Bio-implantable microfiber scaffold and production method therefor

Abstract

The present invention relates to a production method for a bio-implantable microfiber scaffold. The production method may comprise: a first step for producing a two-dimensional microfiber membrane by electrospinning a polymer solution; and a second step for producing a one-dimensional microfiber by rolling or twisting the two-dimensional microfiber membrane. In addition, the present invention relates to a bio-implantable microfiber scaffold.

Description

Bio-implantable microfiber scaffold and method for manufacturing the same

[0001] The present invention relates to a bio-implantable microfiber scaffold and a method for manufacturing the same, and more particularly, to a scaffold formed by forming a two-dimensional microfiber membrane into one-dimensional microfibers and a method for manufacturing the same, and further to a scaffold formed into three dimensions by structuring (braiding, weaving, or knitting) a scaffold formed from one-dimensional microfibers and a method for manufacturing the same.

[0002] The development of artificial organs has evolved through three main approaches: precision machining, tissue engineering (typically organoids), and bioprinting. Precision machining technology offers the advantages of achieving precise designs using 3D modeling and rapid fabrication, enabling patient-specific production. However, limitations in available materials lead to limited biocompatibility, high initial investment costs, and limited fabrication sizes. Tissue engineering utilizes cells and biomaterials to undergo developmental processes (e.g., morphogenesis) similar to those of living tissues, allowing for the formation of structures similar to those of living tissues. This allows for excellent biocompatibility. However, tissue culturing is time-consuming and limits the ability to replicate the functions of living organs. Furthermore, some tissues may raise ethical concerns, and high production costs are also drawbacks. Furthermore, organoids suffer from low structural and functional maturity, poor reproducibility, and the lack of vascular and immune systems, which remain challenging to overcome. Bioprinting offers the advantages of using materials similar to living tissue, offering excellent biocompatibility and the ability to fabricate tissues using a variety of biomaterials. However, current technological limitations make it difficult to perfectly reproduce complex tissue structures, and manufacturing speeds and costs are high. Furthermore, the long-term biosafety of some bioprinting materials has not been sufficiently verified, hindering their practical application. Furthermore, the difficulty in uniformly arranging mature blood vessels when fabricating large organs via bioprinting means restricts its effectiveness to small organs or organs in thin sheet form.

[0003] To overcome the limitations of these conventional technologies, 3D scaffolds (three-dimensional scaffolds) are being developed in the fields of tissue engineering and artificial organs. A 3D scaffold is a three-dimensional structure that supports cells, enabling them to attach, proliferate, and form tissues. 3D scaffolds play a crucial role in the development of artificial organs. Simply culturing cells alone cannot create structures similar to real tissues, nor can they perfectly embody their functions. 3D scaffolds provide a framework for cells to naturally attach and grow, aiding tissue formation and contributing to the realization of structures and functions similar to real human tissues. Furthermore, mechanical strength, porosity (microscopic spaces where cells can migrate and grow), and degradation rate can be adjusted to suit the specific characteristics of the organ, enabling the creation of customized tissues and organs.

[0004] 3D scaffold development plays a crucial role in tissue engineering, providing the structural support essential for cell growth and differentiation. Recently, large-scale 3D scaffolds have attracted significant attention due to their ability to more effectively mimic actual organs. However, existing 3D bioprinting research has limited their scale. This is because artificially forming a vascular network is difficult. Furthermore, as 3D scaffolds grow larger, oxygen and nutrients rely solely on diffusion, hindering the supply of sufficient nutrients to internal cells. Furthermore, metabolic waste excretion is also disrupted, limiting cell survival.

[0005] Accordingly, the present invention presents the possibility of overcoming the problem of delayed vascularization occurring in gel-based bioprinting by developing a bio-implantable microfiber scaffold that can improve mechanical stability and increase long-term cell survival rate, and is expected to establish a standard in the field of artificial organ development and tissue engineering as a new method to solve the existing limitations of large-scale scaffold production.

[0006] One object of the present invention to solve the above-mentioned problems is to provide a method for manufacturing a bio-implantable microfiber scaffold that increases cell viability, reduces cell loss, shortens the time for onset of effect, increases mechanical strength, improves mechanical flexibility, enables customization, promotes cell growth and differentiation, promotes vascularization, enables the production of large scaffolds, and enables rapid production.

[0007] Another object of the present invention to solve the above-mentioned problems is to provide a one-dimensional, thread-shaped porous scaffold capable of being structured into a three-dimensional three-dimensional structure and a method for manufacturing the same.

[0008] Another object of the present invention to solve the above-mentioned problem is to provide a scaffold formed of one-dimensional microfibers by structuring (braiding, weaving or knitting) a scaffold into a three-dimensional structure, and a method for manufacturing the same.

[0009] Another object of the present invention to solve the above-mentioned problems is to provide a bio-implantable microfiber scaffold that increases cell viability, reduces cell loss, shortens the time to onset of effect, increases mechanical strength, improves mechanical flexibility, enables customization, promotes cell growth and differentiation, promotes vascularization, enables the production of large scaffolds, and enables rapid production.

[0010] However, the problem to be solved by the present invention is not limited to this, and may be expanded in various ways without departing from the spirit and scope of the present invention.

[0011] A method for manufacturing a bio-implantable microfiber scaffold according to one embodiment of the present invention for achieving the aforementioned purpose may include a first step of manufacturing a two-dimensional microfiber membrane by electrospinning a polymer solution; and a second step of manufacturing a one-dimensional microfiber by rolling or twisting the two-dimensional microfiber membrane.

[0012] According to one aspect, between the first and second steps, a step of coating the two-dimensional microfiber membrane with parylene may be further included.

[0013] According to one aspect, after coating the two-dimensional microfiber membrane with perylene, a tensioning step may be further included.

[0014] According to one aspect, the rolling or twisting of the two-dimensional microfiber membrane in the second step may be performed by immersing it in water or spraying water using a sprayer.

[0015] According to one aspect, in the second step, the entire two-dimensional microfiber membrane may be twisted by hand or machine to produce one-dimensional microfibers.

[0016] According to one aspect, after the second step, a step of performing a multi-array process using the one-dimensional microfibers manufactured in the second step may be further included.

[0017] According to one aspect, the multi-array process may be a braiding, weaving or knitting process.

[0018] According to one aspect, the first step may further include a step of manufacturing and laminating two or more two-dimensional microfiber membranes.

[0019] According to one aspect, at least one of the two or more two-dimensional microfiber membranes may be an oriented microfiber membrane, and at least one may be an unoriented microfiber membrane.

[0020] According to one aspect, between the first and second steps, a step of loading cells onto the two-dimensional microfiber membrane may be further included.

[0021] According to one aspect, the method may further include preparing at least three of the two-dimensional microfiber membranes and loading hard tissue cells including osteoblasts, soft tissue cells including fibroblasts, or vascular tissue cells including vascular endothelial cells and angiogenesis-inducing substances onto each of the two-dimensional microfiber membranes.

[0022] According to one aspect, in the second step, the method may further include rolling or twisting the two-dimensional microfiber membrane loaded with the hard tissue cells, the two-dimensional microfiber membrane loaded with the soft tissue cells, and the two-dimensional microfiber membrane loaded with the vascular tissue cells, respectively, to produce one-dimensional hard tissue microfibers, one-dimensional soft tissue microfibers, and one-dimensional vascular tissue microfibers, and then structuring the one-dimensional hard tissue microfibers, one-dimensional soft tissue microfibers, and one-dimensional vascular tissue microfibers to produce hard tissue and soft tissue scaffolds.

[0023] According to one aspect, when structuring the one-dimensional hard tissue microfibers, one-dimensional soft tissue microfibers, and one-dimensional vascular tissue microfibers, a gradient can be formed between the hard tissue and the soft tissue by controlling the ratio of the one-dimensional hard tissue microfibers, one-dimensional soft tissue microfibers, and one-dimensional vascular tissue microfibers.

[0024] According to one aspect, the method may further include a step of coating the surface of the manufactured hard and soft tissue scaffolds with a decellularized hydrogel.

[0025] According to one aspect, the step of loading cells onto the two-dimensional microfiber membrane may be performed by a bioink printing method.

