair of supports 29 installed in the chamber 27. The supports 29 are provided with a rotary motor 29a that rotates the cylindrical collector 22 around its central axis. The rotation speed of the collector 22 may be, for example, 0 to 4000 rpm / min. The supports 29 may be electrically grounded.

[0041] In a preferred embodiment, the support 26a is provided with a movement mechanism (not shown) for moving the cylindrical collector 22 in the X and Y directions in the chamber 27. The movement mechanism has a piezoelectric motor, and may be capable of controlling the amount of movement of the cylindrical collector 22 with an accuracy of 200 nm or less.

[0042] In a preferred embodiment, the system 20 further includes a controller 30 that controls the operation of the movement mechanism 25. At least a portion of the controller 30 may be implemented by a computer having a memory for storing instructions and at least one processor for executing instructions.

[0043] The control unit 30 may have a data receiving unit that receives three-dimensional CAD data (e.g., an STL file) of a fiber aggregate to be produced, a calculation unit that calculates the timing and amount of movement of the collector 22 according to the CAD data, and a signal sending unit that sends a control signal according to the calculated timing and amount of movement to the movement mechanism 25. During the discharge of the spinning solution, the movement mechanism 25 moves the collector 22 according to the control signal received from the control unit 30, thereby controlling the geometric shape of the fiber aggregate deposited on the collector 22 to a desired shape defined by the CAD data.

[0044] The present disclosure further provides a method of producing a fiber assembly using the system 20 as described above.

[0045] In one embodiment, the method includes determining a pattern of the target electrodes 22b on the collector based on a desired arrangement, diameter, orientation, and fiber spacing of the fibers in the fiber aggregate. For example, the relationship between the pattern of the target electrodes 22b on the collector 22 and the arrangement, diameter, orientation, and fiber spacing of the fibers of the fiber aggregate to be deposited on the collector 22 may be determined in advance by experiment or simulation, and the determined relationship is stored in a table or database. Next, the pattern of the target electrodes 22b on the collector 22 may be determined based on the arrangement, diameter, orientation, and fiber spacing of the fibers of the fiber aggregate to be manufactured by referring to the table or database. As a variant example, the pattern of the target electrodes 22b on the collector 22 may be determined based on the arrangement, diameter, orientation, and fiber spacing of the fibers in the fiber aggregate to be manufactured using a trained model that has been machine-learned to train the relationship between the pattern of the target electrodes 22b on the collector 22 and the arrangement, diameter, orientation, and fiber spacing of the fibers in the fiber aggregate to be deposited on the collector 22.

[0046] In one embodiment, the method further includes positioning a collector 22 having a target electrode 22b with the determined pattern at a location remote from the nozzle 21 through which the spinning solution is supplied.

[0047] In one embodiment, the method further includes applying a high voltage between the nozzle 21 and the target electrode 22b to charge the spinning solution, ejecting the spinning solution in a charged state toward the target electrode 22b to form fibers, and depositing the fibers as a fibrous aggregate on the collector 22. During ejection of the spinning solution, the moving mechanism 25 moves the collector 22 in accordance with a control signal received from the control unit 30, so that the shape of the fiber aggregate deposited on the collector 22 may be controlled to have a desired shape.

[0048] In the embodiment described above, the nozzle 21 is stationarily disposed within the chamber 27 and the movement mechanism 25 is configured to move the collector 22 in the X, Y and Z directions, respectively, within the chamber 27. In an alternative embodiment, the collector 22 may be stationarily disposed within the chamber 27 and the movement mechanism may be configured to move the nozzle 21 in the X, Y and Z directions, respectively, within the chamber 27. In another alternative embodiment, the movement mechanism may be configured to move both the nozzle 21 and the collector 22 independently within the chamber 27 in the X, Y and Z directions, respectively.

[0049] In an alternative embodiment, the system 20 may include a plurality of nozzles 25. In a preferred embodiment, the plurality of nozzles 25 may have different nozzle diameters. In this case, the spinning solution may be discharged simultaneously from the plurality of nozzles 25, so that the fiber aggregate may include a plurality of types of fibers with different diameters.

[0050] In a preferred embodiment, different spinning solutions or mixtures of a spinning solution, a gas, and a solvent may be supplied to the multiple nozzles 25. In this case, the spinning solution or mixture is simultaneously discharged from the multiple nozzles 25, so that the fiber aggregate can contain multiple types of fibers with different properties.

[0051] In one embodiment, the present disclosure provides, for example, the following invention: [1] A fiber (particularly, a biocompatible fiber, nanofiber, or biocompatible nanofiber) comprising a biocompatible polymer and melanin, or a fiber assembly comprising such a fiber, wherein the polymer may preferably be a biodegradable polymer, more preferably a natural biodegradable polymer, and the fiber or fiber assembly may be dried (e.g., freeze-dried), for example, a fiber comprising a biocompatible polymer and melanin, or a fiber assembly comprising such a fiber. [2] The fiber or fiber assembly described in [1] above, wherein the polymer is a biodegradable polymer. [3] The fiber or fiber assembly described in [1] or [2] above, further comprising serotonin (5-HT). [4] The fiber or fiber assembly according to any one of [1] to [3] above, wherein the biocompatible polymer is polyhydroxyalkane (PHA), preferably polyhydroxybutyrate (PHB), more preferably poly(3-hydroxybutyrate). [5] 1×10 -2 ~1×10 -5 The fiber or fiber aggregate according to any one of the above [1] to [4], having a conductivity of 1.5 S / cm. [6] The fiber or fiber aggregate according to any one of the above [1] to [5], which has a hydrophilic surface (e.g., exhibits a contact angle of 55 degrees or more and less than 90 degrees). [7] The fiber or fiber assembly according to any one of the above [1] to [6], wherein the fiber is coated with laminin and optionally coated with poly-L-lysine. [8] The fiber or fiber assembly according to any one of [1] to [7] above, which satisfies at least one of the following: (i) diameter 100 nm to 2 μm (e.g., 250 nm to 1.5 μm); (ii) has a crystallization temperature of 160°C to 190°C; (iii) having a Young's modulus between 10 MPa and 90 MPa; (iv) exhibits a contact angle of less than 90° (e.g., 55° to 80°); and (v) It exhibits higher biodegradability than fibers that do not contain melanin or serotonin. [8A] A fiber or fiber assembly according to any one of [1] to [7] above, which satisfies at least one of the following: (i) diameter 100 nm to 2 μm (e.g., 250 nm to 1.5 μm); (ii) has a crystallization temperature of 160°C to 190°C; (iii) having a Young's modulus between 10 MPa and 90 MPa; (iv) exhibiting a contact angle of less than 90° (e.g., 55°-80°); (v) 1×10 -2 S / cm~1×10 -5 S / cm, 1×10 -2S / cm~1×10 -4 S / cm, or 1×10 -2 S / cm~1×10 -3 S / cm; and (vi) It exhibits higher biodegradability than fibers that do not contain melanin or serotonin. [9] A scaffold (preferably a transplantable scaffold) or scaffold material, transplantable material or implant comprising a plurality of fibers or fiber aggregates according to any one of the above [1] to [8A] (hereinafter, [1] to [8A] include the above [8]), wherein the fibers in the scaffold, scaffold material, transplantable material or implant may be arranged in a network-like manner.

