Patent Application
20250108146
The present disclosure relates to, for example, a fiber, fibrous assembly, scaffold, scaffold material, implantable material or implant. The present disclosure further relates to, for example, a method of transplanting a graft material comprising the fiber. The present disclosure further relates to, for example, a system for fabricating a fibrous assembly, a method of fabricating a fibrous assembly or producing a fiber.
Neural tissue engineering (NTE) aims to improve regeneration and restoration of normal neural functions by employing a combination of cells, scaffold and biological cues. Electrical stimuli act as a critical physiological signal and help in regulation of proliferation and function of nerve cells (Prabhakaran et al., 2011; Shi et al., 2008). In addition to be electrically conducting, a successful NTE scaffold also needs to mimic the fibrous nature of the native extracellular matrix (ECM) (Y. Chen et al., 2020). Several technologies are available for the fabrication of nanoscale topography, like polymer de-mixing, phase separation, colloidal lithography, chemical etching and self-assembly (Dalby et al., 2002; Qian & Shen, 2005; Wood, 2007). Electrospinning is relatively simple and inexpensive, and has been successfully employed for the generation of nano-/micro-fibrous scaffolds, which mimic fibrous nature of ECM. These fibrous scaffolds are generally non-conducting and are thus, not suitable to propagate electrical stimulus to nerve cells seeded onto these surfaces (Khorshidi et al., 2016; Vimal et al., 2016). On the other hand, electrically conducting polymers (ECP) are not easy to electrospin. PHB is a natural polymer, which has been used to fabricate the implants to promote the guided growth of axons (Young et al., 2002). Melanin is also a bodily pigment, which has the ability to donate or accept electrons and to interact with free radicals and other reactive species due to the presence of unpaired electrons, thus being conductive in nature (Agrawal et al., 2022; Bettinger et al., 2009; Mostert et al., 2012). Melanin can, thus, act additionally as an antioxidant and po-tentially minimize toxin-induced tissue destruction and inflammation. Therefore, we propose the use melanin for the synthesis of nanofiber scaffolds. Interestingly, melanin presence is not limited to the skin; a pool of melanin, known as neuromelanin, is present inside the brain, mainly in the substantia nigra or the locus coeruleus (Gollion et al., 2020). Studies have shown that neuromelanin concentration increases with age, suggesting a role in neuroprotection (neuromelanin can chelate metals and xenobiotics) or senescence (Haining & Achat-Mendes, 2017).
The inventors tested the potential of a new scaffold as a therapeutic approach in regenerative medicine by investigating its behaviors in vitro and in vivo in a mouse model of Spinal Cord Injury (SCI). Pending the success of the aforementioned preliminary investigation phase in animal model, the inventors further explored the effec-tiveness of new scaffolds in patients in collaboration with medical hospitals. In addition, based on the experience in scaffold fabrication with electrospinning, pho-tolithography and 3D printing, the inventors proposed a new electrospinning setup, which allows efficient and reliable 3D printing. This design will provide a new electrospinning setup with upgraded features, like computer aided 3D design, to overcome the limitations of conventional fabrication methods.
The present disclosure provides, for example, a fiber, fibrous assembly, scaffold, scaffold material, implantable material or implant. The present disclosure further provides, for example, a method of transplanting a graft material comprising the fiber. The present disclosure further provides, for example, a system for fabricating a fibrous assembly, a method of fabricating a fibrous assembly or producing a fiber.
The inventors fabricated a novel biomaterial, which can be used for the preparation of implants/conduits for neural tissue engineering. Taking advantage of electrospinning, inventors developed biodegradable and electrically conductive composite polymeric nanofibrous scaffolds, by blending melanin and Poly (3-hydroxybutyrate) (PHB) together with 1.5 mM of 5-hydroxytryptamine (5-HT) at a respective 2:3 ratios. The surface morphology, physio-chemical properties, and conductivity of the resulting fibrous scaffolds were characterized. Our results show that 5-HT/melanin/PHB composite fibers have rough surface with ˜290 nm (mean) diameter. Further, these fibers show higher thermal and mechanical stability with a melting temperature of 179.05° C., a Young's modulus ranging from 10 to 90 MPa, a hydrophilic index of 61.8 +3.4° and a conductivity of 1.3×103 Scm−1. To prove biocompatibility with neural tissues, human motor neurons and mouse sensory neurons were successfully cultured on the composite fibers. In addition, DRG culture on aligned fibers promoted the vectorized growth of axons along the fibers. These results collectively suggest that 5-HT/melanin/PHB composite fibrous scaffolds are a biodegradable conducting and biocompatible material suitable for neural tissue engineering application. In addition, inventors also propose a new grid base design of electrospinning setup, which allows to print computer aided designs of 3D scaffold and hydrogels to generate implants with controlled 3D shape and well-defined parameters, precisely tailored to fit into the site of injury. It is considered that this setup will be highly advantageous for the development of cost effective and personalized regenerative treatment of various tissues.