[0026] In addition, a bio-implantable microfiber scaffold according to one embodiment of the present invention for achieving the aforementioned purpose may be manufactured by a method for manufacturing a bio-implantable microfiber scaffold.

[0027] The technology disclosed in the present invention may have the following effects. However, this does not mean that a specific embodiment must include all or only the following effects, and therefore the scope of the disclosed technology should not be construed as being limited thereby.

[0028] According to the bio-implantable microfiber scaffold and its manufacturing method according to one embodiment of the present invention described above, it can form a structure suitable for cell attachment and growth, providing an environment similar to biological tissue. It also has higher mechanical strength than tissue engineering and bioprinting, while being more flexible than precision machining, thus increasing practicality. Furthermore, the arrangement and density of the microfibers can be adjusted according to the characteristics of the patient's tissue, enabling customized manufacturing, and it can effectively reproduce the functions of actual organs by promoting cell growth and differentiation.

[0029] In addition, according to a bio-implantable microfiber scaffold and a manufacturing method thereof according to an embodiment of the present invention, mechanical stability is excellent, long-term cell survival rate is high, vascularization can be promoted through a porous structure, large-scale scaffold manufacturing is possible, which can accelerate the commercialization of artificial organs, customized manufacturing is easy, cell growth and organization can be optimized, so the function of actual organs can be precisely imitated, and cost reduction and mass production are possible through a manufacturing speed faster than conventional bioprinting. In addition, a bio-implantable microfiber scaffold according to an embodiment of the present invention can be very suitably used not only for artificial organs but also as a surgical suture.

[0030] Moreover, according to the bio-implantable microfiber scaffold and the method for manufacturing the same according to one embodiment of the present invention, compared to a conventional gel-type scaffold or artificial organ injected with a syringe, the cell loss rate can be reduced from about 60% to about 0%, the cell viability can be increased from about 10% to about 90%, and the time for the effect to be expressed can be reduced from about 6 hours to about 30 minutes.

[0031] Therefore, the bio-implantable microfiber scaffold and the method for manufacturing the same according to one embodiment of the present invention can provide an effective treatment method for musculoskeletal diseases and can be expanded into applied research in the medical field.

[0032] Figure 1 is a schematic diagram showing the manufacturing process of a bio-insertable microfiber scaffold according to the present invention.

[0033] Figure 2 is a schematic diagram showing a process for manufacturing a large-area microfiber membrane according to one embodiment of the present invention.

[0034] Figure 3 is a schematic diagram of the production of an artificial hard tissue-soft tissue scaffold through a structuring process of artificial tissue fibers.

[0035] Figure 4 is a schematic diagram showing the diameter of electrospun microfibers according to the present invention.

[0036] Figure 5 is a schematic diagram showing the density of an electrospun microfiber film according to the present invention.

[0037] Figure 6 is a schematic diagram showing the orientation of an electrospun microfiber film according to the present invention.

[0038] Figure 7 is a schematic diagram showing a process for loading hard tissue, soft tissue, vascular tissue cells and effective factors onto an electrospun microfiber film according to the present invention.

[0039] Figure 8 is a schematic diagram of printing bioink containing cells onto electrospun microfibers according to the present invention.

[0040] Figure 9 is a schematic diagram showing the process of manufacturing one-dimensional microfibers by laminating and rolling or twisting an electrospun microfiber film according to the present invention.

[0041] Figure 10 is a schematic diagram of the manufacturing of a large scaffold using one-dimensional microfibers according to the present invention.

[0042] Figure 11 is a schematic diagram showing the control of the fiber ratio of a hard tissue-soft tissue scaffold according to the present invention.

[0043] Figure 12 is a schematic diagram of manufacturing a scaffold by structuring hard tissue fibers and soft tissue fibers simultaneously according to the present invention.

[0044] Figure 13 is a schematic diagram showing stabilizing tissue by coating the surface of a bio-insertable microfiber scaffold according to the present invention.

[0045] Figure 14 is a SEM image at a scale of 1000X showing the effect of polymer solution composition and applied voltage on fiber thickness.

[0046] Figure 15 is a SEM image at a scale of 5000x showing the effect of polymer solution composition and applied voltage on fiber thickness.

[0047] Figure 16 is a SEM image at a scale of 1000× showing the effect of applied voltage on fiber morphology at applied voltages less than 8 kV and greater than 14 kV.

[0048] Figure 17 is a SEM image at a scale of 5000× showing the effect of applied voltage on fiber morphology at applied voltages of less than 8 kV and greater than 14 kV.

[0049] Figure 18 is a SEM image at 1000x and 5000x scale showing the effect of applied voltage on fiber morphology at applied voltages of 8.5kV, 9kV, and 9.5kV.

[0050] Figure 19 is a SEM image at 500x and 5000x scale showing the effect of the distance between the needle and the collector plate on the fiber morphology.

[0051] Figure 20 is a SEM image at 1000x and 5000x scale showing the effect of spinning time on the porous structure and fiber morphology of the fiber.

[0052] Figure 21 is a schematic diagram of a fiber twisting method using a liquid immersion method.

[0053] Figure 22 is an image showing the result of immersing a microfiber membrane according to the present invention in water, lifting it by holding the corners, and then twisting it by holding both ends.

[0054] Figure 23 is an image showing the result of twisting the entire fiber by hand after immersing the microfiber membrane according to the present invention in water.

[0055] Figure 24 is an image showing the result of immersing a microfiber membrane according to the present invention in water, lifting it by holding the vertex, and then twisting it by holding both ends.

[0056] Figure 25 is an image showing the result of twisting the microfiber membrane according to the present invention by immersing it in water, lifting it by holding the vertex, and then twisting the entire fiber by hand.

[0057] Figure 26 is an image showing the result of rolling a microfiber membrane according to the present invention on a petri dish after immersing it in water.

[0058] Figure 27 is a schematic diagram of a fiber rolling or twisting method using a sprayer.

[0059] Figure 28 is an image showing the result of twisting the entire fiber by hand after spraying water from a sprayer in the air on the microfiber membrane according to the present invention.

[0060] Figure 29 is an image showing the result of rolling the fibers on a petri dish after spraying the microfiber film according to the present invention on the petri dish.

[0061] Figure 30 is an image showing the result of spraying the microfiber membrane according to the present invention on a petri dish, lifting it by holding the tip, and then twisting the entire fiber by hand.

[0062] Figure 31 is an image showing the result of spraying the microfiber film according to the present invention on a petri dish, lifting it by holding the corner, and then twisting the entire fiber by hand.

[0063] Figure 32 is an image showing the manufacturing process of a large-scale electrospinning mat.

[0064] Figure 33 shows the braiding process and braiding form.

[0065] Figure 34 is an image of a scaffold manufactured using a one-dimensional microfiber braiding process according to the present invention.

[0066] Figure 35 is an SEM image of a scaffold manufactured using a one-dimensional microfiber braiding process according to the present invention at magnifications of 100 times (left), 150 times (center), and 500 times (right).

[0067] Figure 36 shows the weaving process and weaving form.

[0068] Figure 37 is an image of a scaffold manufactured using a weaving process using one-dimensional microfibers according to the present invention.

[0069] Figure 38 is an SEM image of a scaffold manufactured using a weaving process using one-dimensional microfibers according to the present invention at magnifications of 100 times (left), 150 times (center), and 500 times (right).

[0070] Figure 39 shows the knitting process and knitting form.

[0071] Figure 40 is an image of a scaffold manufactured using a knitting process using one-dimensional microfibers according to the present invention.

[0072] Figure 41 is an SEM image of a scaffold manufactured by a knitting process using one-dimensional microfibers according to the present invention at magnifications of 100 times (left), 150 times (center), and 500 times (right).

[0073] The present invention can be modified in various ways and has various embodiments, and specific embodiments are illustrated in the drawings and described in detail.

[0074] However, this is not intended to limit the present invention to a specific embodiment, but should be understood to include all modifications, equivalents, or substitutes included in the spirit and technical scope of the present invention.

[0075] While terms such as "first" and "second" may be used to describe various components, these components should not be limited by these terms. These terms are used solely to distinguish one component from another. For example, without departing from the scope of the present invention, a first component may be referred to as a "second component," and similarly, a second component may also be referred to as a "first component." The term "and / or" includes any combination of multiple related items described herein or any item among multiple related items described herein.