[10] A scaffold (preferably, an implantable scaffold) or scaffold material, implantable material or implant according to [8] above for use in inducing nerve regeneration, wherein the implantable scaffold or scaffold material, implantable material or implant may be cut, folded or rolled (e.g., in the brain, spinal cord or peripheral nerves).

[11] A method of transplanting a graft material into a subject (e.g., a human subject) in need of a transplant, comprising: implanting the graft material into the subject; wherein the graft material comprises a fiber or fiber aggregate, a scaffold (preferably a transplantable scaffold) or a scaffold material, a transplantable material or an implant, comprising a plurality of fibers according to any one of the above [1] to [8A]; wherein the plurality of fibers may have an aligned pattern in the graft material, and / or may be aligned in a reticular pattern; and wherein the graft material may further comprise nerve cells for nerve regeneration on the surface of the graft, This may induce nerve regeneration in the subject.

[12] The method according to

[11] above, wherein the graft material further comprises nerve cells for nerve regeneration on the surface of the graft, thereby inducing nerve regeneration in the subject.

[0052] In one embodiment, the present disclosure further provides, for example, the following inventions:

[13] A system for producing a fiber assembly, comprising: A nozzle through which the spinning solution is supplied; a collector disposed at a position remote from the nozzle; Equipped with The collector includes a target electrode having a pattern, and when a voltage is applied between the nozzle and the target electrode, the spinning solution is ejected in a charged state toward the target electrode to form fibers and is deposited as a fiber collection on the collector, and the arrangement, diameter, orientation and fiber spacing of the fibers in the fiber collection can be controlled by changing the pattern of the target electrode. [13A] A system for producing a fiber assembly, comprising: A nozzle through which the spinning solution is supplied; a collector disposed at a position remote from the nozzle; Equipped with The collector includes a target electrode having a linear / circular shape arranged in a predetermined geometry and pattern, and when a voltage is applied between the nozzle and the target electrode, the spinning solution is ejected in a charged state toward the target electrode to form fibers and deposited as a fiber aggregate on the collector, and the arrangement, diameter, orientation and fiber spacing of the fibers in the fiber aggregate can be controlled by changing the geometry and pattern of the target electrode.

[14] The system according to

[13] or [13A] above, further comprising a movement mechanism for relatively moving one or both of the nozzle and the collector, and by relatively moving the nozzle and the collector during the discharge of the spinning solution, the shape and size of the fiber aggregate can be controlled. [14A] The system according to

[13] or [13A] above, further comprising a movement mechanism for relatively moving one or both of the nozzle and the collector, wherein the shape and size of the fiber aggregate can be controlled by relatively moving the nozzle and the collector during the discharge of the spinning solution.

[15] The system according to

[14] or [14A] above, The system, wherein the movement mechanism moves one or both of the nozzle and the collector relatively in an X direction and a Y direction, both of the X direction and the Y direction being perpendicular to a discharge direction of the nozzle. [15A] The system according to

[14] or [14A] above, The system, wherein the movement mechanism relatively moves one or both of the nozzle and the collector in an X direction and a Y direction perpendicular to a discharge direction of the nozzle.

[16] The system according to any one of

[14] to [15A] above (hereinafter,

[14] to [15A] include [14A] and

[15] above), The system, wherein the movement mechanism moves one or both of the nozzle and the collector relatively in a Z direction parallel to the ejection direction of the nozzle.

[17] The system according to any one of

[14] to

[16] above (hereinafter,

[14] to

[16] include [14A],

[15] and

[16] above), The system, wherein the movement mechanism has a piezoelectric motor and is capable of controlling the amount of movement of one or both of the nozzle and the collector with an accuracy of 200 nm or less. [17A] The system according to any one of

[14] to

[16] above, The system, wherein the movement mechanism has a piezoelectric motor, and the movement of one or both of the nozzle and the collector can be controlled with an accuracy of 200 nm or less.

[18] The system according to any one of

[14] to [17A] above (hereinafter,

[14] to [17A] include [14A],

[15] , [15A],

[16] and

[17] above), A control unit for controlling the operation of the moving mechanism is further provided. The control unit is a data receiving unit that receives CAD data of a fiber aggregate to be manufactured; a calculation unit that calculates a timing and an amount of movement of one or both of the nozzle and the collector in accordance with the CAD data; a signal transmission unit that transmits a control signal to the movement mechanism according to the calculated movement timing and movement amount; The system has:

[19] The system according to any one of

[13] to

[18] above (hereinafter,

[13] to

[18] include [13A],

[14] , [14A],

[15] , [15A],

[16] ,

[17] and

[18] above), A system comprising a plurality of the nozzles.

[20] The system according to

[19] above, The multiple nozzles each have a different nozzle diameter, and the spinning solution is ejected simultaneously from the multiple nozzles, thereby making it possible to include multiple types of fibers with different diameters in the fiber aggregate. [20A] The system according to

[19] above, The system includes a plurality of nozzles each having a different nozzle diameter, and the fiber aggregate can include a plurality of types of fibers having different diameters by simultaneously ejecting the spinning solution from the plurality of nozzles.

[21] The system according to any one of

[19] to [20A] above (hereinafter,

[19] to [20A] include

[20] above), A system in which a different spinning solution or a mixture of a spinning solution, a gas, and a solvent is supplied to each of the multiple nozzles, and the spinning solution or mixture is simultaneously ejected from the multiple nozzles, thereby making it possible to include multiple types of fibers with different properties in the fiber aggregate. [21A] The system according to any one of

[19] to [20A] above, A system in which a different spinning solution or a mixture of a spinning solution, a gas, and a solvent is supplied to each of the multiple nozzles, and the spinning solution or mixture is simultaneously ejected from the multiple nozzles, so that the fiber aggregate can include multiple types of fibers with different properties.