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The term “fiber” as used herein refers to a material having a thread-like structure. The term “fibrous assembly” as used herein refers to a material formed of a plurality of fibers. The term “nano fiber” as used herein refers to a fiber having a sub-micrometer or less than 1000 nm in diameter.
The term “biocompatible” as used herein refers to having no harmful effect on animals such as mammals, preferably humans.
The present disclosure provides a fiber or fibrous assembly. In an embodiment, the fiber can be a biocompatible fiber, a nano fiber, or a biocompatible nano fiber. In a preferable embodiment, the fiber or the biocompatible fiber may be a fiber of a biodegradable polymer, more preferably be a fiber of a natural biodegradable polymer. In a preferable embodiment, the fiber has a conductivity. The conductivity is preferably sufficient for neurons to attach to and/or proliferate on the fiber. The fibrous assembly contains a plurality of fibers, comprising one or more fibers as mentioned above. In a preferable embodiment, the fibrous assembly consists of the fibers as mentioned above.
In a preferable embodiment, examples of the biocompatible polymers include, for example, but not limited to, polyhydroxy alkane (PHA), preferably polyhydroxy butyrate (PHB), more preferably poly (3-hydroxy butyrate).
In a preferable embodiment, the fiber can further comprise melanin. In a preferable embodiment, the fiber can further comprise serotonin (5-HT). In a more preferable embodiment, the fiber can further comprise serotonin and melanin. In the embodiments, the biodegradability of the fiber may preferably increase. In these embodiments, the fiber is made of a polymer, preferably a biocompatible polymer, and melanin and/or 5-HT. In an embodiment, the biocompatible polymer may be a non-conducting polymer, because melanin and 5-TH can impart a conductivity to the fiber in a dose dependent manner.
In a preferable embodiment, the fiber satisfies at least one, two, three, four, five, or all of:
In a preferable embodiment, the fiber has 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) in diameter.
In a preferable embodiment, the fiber has 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.
In a preferable embodiment, the fiber has 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.
In a preferable embodiment, the fiber has a conductivity sufficient for neurons to proliferate on the fiber. Melanin and 5-HT can impart such a conductivity to a non-conducting fiber such as polyhydroxy alkane (PHA) in a dose dependent manner. Thus, the fiber may preferably comprise a sufficient amount of melanin and/or 5-HT. In a preferable embodiment, the fiber has a conductivity of 1×102 S/cm to 1×105 S/cm, 1×102 S/cm to 1×10+S/cm, or 1×102 S/cm to 1×103 S/cm.
In a preferable embodiment, the fiber comprises melanin and has a conductivity of 1×102 S/cm to 1×105 S/cm, 1×102 S/cm to 1×104 S/cm, or 1×102 S/cm to 1×10 3 S/cm. In a preferable embodiment, the fiber comprises melanin and 5-HT, and has a conductivity of 1×102 S/cm to 1×105 S/cm, 1×102 S/cm to 1×10+S/cm, or 1×10 2 S/cm to 1×103 S/cm.
In a preferable embodiment, the fiber has a hydrophilic surface. In an embodiment, the fiber has a hydrophilic surface exhibiting 90 degree or less, 80 degree or less, 70 degree or less, 60 degree or less, 50 degree or less, 40 degree or less, 30 degree or less, 20 degree or less, or 10 degree or less. In an embodiment, the fiber has a hydrophilic surface exhibiting 10 degree or more, 20 degree or more, 30 degree or more, 40 degree or more, 50 degree or more, 60 degree or more, 70 degree or more, or 80 degree or more. In an embodiment, the fiber has a hydrophilic surface exhibiting 10 degree to 90 degree, 10 degree to 50 degree, 50 degree to 90 degree, or 55 degree to 80 degree, 20 degree to 50 degree.
In a preferable embodiment, the fiber is coated with an extracellular matrix, such as collagen, fibronectin, gelatin, and laminin. An extracellular matrix is known to support attachment of animal cells to a surface coated with the extracellular matrix. In a preferable embodiment, the fiber is coated with poly L-lysine. In a preferable cm-bodiment, the fiber is coated with laminin and poly L-lysine. The coating will allow animal cells such as neurons to proliferate on the fiber or fibrous assembly. In these embodiments, the fiber or fibrous assembly comprising the fiber may be suitable for culturing a cell on its surface.
In an embodiment, the present disclosure provides a scaffold or scaffold material, implantable material or implant, comprising a fiber or fibrous assembly of the present disclosure. In an embodiment, the present disclosure provides a non-woven fabric or web, comprising a fiber or fibrous assembly of the present disclosure. The term “non-woven fabric” or “non-woven web” means an article or sheet that has a structure of individual fibers, which are interlaid, for example, in a reticular manner, but not in an identifiable manner. The scaffold or scaffold material can be used for culturing an organ or a tissue in vitro or in vivo. The scaffold or scaffold material can be suitable and/or used for culturing a neuron or a nerve. The scaffold or scaffold material can be suitable and/or used for inducing nerve regeneration in vivo, or in vitro to obtain a graft material.