[0076] When a component is referred to as being "connected" or "connected" to another component, it should be understood that it may be directly connected or connected to that other component, but that there may be other components intervening. Conversely, when a component is referred to as being "directly connected" or "connected" to another component, it should be understood that there are no other components intervening.

[0077] The terminology used in this application is only used to describe specific embodiments and is not intended to limit the present invention. The singular expression includes the plural expression unless the context clearly indicates otherwise. In this application, it should be understood that the terms "comprise" or "have" indicate the presence of a feature, number, step, operation, component, part, or combination thereof described in the specification, but do not exclude in advance the possibility of the presence or addition of one or more other features, numbers, steps, operations, components, parts, or combinations thereof.

[0078] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and will not be interpreted in an idealized or overly formal sense unless explicitly defined herein.

[0079] Hereinafter, with reference to the attached drawings, preferred embodiments of the present invention will be described in more detail. In order to facilitate an overall understanding in describing the present invention, identical reference numerals will be used for identical components in the drawings, and redundant descriptions of identical components will be omitted.

[0080]

[0081] In the bio-implantable microfiber scaffold of the present invention and its manufacturing method, in order to solve the problems of difficulty in artificially forming a vascular network in conventional 3D bioprinting techniques, difficulty in supplying sufficient materials to internal cells, and difficulty in smoothly excreting metabolites, thereby limiting cell survival, a polyurethane (PU)-based microfiber scaffold was developed using an electrospinning process.

[0082] In one aspect, the electrospinning process of the present invention was set to flow rate (1 mL / hr), perylene thickness (400 nm), needle thickness (22 G), applied voltage (9.5 kV), needle-collector distance (17 cm), and spinning time (60 sec). Polyurethane pellets were dissolved in a mixed solvent of N,N-dimethylformamide (N,N-dimethylformamide, DMF) and tetrahydrofuran (THF), and the ratio of DMF:THF=6:4 and the concentration of 13 wt% of PU were adjusted to form a uniform fiber diameter.

[0083] The lower the concentration, viscosity, and flow rate of the polymer solution, the more advantageous it is for forming nanofibers with a thin diameter. The higher the concentration, viscosity, and flow rate, the more advantageous it is for forming nanofibers with a large diameter. The composition ratio can be appropriately adjusted according to the purpose.

[0084] In one aspect, the material of the electrospun microfiber membrane according to the present invention may include, but is not limited to, polyurethane (PU), polycaprolactone (PCL), polylactic acid (PLA), polyglycolic acid (PGA), polylactide-co-glycolide (PLGA), polyhydroxyalkanoate (PHA), polyvinyl alcohol (PVA), hyaluronic acid (HA), polyethylene glycol (PEG), polycarbonate (PC), polyaniline (PANI), collagen, gelatin, chitosan, alginate, fibrin, decellularized extracellular matrix, conductive polymers such as poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate), PEDOT:PSS, polypyrrole (PPy), etc. It is not.

[0085] Hereinafter, the term "bio-implantable microfiber" in this description may refer to any fiber structure having a diameter generally ranging from nanometers to micrometers that can be inserted into body tissues to perform various functions. However, it should be understood that the unit of the microfiber is not limited thereto. That is, the "microfiber" according to the present invention may be a microfiber or a nanofiber.

[0086]

[0087] In one aspect, in order to convert the microfiber membrane (2D mesh structure) produced in the present invention into a 1D structure, a method of twisting the entire fiber by hand was applied, and a uniform 1D polyfilament structure was formed through an optimized process. Conventional electrospun membranes have small pore sizes and low mechanical strength, making them difficult to apply to large-scale scaffolds. In the present invention, the pore structure of the membrane is controlled by introducing a chemical vapor deposition (CVD) process and the surface is coated (e.g., with a perylene coating) to increase durability, and the porous fiber structure is maintained through a rolling-twisting technique, while simultaneously securing mechanical strength and flexibility. This enables larger scaffolds compared to conventional electrospun scaffolds, and can provide an environment suitable for long-term cell culture and tissue formation while maintaining structural stability.

[0088] In one aspect, in the bio-implantable microfiber scaffold and the manufacturing method thereof according to the present invention, an electrospun two-dimensional microfiber membrane is rolled or twisted to form a one-dimensional polyfilament, and the one-dimensional polyfilament fiber structure is assembled into a three-dimensional structure using a fiber processing technique, thereby manufacturing a CM-grade (centimeter-grade) scaffold having excellent mechanical stability and multi-porosity.

[0089] In one aspect, the present invention provides a one-dimensional, thread-shaped porous scaffold capable of being structured into a three-dimensional three-dimensional structure and a method for manufacturing the same.

[0090] In one aspect, the present invention provides a scaffold formed of one-dimensional microfibers and a method for manufacturing the same by structuring (braiding, weaving, or knitting) the scaffold into a three-dimensional structure. Hereinafter, for the convenience of explanation, the present invention may be described based on 'weaving', but the technical idea of ​​the present invention is not limited thereto, and it should be understood that it includes a fiber-based structuring process using any one or other of the braiding, weaving, or knitting methods described above.

[0091] In one aspect, fiber processing techniques, which are multi-array processes performed on a one-dimensional polyfilament fiber structure, may include, but are not limited to, braiding, weaving, knitting, etc., and various fiber processing techniques or multi-array processes may be performed.

[0092] In one aspect, the porous microfiber membrane or one-dimensional microfiber of the present invention can be coated to impart functions such as chemical resistance, low friction, water repellency, hydrophilicity, cell affinity, cell growth induction, tissue regeneration, infection prevention, antibacterial, thrombosis prevention, hemocompatibility, electrical conductivity, natural decomposition, biocompatibility, and insulation. For example, the porous microfiber membrane or one-dimensional microfiber of the present invention can be coated with PTFE, PVDF, etc. for functions such as chemical resistance, low friction, and water repellency, etc.; can be coated with PEG, PHEMA, etc. for functions such as hydrophilicity and cell affinity, etc.; can be coated with collagen, RGD peptide, fibronectin, etc. for functions such as cell growth induction and tissue regeneration, etc.; can be coated with silver nanoparticles (AgNPs), triclosan, etc. for functions such as infection prevention and antibacterial properties, etc.; can be coated with heparin, superhydrophilic coating, etc. for functions such as thrombosis prevention and blood compatibility, etc.; can be coated with PEDOT:PSS, PPy, PANI, etc. for functions such as electrical conductivity and nerve and muscle function support, etc.; can be coated with PNIPAM (temperature-responsive), self-responsive coating, etc. for functions such as environmental responsiveness, etc.; can be coated with PLGA, PCL, chitosan, etc. for functions such as natural decomposition, etc.; and can be coated with ultra-thin film coating, moisture resistance, biocompatibility, insulation, etc. For functionality, it can be coated with perylene such as Parylene C, Parylene N, etc.

[0093] In one aspect, the porous microfibers or one-dimensional microfibers of the present invention may be coated with parylene. Parylene is a well-known bioinert material. It is chemically and biologically stable, exhibiting excellent biocompatibility and low tissue reactivity. It also exhibits excellent electrical insulation properties and is mechanically flexible yet strong. A microfiber membrane not coated with parylene may have unbonded portions between the microfibers.

[0094] In one aspect, it should be understood that the coating applied to the porous microfiber membrane or one-dimensional microfiber of the present invention can be applied to each of the microfiber membrane, the microfiber bundle, or the one-dimensional microfiber to integrate the joints between the microfibers.

[0095] In one aspect, the coating applied to the porous microfiber membrane or one-dimensional microfiber of the present invention may be performed by a method such as chemical vapor deposition (CVD), physical vapor deposition (PVD), atomic layer deposition (ALD), plasma coating, dip coating, spin coating, etc., but is not limited thereto.

[0096] In one aspect, the porous microfiber membrane or one-dimensional microfiber of the present invention can be subjected to oxygen plasma treatment, and through this oxygen plasma treatment, the surface energy of the microfiber can be increased or adjusted to modify the surface, thereby enhancing cell adhesion.

[0097] In one aspect, the thickness of the coating applied to the porous microfiber membrane or one-dimensional microfiber of the present invention may be 150 nm or more and 4 μm or less, but is not limited thereto.