[22] The system according to any one of the above

[13] to [21A] (hereinafter,

[13] to [21A] include the above [13A],

[14] , [14A],

[15] , [15A],

[16] ,

[17] , [17A],

[18] ,

[19] ,

[20] , [20A] and

[21] ), further comprising a spinning chamber in which the nozzle and the collector are arranged, and at least one of a humidity controller and a temperature sensor in the spinning chamber. [22A] The system according to any one of

[13] to [21A] above, further comprising at least one of a humidity controller and a temperature sensor in the spinning chamber.

[23] The system according to any one of

[13] to [22A] above (hereinafter,

[13] to [22A] include the above [13A],

[14] , [14A],

[15] , [15A],

[16] ,

[17] , [17A],

[18] ,

[19] ,

[20] , [20A],

[21] , [21A] and

[22] ), wherein the collector has a grid pattern, a concentric rectangular pattern, a concentric circular pattern, a continuous planar shape, or a cylindrical shape.

[24] The system according to any one of

[13] to

[23] above (hereinafter,

[13] to

[23] include the above [13A],

[14] , [14A],

[15] , [15A],

[16] ,

[17] , [17A],

[18] ,

[19] ,

[20] , [20A],

[21] , [21A],

[22] and [22A]), wherein the collector is a replaceable or removable collector.

[25] A collector having a target electrode for use in the system described in any of

[13] to

[24] above (hereinafter,

[13] to

[23] include the above [13A],

[14] , [14A],

[15] , [15A],

[16] ,

[17] , [17A],

[18] ,

[19] ,

[20] , [20A],

[21] , [21A],

[22] , [22A] and

[23] ).

[26] A method for producing a fiber assembly, comprising: determining a pattern of target electrodes on the collector based on a desired arrangement, diameter, orientation and fiber spacing of the fibers in the fiber assembly; The collector is electrically conductive and has the target electrode, and is disposed at a position away from a nozzle through which the spinning solution is supplied; A method in which a voltage is applied between the nozzle and the target electrode, so that the spinning solution is discharged in a charged state toward the target electrode to form fibers, and the fibers are deposited on the collector as the fiber aggregate. [26A] A method for producing a fiber assembly, comprising: The collector is disposed at a position away from the nozzle through which the spinning solution is supplied, the collector having a conductive target electrode having a linear / circular shape arranged in a predetermined geometry and pattern; A method in which the spinning solution is discharged in a charged state towards the target electrode by applying a voltage between the nozzle and the target electrode to form fibers and deposit them as the fiber aggregate on the collector, and the arrangement, diameter, orientation and fiber spacing of the fibers in the fiber aggregate can be controlled by changing the geometry and pattern of the target electrode.

[27] A method for producing fibers, comprising the steps of: A collector having a target electrode is disposed at a position away from the nozzle through which the spinning solution is supplied; A voltage is applied between the nozzle and the target electrode, whereby the spinning solution is discharged in an electrically charged state toward the target electrode to form fibers, the spinning solution containing a biocompatible polymer (preferably a biodegradable biocompatible polymer, more preferably a natural biodegradable biocompatible polymer selected from the group consisting of, for example, PHA, PHB, or poly(3-hydroxybutyrate)), chitosan, pectin, gelatin, agar, polycaprolactone, and collagen, the polymer may further carry a drug, a neurohormone or neuropeptide such as serotonin (5-HT), and / or another hormone or peptide such as melanin, (for example, the method can provide a fiber described in any of [1] to [8A] above). [27A] A method for producing fibers, comprising: A collector having a target electrode is disposed at a position away from the nozzle through which the spinning solution is supplied; By applying a voltage between the nozzle and the target electrode, the spinning solution is discharged in an electrically charged state toward the target electrode to form fibers, which are then deposited as a fiber aggregate on the collector, the spinning solution containing a biocompatible polymer (preferably a biodegradable biocompatible polymer, more preferably a natural biodegradable biocompatible polymer selected from the group consisting of, for example, PHA, PHB, or poly(3-hydroxybutyrate)), chitosan, pectin, gelatin, agar, polycaprolactone, and collagen, the polymer may carry a drug, a neurohormone or neuropeptide such as serotonin (5-HT), and / or other hormones or peptides such as melanin, a method (for example, the method can obtain the fibers described in any of [1] to [8A] above).

[28] 1×10 -2 ~1×10 -5 A semiconductor device such as a transistor, an implant, or an electrode, comprising the fiber or fiber assembly according to any one of [1] to [8A] above, which has a conductivity of 100 S / cm. EXAMPLES

[0053] 1. Materials and Methods

[0054] 1.1 Reagents and antibodies

[0055] Synthetic melanin (Sigma M8631-1G), poly[(R)-3-hydroxybutyrate] (Sigma 363502-100G), and serotonin hydrochloride (Sigma H9523-1G), 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) (Sigma-Aldrich, St. USA), 8.0 wt% poly(vinyl alcohol) (Kato Tech Co., Ltd., Japan) were used for the fabrication of scaffolds. All chemicals were supplied by Sigma Aldrich unless otherwise noted. SYLGARD TM A cured PDMS ring for the culture chamber was prepared using a 184 silicon elastomer kit (Dow chemical company, USA). Suppliers of tissue culture media and supplements were specified separately in the methods section. 16% paraformaldehyde (#15710; Electron Microscopy Sciences, Hatfield, PA) was purchased from the same company to fix cells. Anti-βIII tubulin antibody was purchased from GeneTex (#GTX631830), and AlexaFluor594 anti-mouse secondary antibody was purchased from Invitrogen (ThermoFisher Scientific, Japan).