The present disclosure provides a method of transplanting a graft material in a subject (e.g., human subject) in need thereof. The method may comprise transplanting the graft material to the subject. In an embodiment, the graft material comprises the fiber or fibrous assembly, scaffold or scaffold material, implantable material or implant, comprising a plurality of the fibers of the present disclosure; wherein a plurality of the fibers may form a non-woven fabric or be aligned in a reticular manner in the graft material; and wherein the graft material may further comprise a nerve cell for nerve regeneration on a surface of the graft, thereby optionally inducing nerve regeneration in the subject. In an embodiment, the fiber may comprise melanin and 5-HT, and be coated with an extracellular matrix, preferably laminin, and poly L-lysine. In a preferable embodiment, the fiber is made of PHB supplemented with melanin and 5-HT to have a conductivity.
In an embodiment, the fiber or the assembly may be dried, for example, by natural drying or preferably lyophilization. Thus, the present disclosure provides a dried form of the fiber or assembly, preferably, a lyophilized form of the fiber or assembly. In an embodiment, the fiber or the assembly is suitable for attachment of a cell, for example, a neural stem cell; a neuron such as a sensory neuron, an interneuron, and a motor neuron; a ganglion neuron such as a dorsal root ganglia neuron and trigeminal ganglia neuron; and a glia cell such as an astrocyte, an oligodendrocyte, a microglia, an ependiomocyte, a Schwann cell, and satellite cell. In a preferable embodiment, the cell to be attached may be a motor neuron and a ganglia neuron, more preferably a dorsal root ganglia neuron.
The present disclosure further provides a system for fabricating a fibrous assembly.
The nozzle 21 and the collector 22 are located inside a spinning chamber 27. The chamber 27 may be provided with at least one of a humidity controller 27a for measuring the humidity inside the chamber 27 and a temperature sensor 27b for measuring the temperature inside the chamber 27, such that the humidity and/or temperature inside the chamber 27 can be monitored when the spinning liquid is being discharged from the nozzle 21. The chamber 27 may also be provided with an exhaust unit 27c (e.g., a fan) for exhausting the inside of the chamber 27.
In an embodiment, as shown in
In the embodiment shown in
In an embodiment, as shown in
In alternative embodiments, the target electrodes 22b may have a concentric rectangular pattern (see
In an embodiment, the collector 22 is an exchangeable or removable collector. The collector 22 is exchangeably or removably mounted and supported on a stage or mount 26 located inside the chamber 27. The collector 22 may be, for example, but not limited to, detachably fixed to the stage or mount 26 by clips 26a. By exchanging the collector 22 to vary the pattern of the target electrode 22b, the arrangement, diameter, orientation and spacing of fibers in a fibrous assembly deposited on the collector 22 can be controlled. In an alternative embodiment, the collector 22 may be a non-exchangeable collector or non-removable collector. The stage or mount 26 may be electrically grounded.
The nozzle 21 is made of conductive material, for example metal and is arranged above the collector 22 and spaced apart from the collector 22. The tip of the nozzle 21 is directed vertically downward and is coated with an insulator. A metal syringe 21a storing the spinning liquid is fixed to the body of the nozzle 21. A syringe pump 21b for controlling flow rate of the spinning liquid supplied to the nozzle 21 is connected to the syringe 21a. The flow rate of the spinning liquid supplied to the nozzle 21 may be, for example, 0.01 to 1 ml/h. The nozzle 21 is electrically connected to the high voltage power supply 28 through the syringe 21a, and a high voltage of more than 5 kV, for example, 10 kV to 30 kV can be applied between the nozzle 21 and the target electrode 22b.
When the high voltage is applied between the nozzle 21 and the target electrode 22b while the spinning liquid is supplied to the nozzle 21, the spinning liquid is electrically charged, and discharged in an electrically charged state toward the target electrode 22b. The discharged spinning liquid is stretched by the repulsive force of electric charges and the solvent in the liquid evaporates to form fibers (for example, nano fibers), and the formed fibers are deposited as fibrous assembly on the collector 22.
In a preferable embodiment, the stage or mount 26 is provided with a moving mechanism 25 configured to move the collector 22 inside the chamber 27. The moving mechanism 25 may be configured to move the collector 22 on the stage or mount 26 in the X and Y directions (horizontal directions), both of which are perpendicular to the discharge direction of the nozzle 21, or in the Z direction (vertical direction) parallel to the discharge direction of the nozzle 21. The moving mechanism 25 may include a piezo motor, such that the amount of movement of the collector 22 can be controlled with a precision of 200 nm or less.
In an alternative embodiment, both the base 22a and the target electrode 22b may have a cylindrical shape (see
In a preferable embodiment, the columns 26a are provided with a moving mechanism (not shown) configured to move the cylindrical collector 22 inside the chamber 27 in the X and Y directions. The moving mechanism may include a piezo motor, such that the amount of movement of the cylindrical collector 22 can be controlled with a precision of 200 nm or less.