[0098] In one aspect, the thickness of the two-dimensional microfiber membrane of the present invention may be 100 nm or more and 4 μm or less, but is not limited thereto.

[0099] In one aspect, the pore size of the two-dimensional microfiber membrane of the present invention may be 200 nm or more and 8 μm or less, but is not limited thereto.

[0100] In one aspect, the diameter of the one-dimensional microfiber of the present invention may be 30 μm or more and 400 μm or less, but is not limited thereto.

[0101] In one aspect, the pore size of the one-dimensional microfiber of the present invention may be 200 nm or more and 8 μm or less, but is not limited thereto.

[0102]

[0103] Figure 1 is a schematic diagram illustrating a process for manufacturing a bio-implantable microfiber scaffold according to one embodiment of the present invention. In a method for manufacturing a bio-implantable microfiber scaffold according to one embodiment of the present invention, as illustrated on the left side of Figure 1, a porous microfiber membrane (e.g., a porous nanofiber membrane) can be manufactured based on a biocompatible polymer. As a non-limiting example, a porous microfiber membrane can be manufactured by electrospinning polyurethane or a biocompatible polymer, but it should be noted that the present invention is not limited thereto. To this end, a polymer solution having a desired viscosity and concentration is prepared by dissolving the polymer in a solvent, and this is then supplied to a syringe needle-shaped nozzle. After the syringe needle is positioned so as to face a collecting plate, a high voltage is applied between the syringe needle and the collecting plate by a high voltage source, thereby generating an electric field, and the resulting electric force causes the polymer solution to be drawn out in the form of thin fibers. These fibers are formed into microfibers (e.g., nanofibers) as the solvent within them evaporates. These microfibers, released from the syringe needle, are deposited on a collecting plate, allowing the production of a porous microfiber membrane. The optimal final product structure can be achieved by controlling the concentration and viscosity of the polymer solution, as well as the electrospinning environment (voltage, temperature, and humidity).

[0104] As illustrated on the right side of Fig. 1, a porous microfiber membrane can be formed into a twisted structure by rolling or twisting to produce a one-dimensional porous microfiber. A microfiber membrane composed of very thin microfibers may have very weak mechanical strength due to its structure. Therefore, in order to increase the mechanical strength of such a porous microfiber membrane and increase the surface area of ​​the microfiber membrane according to the porous structure so that it can be transplanted into a living tissue, as illustrated on the right side of Fig. 1, the porous microfiber membrane can be formed into a twisted structure to convert it into a one-dimensional porous microfiber. The fibers with increased tensile strength and durability through the twisted structure of such one-dimensional microfibers can be formed with a thinner thickness of individual fibers due to the increased tensile strength and durability, and the ratio of surface area per unit volume can be maximized through the twisted structure bundle of thinner fibers. These nanofibers have an extremely increased surface area per unit volume, which can significantly enhance the passive diffusion behavior of particles within the fibers, thereby increasing reaction efficiency, mass exchange, and cell viability.

[0105] In one aspect, the one-dimensional microfiber formed by twisting the two-dimensional microfiber membrane according to the present invention may be referred to as a 'one-dimensional microfiber' or a 'one-dimensional polyfilament'.

[0106]

[0107] FIG. 2 is a schematic diagram illustrating a process for manufacturing a large-area microfiber membrane (e.g., a nanofiber membrane) according to one embodiment of the present invention. According to one aspect, the microfiber membrane is manufactured into a microfiber membrane having an area greater than a predetermined critical area through a multi-nozzle, so that the microfibers can have a length greater than the predetermined critical length. In order to use the microfibers according to the present invention as a scaffold or artificial organ, microfibers having a length greater than the critical length that can be used as a scaffold are required. To this end, as illustrated in FIG. 2, a large-area microfiber membrane having an area greater than the critical area and ensuring uniformity can be manufactured by controlling the electric field of the multi-nozzle through an electrospinning technique based on a syringe needle-shaped multi-nozzle. As described above, the large-area microfiber membrane can be formed into a microfiber bundle and twisted structure, thereby converting it into one-dimensional microfibers having a length of, for example, 10 cm or more, 30 cm or more, 50 cm or more, or 1 m or more. Accordingly, the one-dimensional microfibers can be used as a scaffold or artificial organ.

[0108]

[0109] Figure 3 is a schematic diagram of the fabrication of an artificial hard tissue-soft tissue scaffold through a structuring process of artificial tissue fibers. As illustrated in Figure 3, the bio-implantable microfiber scaffold and the fabrication method thereof according to the present invention can be used to fabricate scaffolds or artificial organs to be used in hard tissues (bone) and soft tissues (muscle, cartilage). According to the present invention, after processing a two-dimensional microfiber membrane into a one-dimensional microfiber (thread) form, a scaffold that can be used in hard tissues (bone) and soft tissues (muscle, cartilage) can be fabricated. Through this, structural and functional gradient tissues can be realized, and cells suitable for each of hard and soft tissues can be loaded, thereby maximizing tissue engineering utilization.

[0110] According to one aspect, the structured hard and soft tissue constructs based on artificial tissue fibers can include soft tissue fibers (including fibroblasts), hard tissue fibers (including osteoblasts), and angiogenic fibers (including endothelial cells and vascular endothelial growth factor (VEGF)). Soft tissue fibers support muscle tissue formation, and hard tissue fibers are responsible for the growth and structural function of bone tissue. In addition, angiogenic fibers form a vascular network within the tissue to facilitate the supply of nutrients and oxygen, thereby enhancing tissue settlement and functional sustainability after transplantation. These artificial tissue fibers can be assembled through precise structuring processes (e.g., braiding, weaving, knitting, etc.) to realize complex structural and functional gradient tissues including muscle and bone. In particular, by converting a two-dimensional microfiber membrane into a one-dimensional microfiber (thread) and utilizing it for scaffold fabrication, the mechanical strength of the tissue can be increased and biocompatibility can be improved. In addition, by forming structured hard and soft tissues in which hard and soft tissues are connected in stages, functional characteristics similar to natural tissues can be implemented.

[0111]

[0112] Figure 4 is a schematic diagram showing the diameter of electrospun microfibers according to the present invention. As illustrated in Figure 4, in the present invention, the diameter of the microfibers that are electrospun to form a two-dimensional microfiber membrane can be appropriately adjusted to achieve suitable mechanical properties and flexibility. The smaller the diameter of the microfibers, the higher the flexibility, but the pore size decreases rapidly, which may affect whether cells can be loaded. In other words, by optimizing the diameter of the microfibers, it is possible to implement the most ideal structure for the survival of various types of cells (e.g., hard tissue, soft tissue, and vascular cells) and a diameter that does not damage the properties of hard and soft tissues.

[0113]

[0114] Figure 5 is a schematic diagram showing the density of an electrospun microfiber membrane according to the present invention. As illustrated in Figure 5, in the present invention, the density of the microfiber membrane formed by electrospinning into a two-dimensional microfiber membrane can be appropriately controlled to achieve suitable mechanical properties and flexibility. The higher the density of the microfiber membrane, the higher the density of the cells loaded. If the density is too high, the absolute amount of nutrients supplied to the cells may be reduced, which may affect the cell viability. According to the present invention, by appropriately controlling the density of the microfiber membrane, an ideal density that takes both cell viability and transfer rate into account can be realized.

[0115]

[0116] Figure 6 is a schematic diagram illustrating the orientation of an electrospun microfiber membrane according to the present invention. As illustrated in Figure 5, the present invention allows for the design of aligned and randomly structured microfiber composites capable of responding to tensile and compressive forces by controlling the orientation of the electrospun two-dimensional microfiber membrane, thereby enabling the implementation of effective tissue scaffolds. By precisely controlling the electric field during the electrospinning process to adjust the degree of alignment of the electrospun fibers, the microstructure within the tissue fibers can be determined. This can be utilized as a key framework for controlling cell orientation in the future. Most human organs, particularly those related to the musculoskeletal system, have anisotropic structures. To reflect this characteristic, it is important to design a uniaxially oriented tissue scaffold. A uniaxially oriented structure best mimics human anatomy and has the advantage of more accurately reproducing mechanical properties. However, an overly aligned fiber structure drastically reduces the space available for cell loading, which can limit the achievement of ideal tissue mimicry goals. Therefore, the present invention utilizes a multi-axis orientation structure in the production of electrospun microfiber membranes, enabling effective cell loading while also achieving anisotropic mechanical properties. This allows for the production of functionally optimized artificial tissue scaffolds that reflect the complex structural characteristics of human tissue.