[0056] 1.2. Preparation of electrospun fibers

[0057] PHB was used as a mixture to assist electrospinning (5-HT / melanin / PHB and melanin / PHB). The electrospinning setup used for the preparation of random and aligned fibers is shown in Figure 1A. Solutions of PHB (9% w / v) and 5-HT (1.5 mM) with melanin (3% w / v) were prepared in hexafluoro-2-propanol (HFIP). The PHB solution was gently heated in a hot water bath, and the resulting clear solution was stirred overnight for proper mixing. The 5-HT and melanin solutions were then added to the PHB solution and stirred at room temperature for 4 h before being used for electrospinning (Figure 1A). The homogeneously mixed polymer solution was poured into a glass syringe equipped with a 22-gauge flat-tipped needle to prevent the effect of point discharge. To fabricate random fibrous sheets, a syringe pump was used to eject the polymer solution at a flow rate of 0.022 mm / min toward a rotating cylindrical target (5.42 m / min) under a high potential of 12 kV (Figure 1Ai). The syringe movement speed (left-to-right movement) was 5.14 cm / min, and the distance between the needle tip and the cylindrical collector was set to 10 cm. Similarly, to fabricate aligned fibrous sheets, a syringe pump was used to eject the polymer solution at a flow rate of 0.035 mm / min toward a rectangular blade (2 m / min) fixed on a rotating drum target under a high potential (10 k) (Figure 1Aii). The syringe movement speed (left-to-right movement) was 4.15 cm / min, and the distance between the needle tip and the cylindrical collector was set to 8 cm. In both conditions (random / aligned), the relative humidity (48%) and temperature (25 °C) were kept constant during electrospinning. The fibers were collected and dried in a vacuum desiccator overnight to remove the solvent.

[0058] 1.3. Characterization:

[0059] 1.3.1 Surface morphology

[0060] The surface morphology of PHB, melanin / PHB, 5-HT / melanin / PHB and PVA fibers was evaluated using a scanning electron microscope (SEM, JEOL JSM-7900F). Briefly, electrospun fibers were freeze-dried and mounted on conductive carbon tape attached to a copper stub. The samples were sputter-coated with gold for 4 min and then analyzed under SEM at a working distance of 10 mm and an accelerating voltage of 5 kV. The surface morphology and ultrastructure of the fibers were observed at various magnifications, from 500x to more than 20,000x. The mean and standard error of the diameter of the electrospun fibers were determined from the SEM images.

[0061] 1.3.2. Physicochemical and electrical properties

[0062] 1.3.2.1.Chemical composition / functional group analysis The presence of functional groups on the electrospun nanofibers and the powdered PHB, melanin, and 5-HT with KBr was confirmed by Fourier transform infrared spectroscopy (FTIR) (Vertex 80v; Bruker) in transmission mode at 400–4000 cm -1 (MIR range) with a resolution of 2 cm / 4 cm -1 , 64-scan average mode. Furthermore, elemental analysis (carbon, oxygen, nitrogen) was performed using X-ray photoelectron spectroscopy (XPS) on a KRATOS Axis Ultra (SLV Narayana et al. (SLV Narayana et al., 2020)).

[0063] 1.3.2.2 Mapping the elemental composition of fibres Tracing of the elemental composition of the fibers using SEM-EDX was also performed to confirm the successful mixing of melanin and PHB and to map the distribution of melanin in the mixed fibers. Briefly, scaffolds were sputter coated with osmium (Os) for 2 min and visualized using a scanning electron microscope (SEM; JEOL JSM-7900F) fitted with an Oxford energy dispersive X-ray (EDX) detector (Oxford Instruments, UK) at the front and back. SEM micrographs were taken at 500x magnification with a working distance of 10 mm and an accelerating voltage of 10 kV. Aztec-SEM 6.0 (Oxford Instruments, UK) software was used to map the distribution of C, O, and N elements in PHB and mixed 5-HT-melanin-PHB (9% PHB-3% melanin) fibers.

[0064] Thermal properties A differential scanning calorimeter (DSC)-8500 (PerkinElmer) was used for thermal analysis. 2 mg of scaffold samples were placed in an alumina pan, and an empty pan was used as a reference. All samples were first heated at a rate of 10 °C / min in the range of 40–250 °C. The samples were then cooled to 40 °C at 10 °C / min. After each test, the crystallization / melting peak areas were analyzed from the thermographs, and the crystallization (Tc) / melting (Tm) points were determined (Agrawal et al., 2021, 2022).

[0065] 1.3.2.4. Mechanical properties of electrospun fibers

[0066] Atomic force microscopy (AFM) images of the scaffolds were acquired as previously described (Agrawal et al., 2021, 2022). A MultiMode 8 atomic force microscope (Bruker) with a NanoscopeV controller and E-scanner was used. Mechanical characterization of the scaffold nanofibers was performed using PeakForceTapping mode. A RTESPA-150 probe (Bruker) with a nominal spring constant of 5.1 N / m, a nominal frequency of 2 kHz, and a nominal tip radius of 15 nm was used for AFM imaging (Baklaushev et al.). The nanogrid scaffolds were studied in air over an area of ​​5 × 5 μm to determine the force-displacement curves. The low Young's modulus / DMT modulus was calculated automatically by fitting the retraction curves using the Derjaguin, Muller, Toropov (DMT) model (Lagaly, 1988). Images were acquired at a scan rate of 1 Hz and a resolution of 512 × 512 pixels. Raw Young's modulus AFM images were processed using NanoScope Analysis v.1.10 software (Bruker).

[0067] 1.3.2.5. Electrical properties of electrospun fibers

[0068] The dry-state electrical conductivity of the electrospun fibrous meshes of 5-HT / melanin / PHB (80 μm thick) and melanin / PHB (60 μm thick) was measured using a probe station Summit 12K (Cascade Wernersville, PA, USA) equipped with a Keithley SC 4200A parameter analyzer (Tektronix, Oregon, USA). The van der Pauw technique (four-probe technique in which electrical conductivity is measured using two sets of contacts) was used for resistivity measurements.

[0069] 1.4. Surface wettability measurement

[0070] The wettability of the scaffolds was evaluated by static contact angle measurements using an Easy Drop Tensiometer (KRUSS, GmbH) (Agrawal et al., 2021). The contact angle is a quantitative measure of the wettability of a solid by a liquid and is dependent on the surface area, with higher surface energy resulting in lower contact angles (Morouco et al., 2016). A water droplet was cast onto the solid sample surface and the contact angle was measured with drop shape analysis software (KRUSS, GmbH).