In a preferable embodiment, the system 20 further includes a control unit 30 for controlling the operation of the moving mechanism 25. At least part of the control unit 30 may be implemented by a computer having a memory storing instructions and at least one processor configured to execute the instructions.
The control unit 30 may include: a data reception unit configured to receive three-dimensional CAD data (for example, an STL file) of a fibrous assembly to be manufactured; a calculation unit configured to calculate timing and amount of movement of the collector 22 according to the CAD data; and a signal transmitting unit configured to transmit a control signal corresponding to the calculated timing and amount of movement to the moving mechanism 25. During discharge of the spinning liquid, the moving mechanism 25 moves the collector 22 according to the control signal received from the control unit 30, thereby controlling the geometrical shape of the fibrous assembly deposited on the collector 22 to a desired shape defined in the CAD data.
The present disclosure further provides a method for fabricating a fibrous assembly using the system 20 as mentioned above.
In an embodiment, the method includes determining the pattern of the target electrode 22b on the collector based on a desired alignment, diameter, orientation and inter-fiber space of fibers in a fibrous assembly. For example, a relationship between the pattern of the target electrode 22b on the collector 22 and the arrangement, diameter, orientation and spacing of fibers of a fibrous assembly deposited on the collector 22 may be determined in advance through experiments and simulations, and the determined relationship is stored in a table or database. Then, the pattern of the target electrodes 22b on the collector 22 may be determined by reference to said table or database based on the alignment, diameter, orientation and inter-fiber space of fibers in a fibrous assembly to be manufactured. In an alternative example, the pattern of the target electrode 22b on the collector 22 may be determined based on the alignment, diameter, orientation, and inter-fiber space of fibers in the fibrous assembly to be manufactured, using a machine-learned model having trained the relationship between the pattern of the target electrode 22b on the collector 22 and the alignment, diameter, orientation, and inter-fiber space of fibers in the fibrous assembly deposited on said collector 22.
In an embodiment, the method further includes arranging the collector 22 having the target electrode 22b with the determined pattern at a distance from the nozzle 21 configured to be supplied with the spinning liquid.
In an embodiment, the method further includes applying a high voltage between the nozzle 21 and the target electrode 22b and electrically charging the spinning liquid, so as for the spinning liquid to be discharged in an electrically charged state toward the target electrode 22b to form fibers, which are deposited as a fibrous assembly on the collector 22. During discharge of the spinning liquid, the moving mechanism 25 may move the collector 22 according to the control signal received from the control unit 30, thereby controlling the shape of the fibrous assembly deposited on the collector 22 to have a desired shape.
In the embodiment mentioned above, the nozzle 21 is stationary positioned inside the chamber 27, and the moving mechanism 25 is configured to move the collector 22 inside the chamber 27 in the X, Y and Z directions, respectively. In an alternative embodiment, the collector 22 may be stationarily positioned inside the chamber 27 and the moving mechanism may be configured to move the nozzle 21 inside the chamber 27 in the X, Y and Z directions, respectively. In another alternative embodiment, the moving mechanism may be configured to move both the nozzle 21 and the collector 22 independently of each other inside the chamber 27 in the X, Y and Z directions, respectively.
In alternative embodiment, the system 20 may include a plurality of the nozzles 25. In a preferable embodiment, the plurality of nozzles 25 may each have a different nozzle diameter. In this case, by simultaneously discharging the spinning liquids from the plurality of nozzles 25, the fibrous assembly can contain a plurality of types of fibers each having different diameters.
In a preferable embodiment, the plurality of nozzles 25 may each be supplied with a different spinning liquid or a combination of a spinning liquid, a gas and a solvent. In this case, by simultaneously discharging the spinning liquids or combinations from the plurality of nozzles 25, the fibrous assembly can contain a plurality of types of fibers each having different properties.
In an embodiment, the present disclosure provides, for example, the following inventions:
[1] A fiber (in particular, a biocompatible fiber, a nano fiber, or a biocompatible nano fiber), comprising a biocompatible polymer and melanin, wherein the polymer may preferably be a biodegradable polymer, more preferably be a natural biodegradable polymer, or a fibrous assembly comprising the fiber, wherein the fiber or the fibrous assembly may be dried (e.g., lyophilized), for example, a fiber, comprising a biocompatible polymer and melanin, or a fibrous assembly comprising the fiber . . .
[2] The fiber or fibrous assembly according to [1] above, wherein the polymer is a biodegradable polymer.
[3] The fiber or fibrous assembly according to [1] or [2] above, further comprising serotonin (5-HT).
[4] The fiber or fibrous assembly according to any one of [1] to [3] above, wherein the biocompatible polymer is polyhydroxy alkane (PHA), preferably polyhydroxy butyrate (PHB), more preferably poly (3-hydroxy butyrate).
[5] The fiber or fibrous assembly according to any one of [1] to [4] above, having a conductivity of 1×102 to 1×105 S/cm.