[0117]

[0118] Figure 7 is a schematic diagram illustrating a process for loading hard tissue, soft tissue, and vascular tissue cells and effective factors onto an electrospun microfiber membrane according to the present invention. 3D bioink printing technology can be used as a method for loading cells and effective factors onto the microfiber membrane, but is not limited thereto. For example, bioink that introduces an extracellular matrix environment together with fibroblasts, which are representative cells that form soft tissues, and hydrogels decellularized from fibroblasts can be utilized; bioink that introduces an extracellular matrix environment together with osteoblasts, which are representative cells that form hard tissues, and hydrogels decellularized from osteoblasts can be utilized; bioink that introduces an extracellular matrix environment together with fibroblasts, which are representative cells that form vascular tissues, and hydrogels decellularized from vascular endothelial cells can be utilized; and in order to continuously induce the formation of blood vessels, an angiogenesis inducer (VEGF) can be loaded together so that the vascular tissue fibers that uniformly form the inside of the three-dimensional tissue can uniformly secrete angiogenesis inducers throughout the entire tissue.

[0119] Meanwhile, 3D bioink printing is a technology that creates 3D structures using bioinks containing living cells, biomaterials, and growth factors. This technology can be used to create artificial tissues, organs, skin, and blood vessels, and is utilized in various fields such as tissue engineering, organ printing, drug testing, and the cosmetics industry. The core elements of 3D bioprinting are broadly divided into three categories. First, bioinks are composed of cell-based inks, biocompatible hydrogels, and growth factors. Cell-based inks can include stem cells, fibroblasts, osteoblasts, endothelial cells, vascular endothelial cells, and cardiomyocytes, while biocompatible hydrogels, made of gelatin, alginate, and collagen, serve to support cells. Additionally, growth factors (e.g., vascular endothelial growth factor) are proteins used to promote tissue growth. Printing methods are divided into extrusion-based, inkjet-based, and photopolymerization-based. Extrusion-based methods, which extrude viscous bioinks and deposit them layer by layer, are the most widely used. Inkjet-based methods spray liquid bioink in droplets, while photopolymerization-based methods use UV or laser light to harden and form specific areas. These technologies commonly utilize additive manufacturing to gradually build up 3D structures, with printed cells naturally organizing over time. In the present invention, conventional 3D bioink printing technologies, such as those described above, can be used, without limitation, to load cells and effective factors onto microfiber membranes.

[0120]

[0121] Figure 8 is a schematic diagram illustrating the printing of bioink containing cells onto electrospun microfibers according to the present invention. The present invention utilizes the adhesive properties of the bioink to impart adhesive strength to the surfaces of multiple independent tissue fibers, thereby forming a more stable and robust tissue structure. Based on this, single fiber tissues designed in three dimensions using a structuring process can be designed to be more closely connected not only through simple physically structured structures (e.g., braiding, weaving, knitting, etc.) but also through biochemical properties. In particular, the adhesive strength between each single nanofiber can be enhanced by applying an organic film coating to the polymer itself, along with the biochemical bonding strength of the bioink. This enhances the bonding strength between microfibers (nanofibers), thereby enhancing the structural stability of the tissue scaffold and creating a more biocompatible environment. Furthermore, by implementing a mechanism that can rapidly increase mechanical strength even in a very thin and porous membrane, it is possible to manufacture a highly functional scaffold suitable for tissue regeneration and transplantation, maximizing cell adhesion and tissue formation while ensuring mechanical stability required for tissue engineering applications.

[0122] In addition, each of the two-dimensional microfiber membranes loaded with three types of cells (hard tissue cells, soft tissue cells, and vascular tissue cells) can be manufactured into tissue fibers having a diameter of several tens of micrometers through a rolling and twisting process. According to one aspect, the one-dimensional microfibers manufactured according to the present invention or the tissue fibers manufactured therefrom still maintain the internal characteristics of being composed of nanofibers having a nano-scale porous structure, so that the connectivity of pores can be easily controlled even in the deep part of the fiber having a diameter of several tens of micrometers.

[0123]

[0124] Figure 9 is a schematic diagram showing the process of manufacturing one-dimensional microfibers by laminating and rolling or twisting electrospun microfiber films according to the present invention. The electrospun microfiber films according to the present invention can be laminated, and the laminated microfiber films can be of different types or of the same type, and the heterogeneous microfiber films can have different types of polymers and different orientations, densities, pore sizes, microfiber diameters, thicknesses, etc., but are not limited thereto.

[0125] Referring to FIG. 9, the present invention utilizes the advantage of being able to precisely control the internal fiber orientation of a two-dimensional microfiber membrane, and by stacking heterogeneous fiber membranes, for example, oriented fiber membranes and non-oriented fiber membranes, and then rolling or twisting them together, an integrated three-dimensional structure or three-dimensional tissue fiber frame can be implemented, and this can also be manufactured in the form of a one-dimensional microfiber (thread). This method has the advantage of simultaneously implementing an axially aligned surface that does not impede blood flow, especially in vascular tissue fibers, and a random structure (a tangled structure like spaghetti) that can secure the elasticity and mechanical rigidity of blood. In the case of a general 3D printing process, although it is possible to manufacture a very small fiber diameter, there is a limitation in that it is difficult to simultaneously satisfy the material exchange ability and mechanical strength of cells because it has to rely on biopolymers in the form of gels or fluids with very low mechanical strength. In contrast, the porous microfiber-based tissue fibers of the present invention not only facilitate cellular material exchange, but also possess the characteristics of independently controlling mechanical strength and flexibility in response to tensile forces. Therefore, the present invention enables the creation of large, three-dimensional organs (e.g., blood vessels, muscles, cartilage, etc.) beyond simple cell culture scaffolds.

[0126]

[0127] Figure 10 is a schematic diagram of the fabrication of a large-scale scaffold using one-dimensional microfibers according to the present invention. The method for fabricating a structured scaffold according to the present invention allows for the production of pre-prepared, independent component fibers, i.e., fibers for hard tissue components, fibers for soft tissue components, and fibers for blood vessels, by structuring them into artificial tissues at the required time, which is advantageous for the fabrication of three-dimensional artificial organs. Unlike conventional gel-based artificial tissues, the bio-implantable microfiber scaffold according to the present invention utilizes a microfiber porous support that serves as a physical support to distribute cells three-dimensionally. This ensures the structural stability of the tissue and provides an environment in which cells can effectively attach and grow. Furthermore, since capillary-inducing components are uniformly distributed throughout the tissue via the microfibers, a smooth supply of nutrients and oxygen is ensured, creating an environment favorable for the formation of three-dimensional, large-scale tissues. This method plays a crucial role in realizing an environment more similar to actual biological tissue than conventional artificial tissues. In particular, by adjusting the ratio of vascular fibers during the structuring process, the gradient of capillary distribution within the tissue can be easily adjusted. This allows for optimal blood flow supply to specific tissue regions, enabling the creation of customized artificial tissues that can be flexibly adapted to various biological tissue environments.

[0128]

[0129] Figure 11 is a schematic diagram illustrating the control of the fiber ratio of a hard tissue-soft tissue scaffold according to the present invention. As illustrated in Figure 11, when forming a three-dimensional tissue according to the present invention, the physical properties of the tissue can be flexibly controlled by adjusting the hard tissue fiber ratio or the soft tissue fiber ratio.