[0071] 1.5. Degradation Profile

[0072] The swelling and degradation of electrospun fibrous meshes in phosphate-buffered saline (PBS, pH 7.4) were monitored. Briefly, dried electrospun fibrous scaffolds were cut into 10 mm × 10 mm samples. The samples were then placed in a 24-well plate containing 1 ml of PBS (pH 7.4) and incubated in a water bath shaker (30 strokes / min) at 37 °C (Agrawal et al., 2022). To calculate the swelling degree, the samples were immersed in 1 ml of PBS (pH 7.4) at 37 °C for 12 h and then weighed. AFM micrographs were also taken to visually confirm the swelling of the fibers. Similarly, for degradation analysis, after a predefined degradation period (10 days), the scaffolds were rinsed with PBS, dried in an oven for 24 h, and weighed. The initial dry weight of the scaffold (mi), the weight of the swollen nanofibers after removing excess water and surface moisture with filter paper (ms), and the constant residual weight of the scaffold after degradation (mx) were considered for the calculation of the swelling degree (%) and the degraded mass (%) of the scaffold. The swelling degree and the degraded mass (%) of the scaffold were calculated from Eqs. (5) and (6). Swelling degree = (ms-mx / mx)×100 (5) Degraded mass (%)=(mi-mx / mi)×100 (6)

[0073] 1.6.Biocompatibility Testing

[0074] 1.6.1 Ethics Statement All experiments were performed in accordance with the guidelines of the Okinawa Institute of Science and Technology Graduate University (OIST) Genetic Manipulation Procedures. All animal experiments were performed in accordance with Japanese law and the provisions of the OIST Animal Care and Use Committee (Protocol #ACUP-2021-326). The OIST animal facility and animal care and use program are accredited by AAALAC international (Ref. #1551).

[0075] 1.6.2. Preparation of in vitro culture A 24-well plate with a plastic bottom was used for the culture of primary neurons. The scaffold was placed in the well, and a polydimethylsiloxane (PDMS) ring was inserted to fix the scaffold to the bottom of the well. For the fabrication of the polydimethylsiloxane (PDMS) ring, a mold was designed with CAD Rhinocerous3D (V.5, Robert McNeel & Associates) and 3D printed (Object 500 Connex 3, Stratasys, Germany). Then, the PDMS prepolymer and catalyst were thoroughly mixed in a disposable plastic cup in a ratio of 10:1. The mixture was degassed in a vacuum desiccator for 20 min and poured into the mold. The PDMS was cured in an oven at 60 °C for 3 h. Finally, the polymerized PDMS ring was removed from the mold and fixed to the scaffold to fix it to the bottom of the well for neuronal cell culture.

[0076] 1.6.3. Dorsal root ganglion (DRG) / sensory neuron culture

[0077] They were then coated with 0.01% poly-L-lysine (Sigma-Aldrich # P4832-50ml) overnight at 4°C, rinsed with water, and re-coated with laminin (GibcoBRL #23017-015) for 2 hours at 4°C. DRGs were dissected from 2-month-old ICR female mice (Charles River or Nippon CLEA, Japan) and dissociated as previously described ( Agrawal et al., 2021 ; Ben-Yaakov et al., 2012 ; Terenzio et al., 2018 ). Briefly, after dissection, DRGs were dissociated by sequential digestion with 100 U of papain (Sigma-Aldrich, #P4762) in HBSS (GibcoBRL, #14175095), followed by digestion with 1 mg / ml collagenase II (Worthington Biochemical Corporation, #CLS2) and 1.2 mg / ml dispase in HBSS at 37°C for at least 30 min. Ganglia were then treated with HBSS, 10 mM glucose, 5 mM HEPES (Sigma-Aldrich, #H0887), pH 7.35. Cells were harvested by centrifugation at 1000 rpm for 7 min in L15 medium (GibcoBRL #L-5520) with 20% Percoll (Sigma-Aldrich, #P4937) and 2 × 10 4 Cells were plated at a density of 100 cells / scaffold and cultured in F12 medium (GibcoBRL #11765062) for 48 hours / DIV-2.

[0078] 1.6.4. Cultivation of hMN from iPSCs Human induced pluripotent stem cells (hIPSCs) were cultured and differentiated into motor neurons (MNs) according to published protocols (Bossolasco et al., 2018). Human MNs were cultured for 7-DIV before fixation and immunostaining.

[0079] 1.6.5. Immunohistochemistry of cultured neurons

[0080] DRG neurons and MNs were cultured as above for 2 days and 7 days, respectively, and then fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 30 min at room temperature. Nonspecific antibody binding was blocked by incubation with 2% normal goat serum and 0.1% Triton X-100 in PBS for 30 min. To visualize axonal networks, neurons were incubated with anti-βIII-tubulin antibody (1:1000 dilution in PBS) overnight at 4 °C. Afterwards, cells were washed three times with PBS and incubated with anti-mouse Alexa Fluor 594-conjugated secondary antibody (1:500 dilution in PBS) for 1 h at room temperature. Fluorescence imaging was performed on a confocal laser scanning microscope LSM900 (Carl Zeiss AG, Germany) using a 63× oil immersion objective (Plan-Apochromat DIC M27, NA = 1.40). Images were acquired with ZenBlue 3.1 (Carl Zeiss AG) at a resolution of 512 × 512 pixels and a pixel dwell time of 2.05 μs. 3 × 3 tile and z-stack (1 μm step) images were scanned to encompass the entire grid scaffold in the xyz plane. After stitching the tiles, the resulting image had a final xy resolution of 1433 × 1434 pixels.

[0081] 1.7.Statistical analysis

[0082] Statistical analysis was performed using GraphPad Prism 9 Software (GraphPad, USA). ANOVA with Tukey's post hoc test was used for multiple comparisons, and unpaired t-test was used for comparison between two groups. Differences were considered significant if the error probability was less than 5%. All data are expressed as mean ± sem, and error bars indicate the standard error of the mean. p<0.05, ***p<0.01, ***p<0.001, ***P<0.0001.

[0083] 2.Results

[0084] 2.1 Surface properties, morphology, and diameter analysis of nanofibers

[0085] Scanning electron microscopy (SEM) and helium ion scanning microscopy (HIM) revealed the ultrastructure of the nanofiber scaffolds (Figure 1B-1F). Both the 5-HT-melanin and PHB composite nanofibers (Figure 1D) and the PHB-mixed melanin nanofibers (Figure 1C) had rough surfaces, in contrast to the PVA and PHB fibers, which had smooth surfaces (Figure 1B and 1E). Furthermore, the data showed that both random and aligned fibers were produced using electrospun (Figure 1B-1E; left and middle panels). Direct measurement of fiber diameter by SEM showed that the mean diameters of the PVA fibers (0.20 ± 0.007 μm) and 5-HT+melanin+PHB fibers (0.29 ± 0.03 μm) were smaller (p****) than the melanin-mixed PHB fibers (1.28 ± 0.08 μm) and PHB fibers (3.18 ± 0.07 μm) (Figure 1F).