[6] The fiber or fibrous assembly according to any one of [1] to [5] above, having a hydrophilic surface (e.g., exhibiting a contact angle of 55 degree or more to less than 90 degree).
[7] The fiber or fibrous assembly according to any one of [1] to [6] above, wherein the fiber is coated with laminin and optionally poly L-lysine.
[8] The fiber or fibrous assembly according to any one of [1] to [7] above, wherein the fiber satisfies at least one of:
[8A] The fiber or fibrous assembly according to any one of [1] to [7] above, wherein the fiber satisfies at least one of:
[9] A scaffold (preferably an implantable scaffold) or scaffold material, implantable material or implant, comprising a plurality of the fibers or fibrous assembly according to any one of [1] to [8A] above (hereinafter [1] to [8A] includes [8] above), wherein the fibers may be aligned in a reticular manner in the scaffold or scaffold material, implantable material or implant.
[10] The 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 is optionally cut, folded, or rolled (e.g., in brain, spinal cord, or peripheral nerve).
[11] A method of transplanting a graft material in a subject (e.g., human subject) in need thereof, comprising:
[12] The method according to above, wherein the graft material further comprises a nerve cell for nerve regeneration on a surface of the graft, thereby inducing nerve regeneration in the subject.
In an embodiment, the present disclosure further provides, for example, the following inventions:
[9] A System for Fabricating a Fibrous Assembly Comprising:
[13A] A system for fabricating a fibrous assembly comprising:
[14] The system according to or [13A] above, further comprising a moving mechanism configured to move one or both of the nozzle and the collector relative to each other, such that a shape and size of the fibrous assembly can be controlled by moving the nozzle and the collector relative to each other during discharge of the spinning liquid.
[14A] The system according to or [13A] above, further comprising a moving mechanism configured to move one or both of the nozzle and the collector relative to each other, so that a shape and size of the fibrous assembly may be controlled by moving the nozzle and the collector relative to each other during discharge of the spinning liquid.
[15] The system according to or [14A] above, wherein
[15A] The system according to or [14A] above, wherein
[16] The system according to any one of to [15A] above (hereinafter to [15A] includes [14A] and above), wherein
[16] The system according to any one of to above (hereinafter to includes [14A], and [15A] above), wherein
[17A] The system according to any one of to above, wherein
[18] The system according to any one of to [17A] above (hereinafter to [17A] includes [14A], [15], [15A], and above), further comprising a control unit
[19] The system according to any one of to above (hereinafter to includes [13A], [14], [14A], [15], [15A], [16], and [17A] above), comprising a plurality of the nozzles.
[20] The system according to above, wherein
[20A] The system according to above, wherein
[21] The system according to any one of to [20A] above (hereinafter to [20A] includes above), wherein
[21A] The system according to any one of to [20A] above, wherein
[22] The system according to any one of to [21A] above (hereinafter to [21A] includes [13A], [14], [14A], [15], [15A], [16], [17], [17A], [18], [19], [20], [20A] and [21] above), further comprising: a spinning chamber the nozzle and the collector are located therein; and at least one of a humidity controller and a temperature sensor inside the spinning chamber.
[22A] The system according to any one of to [21A] above, further comprising at least one of a humidity controller and a temperature sensor inside a spinning chamber.
[23] The system according to any one of to [22A] above (hereinafter to [22A] includes [13A], [14], [14A], [15], [15A], [16], [17], [17A], [18], [19], [20], [20A], [21], [21A] and above), wherein the collector has a grid pattern, a concentric rectangular pattern, a concentric circular pattern, a solid planar shape, or a cylindrical shape.
[24] The system according to any one of to above (hereinafter to includes [13A], [14], [14A], [15], [15A], [16], [17], [17A], [18], [19], [20], [20A], [21], [21A], and [22A] above), wherein the collector is an exchangeable or removable collector.
[25] A collector with a target electrode for use in the system according to any one of to above (hereinafter to includes [13A], [14], [14A], [15], [15A], [16], [17], [17A], [18], [19], [20], [20A], [21], [21A], [22], [22A] and above).
[26] A method for fabricating a fibrous assembly comprising:
[26A] A method for fabricating a fibrous assembly comprising:
[27] A method of producing a fiber, comprising:
[27A] A method of producing a fiber, comprising:
[28] A semiconductor device such as a transistor, an implant, or an electrode, comprising the fiber or fibrous assembly according to any one of [1] to [8A] above having a conductivity of 1×102 to 1×105 S/cm.