[0130]

[0131] Figure 12 is a schematic diagram illustrating a method for manufacturing a scaffold by structuring hard tissue fibers and soft tissue fibers simultaneously according to the present invention. As illustrated in Figure 12, the present invention can manufacture a scaffold that can combine the mechanical properties of both hard tissue fibers and soft tissue fibers by structuring them simultaneously. This allows for the harmonious combination of properties of different tissues to reproduce a functional structure similar to human tissue. In particular, by gradually adjusting the composition ratio of hard tissue fibers and soft tissue fibers within a single tissue, the gradient structure of hard and soft tissues can be uniformly and perfectly implemented. Through this structural adjustment, mechanical strength and flexibility can be optimized according to the required properties of each part of the tissue. Furthermore, according to the present invention, even when the volume of hard and soft tissues increases, the vascular guidance fibers can be structured together to control the distribution of blood vessels within the tissue, thereby designing a scaffold to ensure smooth supply of nutrients and oxygen. Depending on the need, for bone-muscle tissue, the vascular gradient can be set so that the bone region has fewer blood vessels and the muscle region has more blood vessels. For bone-cartilage tissue, the vascular structure can be optimized so that the cartilage region has fewer blood vessels and the bone region has more blood vessels. The hard-soft tissue scaffold according to the present invention can effectively mimic the bone-muscle and bone-cartilage structures and can be applied to various tissue regeneration and transplantation. In addition, the fiber structure can be utilized in the form of a suture, providing an optimized shape during surgery and highly practical in the medical field.

[0132]

[0133] Figure 13 is a schematic diagram illustrating the stabilization of tissue by coating the surface of a bio-implantable microfiber scaffold according to the present invention. By coating the surface of a bio-implantable microfiber scaffold manufactured according to the present invention, tissue can be stabilized and immune and adhesion responses can be suppressed. As a surface coating of the bio-implantable microfiber scaffold manufactured according to the present invention, decellularized hydrogel coating, polyethylene glycol (PEG) coating, poly(2-hydroxyethyl methacrylate) (PHEMA) coating, alginate coating, heparin coating, chitosan coating, superhydrophilic coating, extracellular matrix (ECM) coating, anti-inflammatory nanoparticle coating, immune tolerant protein coating, polylactic-co-glycolic acid (PLGA) coating, polycaprolactone (PCL) coating, etc. can be performed, and decellularized hydrogel coating is preferably performed, but is not limited thereto.

[0134]

[0135] Hereinafter, a bio-insertable microfiber scaffold and a method for manufacturing the same according to the present invention will be described with reference to examples.

[0136]

[0137] 1. Example 1: Optimization of the electrospinning process

[0138]

[0139] (1) Example 1-1: Electrospinning process and variables

[0140] Electrospinning variables include solution parameters, process parameters, and ambient parameters. Solution variables include concentration, viscosity, molecular weight, surface tension, and conductivity. Process variables include applied voltage, flow rate, tip-to-collector distance (needle-to-collector distance), nozzle diameter, and collector. Environmental variables include temperature and humidity.

[0141] To maintain constant environmental variables such as temperature and humidity, the experiment was conducted under constant temperature and humidity conditions at a temperature of 20℃ and humidity of 50%.

[0142] Electrospinning is a process that uses a high-voltage electric field to spin a solution into the form of fine fibers. In this study, a syringe pump and a high-voltage source were used to induce stable nanofiber formation.

[0143] First, a polyurethane (PU) solution is injected into a syringe equipped with a 22G needle, and the solution is discharged at a constant flow rate of 1 mL / hr using a syringe pump. The 22G needle can be changed to a 16G to 38G needle depending on the experimental purpose and electrospinning conditions, and the needle diameter can affect solution flow and fiber formation.

[0144] The syringe needle tip is placed 17 cm apart from the metal collecting plate, and a high-voltage source is used to apply 9.5 kV to the needle and ground the collecting plate. When the high voltage is applied, the electric field formed at the needle tip induces charges in the solution, resulting in an interaction between surface tension and the electric field. When a constant voltage is applied, the solution forms a Taylor cone shape at the needle tip, which gradually lengthens before overcoming surface tension and spraying out in the form of fine fibers.

[0145] At this time, the radiated fibers are accelerated by high voltage, and the solvent in the air quickly evaporates and solidifies. The fibers that reach the metal plate collector (collecting plate) are randomly arranged under the influence of electric charge, or

[0146] When a rotary collector is used in electrospinning, fibers can be aligned in a specific direction. While conventional electrospinning involves randomly depositing fibers on a static collector, the use of a rotary collector allows for the alignment of fibers in a specific direction or the formation of specific structures. As the rotation speed increases, the fiber orientation increases, enabling the formation of specific structures (e.g., linear, spiral, multilayer, etc.). Examples of rotary collectors include, but are not limited to, cylindrical rotary collectors, disc-shaped rotary collectors, and magnetic rotary collectors.

[0147] To facilitate the separation of microfibers from the collector, a non-adhesive silicone-coated paper is placed on the metal plate. This substrate can be made of various materials and shapes depending on experimental conditions, facilitating the collection and post-processing of microfibers.

[0148] Through this process, microfibers with uniform diameter and regular arrangement are formed, and optimal spinning conditions can be secured by controlling variables such as electric field, spinning distance, and flow rate.

[0149]

[0150] (2) Example 1-2: Optimization of process variables to ensure uniform fiber thickness

[0151] In this example, process variables were reestablished and adjusted to ensure uniform fiber thickness. To achieve this, solution and process variables were established, and the impact of each variable was analyzed to derive optimal conditions.

[0152] First, experiments were conducted to optimize solution variables by adjusting the concentration of PU (wt%) and the ratio of N,N-dimethylformamide (DMF) and tetrahydrofuran (THF). The PU concentration was set as variables at 10 wt%, 13 wt%, and 15 wt%, and the ratios of DMF and THF were set as 5:5, 6:4, and 7:3, setting a total of nine experimental conditions.

[0153] In this example, the fixed variables were the distance between the needle and the collector plate (15 cm), the spinning time (60 s), and the flow rate (1 mL / hr), and the experimental variables were the solution composition (PU, DMF:THF) and the applied voltage at which a Taylor cone was stably formed in each solution. As a result of the experiment, the optimal solution conditions for forming the most uniform fiber thickness and stable Taylor cone were obtained at 13 wt% PU, a DMF:THF ratio of 6:4, and a voltage of 9.5 kV.

[0154] The results are shown in Figures 14 and 15.

[0155] Figure 14 is a SEM image at a scale of 1000X showing the effect of polymer solution composition and applied voltage on fiber thickness.

[0156] Figure 15 is a SEM image at a scale of 5000x showing the effect of polymer solution composition and applied voltage on fiber thickness.

[0157]

[0158] Next, experiments were conducted to determine stable nanofiber formation according to voltage. The fixed variables were solution conditions (PU 13 wt%, DMF:THF ratio 6:4), the distance between the needle and the collector (15 cm), the spinning time (60 s), and the flow rate (1 mL / hr), while the applied voltage was the experimental variable. First, experiments were conducted by setting the voltage range to less than 8 kV, 8 kV, 10 kV, 12 kV, 14 kV, and greater than 14 kV.

[0159] As a result of the experiment, at voltages lower than 8 kV, spinning did not occur and the solution flowed out, and when it exceeded 14 kV, the syringe needle inlet was blocked, preventing smooth electrospinning. Accordingly, after confirming that uniform spinning occurred in the range of 8 to 10 kV, additional experiments were conducted in 0.5 kV increments for more precise experiments. As a result of comparing the conditions of 8.5 kV, 9 kV, and 9.5 kV, it was confirmed that the most uniform fiber diameter and stable spinning were achieved at 9.5 kV. Based on this, the optimal spinning voltage was set to 9.5 kV under the conditions of 13 wt% PU and DMF:THF = 6:4.

[0160] The results are shown in Figures 16 to 18.

[0161] Figure 16 is a SEM image at a scale of 1000× showing the effect of applied voltage on fiber morphology at applied voltages less than 8 kV and greater than 14 kV.

[0162] Figure 17 is a SEM image at a scale of 5000× showing the effect of applied voltage on fiber morphology at applied voltages of less than 8 kV and greater than 14 kV.

[0163] Figure 18 is a SEM image at 1000× and 5000× scale showing the effect of applied voltage on fiber morphology at applied voltages of 8.5 kV, 9 kV, and 9.5 kV. Here, the SEM images in the upper row are at 1000× scale, and the SEM images in the lower row are at 5000× scale.

[0164]

[0165] Through this experiment, we derived the optimal process variables that can form a uniform fiber thickness by optimizing the solution composition and electrospinning conditions, and confirmed that the reproducibility of nanofiber production using electrospinning can be increased based on this.

[0166]

[0167] (3) Example 1-3: Optimization of process conditions to achieve sustainable pore size (400 nm) for cell culture

[0168] In this example, electrospinning process conditions were optimized to ensure a sustainable pore size (400 nm) for cell culture. Specifically, the optimal conditions were derived by analyzing changes in fiber formation and porous structure according to the distance between the needle and the collecting plate and the spinning time.