[0086] 2.2.Physicochemical properties

[0087] The physicochemical properties of the scaffolds were investigated by measuring their chemical, thermal, mechanical, electrical properties, hydrophilicity, etc. These properties determine the material's stability in vivo, biocompatibility, biodegradability, and ability to maintain the intended structure over a period of time to support cell and tissue growth.

[0088] First, chemical analysis of the scaffolds was performed by FT-IR. The UV-Vis transmission spectra of pure PHB, melanin and 5-HT powders were determined to be 3436.88 cm−1, respectively. -1 aliphatic –OH stretching peak at 3373.24 cm (Figure 7A); -1 The aromatic –OH stretching peak at 3500–3100 cm (Figure 7B) and -1 The 5-HT sample showed the presence of an aromatic –OH stretching peak (Figure 7C) that was merged between 3361.66 cm -1 and 3249.80 cm -1 (Figure 7C) showed primary and secondary amine peaks, whereas melanin showed peaks at 3215.08 cm -1(Figure 7B). Furthermore, powdered PHB and 5-HT had peaks at 2970.14 cm, respectively. -1 and 2933.50 cm -1 sp 3 A –CH stretching peak was observed (Figures 7A and 7C). For PHB and melanin, the peak was 1724.22 cm -1 and 1718.44 cm -1 A carboxyl-C=O stretching peak was observed (Figures 7A and 7B).

[0089] In addition, the FT-IR spectral characteristics of PHB, melanin-mixed PHB, 5-HT-melanin and PHB composite fibers, and PVA fibers were also revealed. -1 Carboxylic acid-C=O stretching peak at 2979.45 cm -1 sp 3 The -CH stretching peak is present, and the peak is ~4 cm in the PHB powder due to the reaction with HFIP. -1 and ~9 cm -1 (Fig. 2A). For melanin and PHB composite fibers, the shift was 3220.51 cm -1 A broad primary amine peak at 2977.52 cm -1 sp 3 -CH stretching peak, 1722.10 cm -1 A carboxylic acid-C=O stretching peak was observed at 3210.87 cm, and a significant shift in the peak value was observed in the PHB fibers and melanin powder (Figure 2B). Similarly, in the 5-HT-melanin and PHB composite fibers, a peak at 3210.87 cm was observed. -1 Broad and structured peaks of primary and secondary amines at 2977.52 cm -1 sp 3 -CH stretching peak, 1722.10 cm -1 The carboxylic acid-C=O stretching peak was confirmed at 3338.14 cm for the PHB fibers and 5-HT powder, and the peak value was significantly shifted in the PVA fibers (Fig. 2C). -1 A broad and strong peak of -OH at 2938.95 cm -1 sp 3 -CH stretching peak, 1735.60 cm -1An amide-C=O stretching peak was observed (Figure 2D) (Goudappagouda et al.).

[0090] Furthermore, elemental composition analysis by X-ray photoelectron spectroscopy (XPS) of PHB, melanin + PHB + 5-HT, melanin + PHB, and PVA are shown in Figures 2E-Gi. For all polymers, the core levels of C 1s (Figure 2E) and O 1s (Figure 2F) were similar, with peaks located at ~284 eV and ~532 eV, respectively. However, the N 1s peak located at 400.0 eV (Figure 2G) was only present in the melanin-PHB mixed fibers and the 5-HT-melanin-PHB composite fibers (Figure 2Gi), suggesting the presence of pyrrole-N groups.

[0091] We also tracked the fiber composition by SEM-EDX analysis (Fig. 7D-E). We focused on the localization of nitrogen, which is present in both functional groups of 5-HT and melanin but not in PHB, and mapped the distribution of 5-HT and melanin in the blended fibers. We compared the fibers obtained by electrospinning PHB (control sample) (Fig. 8D) and the 5-HT-PHB-melanin blend (Fig. 8E). Indeed, characteristic peaks of carbon (Kα 277 eV) and oxygen (Kα 525 eV) were detected in both samples, whereas nitrogen (Kα 392 eV) was present only in the 5-HT-melanin-PHB fibers, explaining the percentage of nitrogen atoms in the composition of the blend of 0.7% (Fig. 8Ei).

[0092] Next, the thermal properties of the scaffolds were measured using DSC (Figure 3A-E). Figure 3B-3E show the melting temperature (T m ) during the heating cycle. No peak was observed during the cooling cycle, suggesting that the polymer remains in the same state after melting (Goudappagouda et al., 2019, 2020). The calculated T for 5-HT-melanin and PHB composite fibers, melanin-mixed PHB fibers, and PHB and PVA fibers mwere 179.05 °C (Figure 3B), 178.45 °C (Figure 3C), 175.84 °C (Figure 3D), and 194.4 °C (Figure 3E), consistent with a thermally stable material. Thus, our fibers are suitable candidates for use as implants or conduits in vivo.

[0093] To measure the mechanical strength of the scaffolds, nanoindentation atomic force microscopy (AFM) was used to generate Young's modulus distribution maps simultaneously with topographical imaging to map the anisotropic distribution of mechanical strength (Figures 3F-3I). The Young's modulus values ​​varied depending on the fiber structure, with the center of the fiber exhibiting higher values ​​than its periphery (Figures 3Fi, Gi, Hi, Ii). The calculated Young's moduli (Y) of 5-HT melanin·PHB composite nanofibers (Figure 3Fi), melanin-mixed PHB nanofibers (Figure 3Gi), PHB nanofibers (Figure 3Hi), and PVA nanofibers (Figure 3Ii) ranged from 10 MPa to 90 MPa, suggesting that the nanofibers could maintain their spatial structure and mechanical properties and be suitable as implants for CNS and soft tissue engineering.