1. Materials and methods
Synthetic Melanin (Sigma M8631-1G), Poly [(R)-3-hydroxybutyric acid (Sigma 363502-100G), and Serotonin hydrochloride (Sigma H9523-1G), 1, 1, 1, 3, 3, 3-Hexafluoro-2-propanol (HFIP) (Sigma-Aldrich, St. Louis, USA), poly (vinyl alcohol) 8.0 wt % (Kato tech, Japan) were used for the fabrication of scaffolds. All chemicals were supplied by Sigma Aldrich, unless stated otherwise. SYLGARD™ 184 silicon elastomer kit (Dow chemical company, USA) was used to prepared cured PDMS ring for the culture chamber. Suppliers for tissue culture media and sup-plements are individually specified in the method section. 16% paraformaldehyde (#15710; Electron Microscopy Sciences, Hatfield, PA) was purchased from the for the fixation of the cells. Anti-BIII tubulin antibody was purchased from GeneTex (#GTX631830) and AlexaFluor 594 anti-mouse secondary antibody was purchased from Invitrogen (ThermoFisher Scientific, Japan).
PHB was used as a blend to assist electrospinning (5-HT/melanin/PHB and melanin/PHB). The electrospinning setup used in the preparation of random and aligned fibers is shown in
Surface morphology of PHB, melanin/PHB, 5-HT/melanin/PHB and PVA fibers was evaluated using scanning electron microscopy (SEM, JEOL JSM-7900F). Briefly, electrospun fibers were lyophilized and mounted onto a conducting carbon tape attached on a copper stub. Samples were sputter coated with gold for 4 min followed by analysis under SEM at a working distance of 10 mm and an accelerating voltage of 5 kV. The surface morphology and ultra-structure of the fibers was observed at various magnifications ranging from 500X to >20000X. The average and standard error of the mean of the diameter of electrospun fibers was determined from the SEM images.
Functional group presence on electrospun nanofibers and powder forms of PHB, melanin and 5-HT (with KBr) were performed with a Fourier-transform infrared spectroscopy (FTIR) machine (Vertex 80v; Bruker) in transmission mode over a range of 400-4000 cm−1 (MIR range) at the resolution 2 cm/4 cm−1 with 64 scans averaging mode. Further, elemental analysis (carbon, oxygen and nitrogen) was performed using X-ray photoelectron spectroscopy (XPS) on a KRATOS Axis Ultra has (S L V Narayana et al., 2020).
Tracing of the fibers' elemental composition using SEM-EDX was also performed to confirm successful blending of melanin and PHB and map the distribution of melanin in blended fibers. Briefly, scaffolds were sputter coated with Osmium (Os) for 2 min and visualized using a scanning electron microscope (SEM; JEOL JSM-7900F), equipped with a front and rear Oxford energy-dispersive X-ray (EDX) detector (Oxford Instruments, UK). SEM micrographs were captured at a working distance of 10 mm and an accelerating voltage of 10 kV at 500x. Aztec-SEM 6.0 (Oxford Instruments, UK) software was used to map the distribution of C, O and N elements in PHB and blended 5-HT-melanin-PHB (9% PHB-3% melanin) fibers.
Differential scanning calorimetry (DSC)-8500 (PerkinElmer) was used for the thermal analysis. Scaffold samples of 2 mg were placed in alumina pans and empty pans were used as a reference. All samples were first heated at a range of 40 to 250° C. with a rate of 10° C./min. Afterwards, the samples were cooled to 40° C. at 10° C./min. After each test, the crystallization/melting peak region from the thermograph was analysed to determine the crystallization (Tc)/melting (Tm) point (Agrawal et al., 2021, 2022).
Atomic Force Microscope (AFM) images of the scaffolds were acquired as previously described (Agrawal et al., 2021, 2022), using a MultiMode 8 Atomic Force Microscope with a Nanoscope V controller and E scanner (Bruker). Mechanical characterization of the scaffold nanofibers was performed using a PeakForceTapping mode. AFM imaging was conducted with RTESPA-150 probe (Bruker) with a nominal spring constant of 5.1 N/m, nominal frequency of 2 kHz and a nominal tip radius of 15 nm (Baklaushev et al., 2019). Nanogrid scaffolds were studied in air over the area of 5×5 μm to determine force-displacement curves. The reduced Young's Modulus/DMT modulus, is automatically calculated by fitting the retract curve using the Derjaguin, Muller, Toropov (DMT) model (Lagaly, 1988). Images were obtained at a scan rate of 1 Hz and 512×512 pixels' resolution. The raw Young's modulus AFM images were processed using the NanoScope Analysis v.1.10 software (Bruker).
Conductivity of the electrospun fibers mesh of 5-HT/melanin/PHB (80 μm thickness) and melanin/PHB (60 μm) under dry condition was determined by using a probe station Summit 12K(Cascade Wernersville, PA, USA) equipped with a Keithley SC 4200A parameters analyser (Tektronix, Oregon, USA). We used van der Pauw method for resistivity measurement (4-probes method in which two pairs of contacts are used to measure the conductivity).
The wettability of the scaffold was evaluated by static contact angle measurement using an Easy Drop tensiometer (KRUSS, GmbH) (Agrawal et al., 2021). The contact angle is a quantitative measure of the wetting properties of a solid by liquid and is dependent on the surface area, with higher surface energies being associated with lower contact angles (Morouco et al., 2016). A water droplet was poured onto the surface of solid samples and the contact angle was measured by Drop Shape analysis software (KRUSS, GmbH).