[0169] To evaluate the effect of the distance between the needle and the collector plate on fiber formation, experiments were conducted with distances of 10 cm, 15 cm, 17 cm, and 20 cm. At this time, the fixed variables were set to the solution concentration (PU 13 wt%, DMF:THF=6:4), applied voltage (9.5 kV), spinning time (60 s), and flow rate (1 mL / hr), and the experimental variable was the distance between the needle and the collector plate.

[0170] Experimental results showed that at a distance of 10 cm, the fibers were formed in clusters, had irregular diameters, and were entangled with each other, making it difficult to confirm the porous structure. At a distance of 20 cm, the fiber diameter decreased significantly and the pore size tended to increase. At a distance of 15 cm, relatively uniform fibers were formed, but the most stable pore structure and uniform fiber diameter were confirmed at 17 cm. Therefore, the optimal distance between the needle and the collector plate was set at 17 cm to ensure uniform fiber formation and an appropriate pore size.

[0171] The results are shown in Figure 19.

[0172] Figure 19 is an SEM image at 500x and 5000x magnification showing the effect of the distance between the needle and the collector plate on the fiber morphology. For each needle-to-collecting plate distance (10 cm, 15 cm, 17 cm, 20 cm), the left side is an SEM image at 500x magnification, and the right side is an SEM image at 5000x magnification. At a needle-to-collecting plate distance of 10 cm, the pore size could not be measured, at 15 cm the pore size was approximately 10 to 12 μm, at 17 cm the pore size was approximately 14 to 16 μm, and at 20 cm the pore size was approximately 17 to 19 μm.

[0173]

[0174] In addition, to analyze the change in porous morphology according to the spinning time, electrospinning was performed for 30, 60, 90, and 120 seconds. At this time, the fixed variables were set as the solution concentration (PU 13 wt%, DMF:THF=6:4), applied voltage (9.5 kV), distance between the needle and the collecting plate (17 cm), and flow rate (1 mL / hr), and the experimental variable was set as the spinning time.

[0175] Experimental results showed that when spinning for 30 seconds, the fiber density was low, resulting in uneven pore formation. Over 120 seconds, excessive fiber accumulation resulted in pore blockage. Under 60 and 90-second conditions, a uniform porous structure was formed. In particular, when spinning for 90 seconds, the pore size was uniform and a uniform fiber arrangement was confirmed.

[0176] The results are shown in Figure 20.

[0177] Figure 20 is a SEM image at 1000X and 5000X scales showing the effect of spinning time on the porous structure and fiber morphology of the fibers. For each spinning time (30 s, 60 s, 90 s, 120 s), the left side is the SEM image at 1000X scale, and the right side is the SEM image at 5000X scale. The pore size at a spinning time of 30 s was 19 to 22 µm, the pore size at 60 s was approximately 10 to 12 µm, the pore size at 90 s was approximately 8 to 10 µm, and the pore size at 120 s was approximately 4 to 7 µm.

[0178]

[0179] This experiment confirmed that the distance between the needle and the collection plate (17 cm) and the spinning time (60 seconds) were optimal for creating a sustainable pore size for cell culture. Based on this, optimal process parameters can be derived for the production of cell culture scaffolds using the electrospinning process, and it is expected that future research in tissue engineering and biomedical applications will be utilized.

[0180]

[0181] (4) Example 1-4: Optimized electrospinning process conditions

[0182] As shown in Table 1 below, the electrospinning process conditions were optimized in this experiment to form uniform nanofibers. The optimal solution composition was set to 13 wt% PU, the DMF:THF ratio was 6:4, and the distance between the needle and the collector plate was adjusted to 17 cm. In addition, the flow rate was set to 1 mL / hr and the spinning time was set to 60 s to ensure stable fiber formation during the spinning process. The optimal applied voltage to obtain the most uniform fiber diameter was determined to be 9.5 kV. The needle diameter used was 22 G, and the parylene coating thickness was maintained at 400 nm, the same as before. It was confirmed that these optimized process conditions could form a uniform nanofiber structure for neural cell culture.

[0183] Variable optimized conditions Solution composition PU 13 wt%, DMF:THF 6:4 Needle-to-collector plate distance 17 cm Applied voltage 9.5 kV Radiation time 60 s Flow rate 1 mL / hr Needle diameter 22 G Perylene thickness 400 nm

[0184]

[0185] 2. Example 2: Twisting or rolling process for producing 1D polyfilament

[0186] In this embodiment, 'twisting' and 'rolling' may be used interchangeably.

[0187] After completing the optimization of the electrospinning process, a single fiber twisting methodology was established using a 6 cm x 1 cm specimen (microfiber membrane) with a 400 nm perylene coating. To this end, experiments were conducted using a water-based method (liquid immersion method) and a spray-based method, and the uniformity of the fiber twisting was evaluated. The reason for comparing the two methods is that the cell attachment method varies depending on the cell characteristics. The spray-based method is suitable for highly adherent cells such as keratinocytes, fibroblasts, and mesenchymal stem cells, whereas the liquid immersion method is effective in helping the anchorage of cells with low adherence such as endothelial cells and cardiomyocytes. Therefore, the goal was to find the optimal twisting method that would maintain a uniform fiber morphology while also enabling good cell attachment.

[0188]

[0189] (1) Example 2-1: Five twisting experiments using the liquid immersion method

[0190] ① After immersing the microfiber membrane according to the present invention in water, lift it by holding the edge, then twist it by holding both ends.

[0191] ② After soaking the microfiber membrane according to the present invention in water, twist the entire fiber by hand.

[0192] ③ After immersing the microfiber membrane according to the present invention in water, lift it by holding the vertex, then twist it by holding both ends.

[0193] ④ After soaking the microfiber membrane according to the present invention in water, lift it by holding the tip, and twist the entire fiber by hand.

[0194] ⑤ After soaking the microfiber membrane according to the present invention in water, roll it on a petri dish.

[0195]

[0196] The results of Example 2-1 are shown in Figures 21 to 26.

[0197] Figure 21 is a schematic diagram of a fiber rolling or twisting method using a liquid immersion method.

[0198] Figure 22 shows the results of immersing a microfiber membrane according to the present invention in water, lifting it by holding a corner, and then twisting it by holding both ends (① of Example 2-1). The result is that the twisting is irregular. It is believed that excessive moisture increases the cohesiveness of the water, resulting in an uneven distribution of force and asymmetrical deformation of the fibers.

[0199] Figure 23 shows the results of twisting the entire fiber by hand after immersing the microfiber membrane according to the present invention in water (② of Example 2-1), which showed regular and continuous twisting. The upper right and lower right of Figure 23 are SEM images of the primary microfiber according to the present invention at magnifications of 100x and 5000x, respectively.

[0200] Figure 24 shows the results of immersing a microfiber membrane according to the present invention in water, lifting it by its vertices, and then twisting it by holding both ends (③ of Example 2-1). The result is that the twisting is irregular. It is believed that excessive moisture increases the cohesiveness of the water, resulting in an uneven distribution of force and asymmetrical deformation of the fibers.

[0201] Figure 25 shows the results of twisting the microfiber membrane according to the present invention by soaking it in water, lifting it by holding the vertex, and twisting the entire fiber by hand (④ of Example 2-1), which showed regular and continuous twisting. The upper right and lower right of Figure 25 are SEM images of the primary microfiber according to the present invention at magnifications of 100x and 5000x, respectively.

[0202] Figure 26 shows the result of rolling the microfiber membrane according to the present invention on a petri dish after immersing it in water (⑤ of Example 2-1). Since it could not be removed from the petri dish, it was not possible to produce one-dimensional microfibers.

[0203]

[0204] As a result of the experiment, it was confirmed that when the microfiber membrane according to the present invention was twisted by holding both ends and when rolling, the fibers were not rolled uniformly, whereas when the entire fiber was twisted by hand, the fibers were rolled very uniformly.

[0205]

[0206] (2) Example 2-2: Five twisting experiments using a sprayer method

[0207] ① For the microfiber membrane according to the present invention, after spraying water from a sprayer in the air, twist the entire fiber by hand.