[0094] To confirm the electrical properties of the electrospun fibers, we measured the resistivity (ρ) and conductance (σ) of the scaffolds containing mixed 5-HT and melanin. The data from the four-probe method showed that the resistivity of the fibrous scaffold (7.71 × 10 3 ohm-cm) and conductance (1.3×10 -3The wettability (S / cm) of the 5-HT and melanin mixed scaffold surface was measured. This is an important parameter that determines its suitability as a biological support, and was determined using a tensiometer by quantitatively measuring the contact angle (Figure 4B-Bi). Surfaces with a contact angle smaller than 90° are considered hydrophilic (Agrawal et al., 2021; W et al., 2020). Figure 4B and 4Bi suggested that the observed mean ± sem values ​​of the contact angle of the 5-HT-melanin and PHB composite scaffold (61.8 ± 3.4°) and the melanin mixed PHB scaffold (60.6 ± 2.5°) were higher than the conventional laminated quartz glass surface (~53°) and the PVA scaffold (24.3 ± 2.03°) (Ito et al., 2018), but lower than the PHB scaffold (100.03 ± 1.2°). Nevertheless, this value was less than 90°, suggesting that the PHB scaffold mixed with conductive 5-HT and melanin was hydrophilic.

[0095] 2.4. Swelling and degradation of fibers

[0096] The degree of swelling (swelling capacity (%)) over a 12-hour period was measured for PHB and 5-HT-melanin-PHB scaffolds (Figure 8A-B). The results showed that the swelling capacity (%) of 5-HT-melanin-PHB fibers after 12 hours of incubation in phosphate-buffered saline (PBS) was significantly higher (**p<0.01) than that of PHB fibers (259.75±11.74) (Figure 8B). The degradation of PHB and 5-HT-melanin-PHB fibers was also analyzed by measuring the weight loss after immersion in PBS for 10 days (Figure 8C and D). The preliminary degradation data plot over 10 days (Figure 8E) suggested that mixing 5-HT with melanin increased the biodegradation of the scaffolds compared to the PHB scaffolds (Figure 8C) (Figure 8D) (***p<=0.0001).

[0097] 2.5.Biocompatibility

[0098] DRG and hMN neurons are relevant in peripheral injury, spinal cord injury, and neuronal degeneration, so we tested the biocompatibility of 5-HT-melanin with PHB composite scaffolds (Figure 5). Adult DRG neurons were dissected from 2-month-old mice, and hMNs were derived from human induced pluripotent stem cells (IPS cells) and cultured on the scaffolds. For culture, scaffolds measuring 1 cm × 1 cm × 30 μm (length × width × height) and control glass surfaces were coated with two ECM proteins, poly-L-lysine and laminin, which are important for the adhesion and proliferation of these neuronal types in culture. DRG neurons were then seeded on the scaffolds (random and aligned; glass surfaces (Figure 5A)) and cultured for 48 h, after which hMNs were allowed to grow for 7 days in vitro (DIV) (Figure 5B). Cells were then fixed, stained with β-III tubulin antibody, and analyzed by confocal microscopy. As a result, DRGs neurons could grow on our scaffolds and extend their neurites arbitrarily when cultured on glass surfaces and random fibrous scaffolds (Figure 5A and Ai); whereas neurons cultured on aligned fibers showed vectorial growth along the fibers (Figure 5Aii). Similarly, hMNs could grow on our scaffolds as well as on conventional substrates such as laminated quartz glass (Figure 5B and Bi). No significant effect on neuronal viability or signs of axonal stress were observed when comparing nanofibers to flat control surfaces. Thus, our data support the biocompatibility of the 5-HT and melanin mixed PHB scaffolds and their suitability for mammalian cell culture and as implants.

[0099] 3. Design of advanced fabrication equipment

[0100] We are developing conductive, biocompatible, and biodegradable implants for tissue engineering purposes to address long-term medical problems such as SCI, nerve injury (NI), and neo-tissue formation for the treatment of deep wounds (>5 mm) that are usually formed after accidents or during critical operations such as cortical brain surgery and open-heart surgery. Researchers have found that the combined use of biomaterials with and without stem cells may replace traditional inefficient ways of addressing medical problems. Recent advances in manufacturing techniques, such as two-photon polymerization (2PP) laser lithography devices, have made it possible to fabricate 2D scaffolds and circuits with high precision of hundreds of nanometers. However, when it comes to fabricating 3D structures with dimensions from nanometers to several millimeters, with user-defined shapes / sizes and specific textures, with high precision, in a short time, and cost-effectively, it remains a great challenge for the scientific community to develop such tools and state-of-the-art technologies. On the one hand, conventional electrospinning devices enable faster fabrication but are mainly limited to the development of 2D scaffolds (with limited thickness in the mm to cm range) and do not allow control over scaffold parameters such as fiber diameter and fiber spacing. On the other hand, 3D lithography allows precise control of parameters (~200 nm) but limits the size of the scaffolds (only a few millimeters) (Agrawal et al., 2021). Moreover, it is a laborious and expensive method and requires relatively long printing times. Considering all the drawbacks of existing fabrication techniques, we decided to develop a hybrid nanofabrication system that merges the principles of electrospinning (ES) and 3D bioprinting based on conductive grid patterns to overcome the above limitations. Figure 6A-D shows the proposed design of the hybrid fabrication system. Figure 6A shows the detailed grid-based technique of the proposed fabrication apparatus. By changing the parameters of the geometry of the grid, the type of collector, and the grid spacing mounted on the piezo-enabled (X, Y, Z) stage (Figure 6B-D), the fabrication of user-defined computer-aided designs in ES mode is possible, which is not possible with existing ES apparatus.Moreover, 3D bioprinting technology can be easily integrated into this system by controlling the needle / nozzle size, the flow rate of the polymer solution, and the flow rate of CO2 / air through the polymer solution. This would make this device a cost-effective 3D fabrication technology. This device also enables the fabrication of 3D hydrogels for therapeutic purposes. Moreover, the integration of multi-nozzles allows the fabrication of complex 3D structures with high precision using semi-solid viscous solutions, giving it an advantage over existing fabrication techniques.