Electrospun fibrous meshes swelling and degradation in phosphate buffered saline (PBS, pH 7.4) was monitored. Briefly, dried electrospun fibers scaffolds were cut into 10 mm×10 mm samples. Subsequently, samples were 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 degree of swelling, samples were immersed in 1 ml of PBS (pH 7.4) at 37° C. for 12 h, and subsequently weighed. AFM micrographs were also taken to visually confirm the swelling of fibers. Similarly, for the degradation analysis, after the pre-determined degradation period (10 days), the scaffolds were rinsed in PBS and dried in an oven for 24 h and weighted. For the calculation of the degree of swelling (%) and degraded mass (%), we considered the initial dry weight of the scaffold (mi), the weight of the swelled nanofibers after removing excess water and surface moisture with a filter paper (ms), and the constant residual weight of the scaffolds after degradation (mx). The degree of swelling and degraded mass (%) of the scaffolds were calculated from equation (5) and (6).
All experiments were carried out following the guidelines of the Okinawa Institute of Science and Technology Graduate University (OIST) genetic manipulation procedures. All animal experiments have been performed in the accordance to Japanese laws and to the regulations of the OIST animal care and use committee (protocol #ACUP-2021-326) OIST animal facilities and animal care and use program are accredited by AAALAC international (Ref. #1551).
We used plastic bottom 24 well plate for primary neural cell culture. Scaffolds were placed inside the well and a Polydimethylsiloxane (PDMS) ring was inserted to fix the scaffold at the bottom of the well. For the fabrication of Polydimethylsiloxane (PDMS) rings, molds were designed in CAD Rhinoccrous3D (V.5, Robert McNeel & Associates) and 3D printed (Object 500 Connex 3, Stratasys, Germany). PDMS prc-polymer and catalyst were then mixed thoroughly with a 10:1 ratio in a disposable plastic cup. The mixture was degassed in a vacuum desiccator for 20 minutes and poured inside the molds. The PDMS was cured in an oven at 60° C. for 3 h. Finally, polymerized PDMS rings were extracted from the molds and fixed on the scaffold to secure it at the bottom of the well for neuronal culture.
The structure was then coated with 0.01% Poly-L-lysine (Sigma-Aldrich #P4832-50 ml) overnight at 4° C., rinsed with water and coated again with Laminin (GibcoBRL #23017-015) for 2 h at 4° C. DRGs were dissected from 2 months old ICR female mice (Charles River or Japan 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 at 37° C. in HBSS for at least 30 minutes. The ganglia were then triturated in HBSS, 10 mM Glucose, and 5 mM HEPES Sigma-Aldrich, #H0887), pH 7.35. Cells were recovered by centrifugation through 20% Percoll (Sigma-Aldrich, #P4937) in L15 medium (GibcoBRL #L-5520) at the speed of 1000 rpm for 7 min, plated at a density of 2×10+ cells/scaffold and grown in F12 medium (GibcoBRL #11765062) for 48 hours/DIV-2.
1.6.4. Culture of hMNs from IPS Cells
We adapted a published protocol (Bossolasco et al., 2018) for the culture and differ-entiation of human inducible pluripotent stem cells (hIPSCs) into motor neurons (MNs). Human MNs were cultured for 7-DIV prior fixation and immunostaining.
DRG neurons were cultured for 2 days and MNs for 7 days as described above before fixation with 4% paraformaldehyde in phosphate buffer 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 the axonal network, neurons were incubated overnight at 4° C. with anti-βIII-tubulin antibody (1:1000 dilution in PBS). Cells were then washed 3 times with PBS and incubated with anti-mouse Alexa Fluor 594 conjugated secondary antibody (1:500 dilutions in PBS) for 1 h at room temperature. Fluorescence imaging was performed on a confocal laser scanning microscope LSM900 (Carl Zeiss AG, Germany) using 63x oil immersion objective (Plan-Apochromat DIC M27, NA=1.40). Images were acquired in ZenBlue 3.1 (Carl Zeiss AG) with a 512×512 pixel resolution and a 2.05 ρs pixel dwell time. A 3×3 tiles and z-stacks (with 1 μm steps) image was scanned to encompass the entire grid scaffold on xyz planes. After stitching tiles, the resulting image had a final xy resolution of 1433×1434 pixels.
GraphPad Prism 9 Software, (GraphPad, U.S.A.) was used for statistical analysis. ANOVA with Tukey's post hoc test was performed for multiple comparisons and unpaired t-test was performed for the comparison between 2 groups. Differences were considered significant if the probability of error was less than 5%. All the data were expressed as mean±s.c.m., error bar indicates standard error of mean. * P<0.05, ** P<0.01, *** P<0.001, and **** P<0.0001.