[0101] discussion

[0102] Each year, millions of people worldwide suffer from SCI ( Kang et al., 2017 ), with 15–40% of cases involving PNI ( S. Chen et al., 2015 ). Over the past decade, regenerative medicine has seen rapid and promising advances in neural tissue engineering and stem cell therapy ( McMurtrey, 2015 ; Rajabzadeh et al., 2019 ). Effective therapeutic strategies for the treatment of SCI and PNI are still limited (Cristante et al., 2012; Hussain et al., 2020) and mainly include the use of spinal cord stabilizing implants, autografts, and systemic injections of growth factors (Jendelova, 2018). Loss of function of motor and sensory neurons, neuronal degeneration, and dimensional mismatch between the injured nerve and the graft are known drawbacks of these approaches. A meta-analysis of more than 70 preclinical studies suggests that the combination of cell therapy with various scaffolds improves functional recovery compared to cell therapy alone (Baklaushev et al., 2019). Nevertheless, designing an ideal nanofiber scaffold that would provide an attractive clinical alternative to neural autografts and semiconductor implants for SCI remains a major challenge (Baklaushev et al., 2019; Boni et al., 2019). (e.g., 2018). We fabricated 5-HT-melanin mixed PHB composite conductive scaffolds for neural tissue engineering. SEM analysis confirmed that we successfully fabricated 5-HT and melanin mixed PHB fibers with a diameter of ~290 nm (Fig. 1f). Recently, efforts toward the development of biodegradable and conductive scaffolds have been intensified (Agrawal et al. 2022). The addition of melanin and 5-HT not only made the nanofibers more biodegradable but also made them conductive (Fig. 4A). The conductivity of the scaffold under physiological conditions provides electrical cues to promote and align neurite growth. Furthermore, the combined effect of the rough surface, conductivity, and laminin coating of the fibers promoted the adhesion and survival of mouse sensory neurons (Agrawal et al. 2022) and human motor neurons (Fig. 5).

[0103] We also fabricated aligned fibers to create mechanical constraints to promote vectored growth of axons (Figure 1B-1E; middle panels) (Agrawal et al., 2021). Indeed, aligned fibers can promote vectored growth of remaining axons at the injury site (Figure 5Aii) and aid in functional repair of damaged neural tissue (Hadlock et al., 2000). Thus, fabrication of aligned fibers was performed in preparation for implant engineering for in vivo use, where guiding axonal growth along the main axis would be advantageous. Taken together, our study shows that aligned / random scaffolds / conduits / implants of 5-HT, melanin, and PHB have the advantage of promoting repair of damaged neural tissue at the injury site as a therapeutic approach to promote tissue regeneration. Finally, we propose the design of a hybrid grid-based fabrication system setup that allows for 3D scaffold fabrication that overcomes the limitations of existing fabrication systems (Figure 6). We believe that this setup is a cost-effective way to fabricate aligned 3D scaffolds for patient-specific fabrication of medical implants that will be beneficial for clinical applications.

Claims

1. A system for producing a fiber assembly, comprising: a nozzle to which the spinning solution is supplied; a collector positioned at a distance from the nozzle; Equipped with The collector includes a target electrode having a pattern, and when a voltage is applied between the nozzle and the target electrode, the spinning solution is discharged in a charged state toward the target electrode to form fibers, which are deposited as a fiber aggregate on the collector, and the arrangement, diameter, orientation, and fiber spacing of the fibers in the fiber aggregate can be controlled by changing the pattern of the target electrode.

2. 2. The system according to claim 1, further comprising a movement mechanism for relatively moving one or both of the nozzle and the collector, wherein the shape and size of the fiber aggregate can be controlled by relatively moving the nozzle and the collector while the spinning solution is being discharged.

3. The system of claim 2 , wherein the movement mechanism moves one or both of the nozzle and the collector relatively in an X direction and a Y direction, both of the X direction and the Y direction being perpendicular to the ejection direction of the nozzle.

4. The system according to claim 2 , wherein the movement mechanism moves one or both of the nozzle and the collector relatively in a Z direction parallel to the ejection direction of the nozzle.

5. The system according to claim 2 , wherein the movement mechanism has a piezoelectric motor and is capable of controlling the movement of one or both of the nozzle and the collector with an accuracy of 200 nm or less.

6. a control unit for controlling the operation of the movement mechanism; The control unit a data receiving unit that receives CAD data of a fiber aggregate to be manufactured; a calculation unit that calculates the timing and amount of movement of one or both of the nozzle and the collector according to the CAD data; a signal transmitting unit that transmits a control signal to the movement mechanism according to the calculated movement timing and movement amount; The system of claim 2 , comprising:

7. The system according to any one of claims 1 to 6, comprising a plurality of the nozzles.

8. The system according to claim 7 , wherein the plurality of nozzles have different nozzle diameters, so that the fiber aggregate can contain a plurality of types of fibers with different diameters.

9. The system according to claim 7, wherein different spinning solutions or mixtures of spinning solutions, gases, and solvents are supplied to the plurality of nozzles, respectively, and the spinning solutions or mixtures are simultaneously ejected from the plurality of nozzles, thereby making it possible to include multiple types of fibers with different properties in the fiber aggregate.

10. The system according to any one of claims 1 to 6, further comprising: a spinning chamber in which the nozzle and the collector are disposed; and at least one of a humidity controller and a temperature sensor in the spinning chamber.

11. The system of any one of claims 1 to 6, wherein the collector has a grid pattern, a concentric rectangular pattern, a concentric circular pattern, a continuous planar shape, or a cylindrical shape.

12. The system according to any one of claims 1 to 6, wherein the collector is a replaceable or removable collector.

13. A collector having a target electrode for use in a system according to any one of claims 1 to 6.

14. A method for producing a fiber assembly, comprising: determining a pattern of target electrodes on the collector based on a desired arrangement, diameter, orientation and fiber spacing of the fibers in the fiber assembly; the collector, which is electrically conductive and has the target electrode, is disposed at a position away from a nozzle through which the spinning solution is supplied; a voltage is applied between the nozzle and the target electrode, whereby the spinning solution is discharged in a charged state toward the target electrode to form fibers, which are then deposited on the collector as the fiber aggregate.

15. 1. A method for producing a fiber, comprising: a collector having a target electrode disposed at a position away from a nozzle through which the spinning solution is supplied; A method in which the spinning solution is discharged in a charged state toward the target electrode by applying a voltage between the nozzle and the target electrode to form a fiber, and the spinning solution includes a biocompatible polymer.

16. 16. The method of claim 15, wherein the biocompatible polymer is selected from the group consisting of PHA, PHB, or poly(3-hydroxybutyrate), chitosan, pectin, gelatin, agar, polycaprolactone, and collagen.

17. 16. The method of claim 15, wherein the biocompatible polymer further carries one or more of a neurohormone, a neuropeptide, a drug, serotonin (5-HT), melanin, other hormones, or other peptides.

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