Scanning Electron Microscope (SEM) and Helium Ion Scanning Microscopy (HIM) revealed the ultrastructure of the nanofiber scaffolds (
The physio-chemical properties scaffolds were investigated by measuring the chemical, thermal, mechanical, electrical properties and hydrophilicity. These properties determine the stability, biocompatibility and biodegradability of materials in vivo and its ability to maintain its intended structure over a period of time to support the growth of cells or tissues.
First, we performed chemical analysis of the scaffolds with FT-IR. UV-vis trans-mittance spectra of pure PHB, melanin and 5-HT powders show the presence of characteristic aliphatic-O—H stretching peak at 3436.88 cm−1 (
We also characterize the FT-IR spectra for PHB, melanin blended PHB, 5-HT-melaninand PHB composite fibers and PVA fibers. We observed the presence of carboxylic acid-C═O stretching peak at 1720.37 cm−1, sp3-C—H stretching peak at 2979.45 cm−1 in PHB fibers with a shift of ˜4 cm−1 and ˜9 cm−1 with PHB powder form due to the reaction with HFIP(
Moreover, elemental composition analysis with X-ray photoelectron spectroscopy (XPS) of PHB, melanin+PHB+5-HT, melanin+PHB, and PVA are shown in
We also traced fiber composition by SEM-EDX analysis (
We then determined the thermal properties of our scaffolds by DSC (
To measure the mechanical strength of our scaffolds, we used nanoindendation atomic force microscopy (AFM) to generate maps of Young's modulus distribution concurrently with topographical imaging to map the anisotropic distribution of mechanical strength (
We also measured the resistivity (p) and conductance (o) of the 5-HT and melanin blended scaffold to check the electrical properties of electrospun fibers. Data from the 4-probe method suggested that the resistivity of the fibrous scaffold (7.71×103 ohm-cm) and conductivity (1.3×103 S/cm) lies within the range of semiconductor materials (
We measured the degree of swelling (swelling capacity (%)) of PHB and 5-HT-melanin-PHB scaffolds over a period of 12h (
Because of the relevance of DRG and hMNs neurons in peripheral injury, spinal cord injuries and neuronal degeneration, we tested their biocompatibility with 5-HT-melanin and PHB composite scaffold (
We are developing electrically conducting, biocompatible and biodegradable implants for the purpose of tissue engineering to address the long-lasting medical problems such as SCI, nerve injuries (NI) and neo-tissue formation for the treatment of the deep wound (>=5 mm) usually formed after the accidents or during critical surgeries such as cortical brain surgery and open-heart surgery. Researchers found that the combined use of biomaterials with and without the use of stem cells have shown the potential to replace the conventional inefficient methods to deal said medical problems. Recent advancements in the fabrication technology enabled us to fabricate 2D scaffolds and circuitries with a high precision range from few hundreds of nanometers such as 2-photon polymerization (2-PP) laser lithography system. However, when it comes to the cost-effective fabrication of 3D structures from nanometer to few millimeters dimension with user defined shape/size and specific texture and with the high level of precision, over a short-time period; developing such a tool or state of the art technology remains a big challenge to the scientific community. On one hand, conventional electrospinning setup allows faster fabrication but is mainly limited to development of 2D scaffolds (ranging from mm to cm with limited thickness) and doesn't allow any control over scaffolding parameters such as fiber diameter and inter-fiber space. On the other hand, 3D-lithography allows precise control over parameter (˜200 nm) but the size of scaffold is restricted (only few millimeters) (Agrawal et al., 2021). Further, it is a laborious and expensive method, and requires relatively longer print time. Considering all the shortcomings of existing fabrication techniques, we decided to develop a hybrid nanofabrication system amal-gamating the principles of electrospinning(ES) and 3D-bioprinting based on a conducting grid pattern to overcome the said limitations.
Every year several million people worldwide suffer from SCI (Kang et al., 2017), 15-40% of the cases also involving PNI (S. Chen et al., 2015). Over the last decade, regenerative medicine has achieved rapid and promising advancements 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), mainly including the use of implants to stabilize the spinal cord, auto grafting, and systemic injection of growth factors (Jendelova, 2018). Motor/sensory neuron loss of function, neuronal degeneration and mismatch of damaged nerve and graft dimensions are known drawbacks of these ap-proaches. Meta-analysis of more than 70 preclinical studies suggests that combination of cell therapy with various scaffolds improves function restoration when compared to cell therapy alone (Baklaushev et al., 2019). Nonetheless, it is still a major challenge to engineer an ideal nanofibrous scaffold that provides an attractive clinical alternative to nerve auto grafts and semiconductor implants for SCI (Baklaushev et al., 2019; Boni et al., 2018). We fabricated 5-HT-melanin blended PHB composite conductive scaffolds for neural tissue engineering. SEM analysis confirmed successful fabrication of ˜290 nm (
We also fabricated aligned fibers for creating a mechanical constraint to promote the vectorized growth of axon (
| Number | Date | Country | Kind |
|---|---|---|---|
| 2022-005711 | Jan 2022 | JP | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2023/001383 | 1/18/2023 | WO |