[0208] ② For the microfiber membrane according to the present invention, after spraying on a petri dish, the fibers are rolled on the petri dish.

[0209] ③ For the microfiber film according to the present invention, after spraying it on a petri dish, lift it by holding the tip, and then twist the entire fiber by hand.

[0210] ④ For the microfiber film according to the present invention, after spraying it on a petri dish, lift it by holding the corner, and then twist the entire fiber by hand.

[0211]

[0212] The results of Example 2-2 are shown in Figures 27 to 31.

[0213] Figure 27 is a schematic diagram of a fiber rolling or twisting method using a sprayer.

[0214] Figure 28 shows the result of twisting the entire fiber by hand after spraying water from a sprayer in the air on the microfiber membrane according to the present invention (① of Example 2-2), and regular and continuous twisting was observed.

[0215] Fig. 29 shows the results of spraying the microfiber membrane according to the present invention onto a petri dish and then rolling the fibers on the petri dish (② of Example 2-2). Since the fibers could not be removed from the petri dish, it was concluded that one-dimensional microfibers could not be produced. The upper right and lower right of Fig. 28 are SEM images at 100x and 5000x magnifications, respectively, of the primary microfibers according to the present invention.

[0216] Figure 30 shows the results of spraying the microfiber membrane according to the present invention on a petri dish, lifting it by grasping the tip, and then twisting the entire fiber by hand (③ of Example 2-2), which showed regular and continuous twisting. The upper right and lower right of Figure 30 are SEM images of the primary microfiber according to the present invention at magnifications of 100x and 5000x, respectively.

[0217] Figure 31 shows the results of spraying the microfiber membrane according to the present invention on a petri dish, lifting it by holding the corner, and then twisting the entire fiber by hand (④ of Example 2-2), which showed regular and continuous twisting. The upper right and lower right of Figure 31 are SEM images of the primary microfiber according to the present invention at magnifications of 100x and 5000x, respectively.

[0218]

[0219] Experimental results confirmed that the fibers were rolled uniformly in all experiments except for the case where the microfiber membrane according to the present invention was rolled on a petri dish. In particular, the most effective methods were spraying water in mid-air and then twisting, or lifting the membrane by the corner and then twisting.

[0220]

[0221] Through this, we were able to conclude that the optimal twisting method for producing one-dimensional polyfilaments is to twist the entire fiber by hand, which is the most suitable method for forming a uniform structure when considering uniform fiber formation and cell attachment.

[0222]

[0223] 3. Example 3: Scaffold fabrication using a multi-array process of one-dimensional polyfilaments and braiding, weaving, and knitting.

[0224] (1) Example 3-1: Manufacturing process of large-scale electrospinning mat

[0225] To produce large-scale electrospun mats, a G-code was generated and executed using a 3-axis stage for electrospinning, which moved the stage in an L-shape while spinning. Under these set conditions, electrospinning was performed for 6 minutes to produce a mat measuring 20 cm Х 10 cm.

[0226] After the mat was manufactured, the manufactured mat was hung on a square-shaped window frame and levitated in the air, and then transferred to a parylene deposition machine in a vacuum environment. Thereafter, a perylene coating was performed using a chemical vapor deposition method, and then a tensile force was applied to prevent the mat from breaking.

[0227] Additionally, a process of preparing one-dimensional polyfilaments by spraying water from a sprayer in the air and then twisting the entire fiber by hand (method ① of Example 2-2 above) was also performed at this stage. This laid the foundation for utilizing a sample radiated in a uniform pattern and a twisted single fiber in a multi-array process.

[0228] Figure 32 is an image showing the manufacturing process of a large-scale electrospun mat. The upper left is an image showing a three-axis stage for electrospinning, the upper right is an image showing a manufactured large-scale electrospun mat, the lower left is an image showing an electrospun mat that has been subjected to perylene coating and tensioning, and the lower right is an image showing a primary polyfilament manufactured by rolling and twisting the electrospun mat that has been subjected to perylene coating and tensioning.

[0229]

[0230] (2) Example 3-2: Scaffold production using braiding

[0231] The braiding process is similar to braiding hair, interweaving three prepared one-dimensional polyfilaments to form a structure. The tightly woven fibers provide high mechanical strength and structural stability, while maintaining consistent spacing while maintaining flexibility. These properties enable the creation of complex structures and make them useful in tissue engineering research that requires both strength and flexibility, such as vascular regeneration or tendon and ligament tissue regeneration.

[0232] Figure 33 shows the braiding process and braiding form.

[0233] Figure 34 is an image of a scaffold manufactured using a one-dimensional microfiber braiding process according to the present invention.

[0234] Figure 35 is an SEM image of a scaffold manufactured using a one-dimensional microfiber braiding process according to the present invention at magnifications of 100 times (left), 150 times (center), and 500 times (right).

[0235]

[0236] (3) Example 3-3: Scaffold production using weaving

[0237] The weaving process is similar to weaving fabric, forming a woven structure by interlacing multiple one-dimensional polyfilaments in a orthogonal direction. This woven structure not only offers high mechanical strength but also allows for tissue formation while maintaining consistent spacing, providing a porous environment. This facilitates cell attachment and tissue formation, making it ideal for applications requiring high strength, such as bone tissue regeneration, or for skin regeneration and the production of cell culture scaffolds, which require a uniform structure.

[0238] Figure 36 shows the weaving process and weaving form.

[0239] Figure 37 is an image of a scaffold manufactured using a weaving process using one-dimensional microfibers according to the present invention.

[0240] Figure 38 is an SEM image of a scaffold manufactured using a weaving process using one-dimensional microfibers according to the present invention at magnifications of 100 times (left), 150 times (center), and 500 times (right).

[0241]

[0242] (4) Example 3-4: Scaffold production using knitting

[0243] The knitting process, similar to knitting, involves weaving one-dimensional polyfilaments into loops to form a continuous structure. These loop-woven fibers possess high flexibility and elasticity, and the strong inter-fiber bonds allow the tissue to stretch and deform. These properties are particularly suitable for research fields such as cardiac patches, cartilage tissue, and muscle tissue regeneration. Because the heart is an organ that continuously contracts and relaxes, the knitting process, which possesses high elasticity and stretchability, is highly effective in producing cardiac patches. Similarly, the knitting process can be useful for cartilage and muscle tissue.

[0244] Figure 39 shows the knitting process and knitting form.

[0245] Figure 40 is an image of a scaffold manufactured using a knitting process using one-dimensional microfibers according to the present invention.

[0246] Figure 41 is an SEM image of a scaffold manufactured by a knitting process using one-dimensional microfibers according to the present invention at magnifications of 100 times (left), 150 times (center), and 500 times (right).

[0247]

[0248] As described above, the bioimplantable microfiber scaffold and its manufacturing method according to the present invention provide a structure that optimizes cell attachment and growth. This structure maintains higher mechanical strength than tissue engineering and bioprinting while offering greater flexibility than machining, thereby enhancing practicality. Furthermore, the arrangement and density of the microfibers can be adjusted to suit the patient's tissue characteristics, enabling customized manufacturing. Furthermore, by promoting cell growth and differentiation, the scaffold can effectively replicate the functions of actual organs.

[0249] Furthermore, the bio-implantable microfiber scaffold according to the present invention and its manufacturing method exhibit excellent mechanical stability, high long-term cell viability, and can promote vascularization through its porous structure. This enables the production of large-scale scaffolds, accelerating the practical application of artificial organs. Furthermore, the scaffold facilitates customized manufacturing and optimizes tissue organization, enabling faster production speeds than conventional bioprinting methods, resulting in cost savings and mass production. Furthermore, the bio-implantable microfiber scaffold according to the present invention can be utilized not only for artificial organs but also as a surgical suture, expanding its scope of application.

[0250] In addition, according to the bio-implantable microfiber scaffold and the manufacturing method thereof according to the present invention, a two-dimensional microfiber membrane can be processed into a one-dimensional microfiber (thread) form and then used to manufacture scaffolds for hard tissue (bone) and soft tissue (muscle, cartilage), and a complex gradient tissue including muscle and bone can be implemented through a precision structuring process. In particular, by applying a multi-axial orientation structure, cells can be effectively loaded while implementing anisotropic mechanical properties, thereby reflecting structural properties similar to actual human tissue. In addition, according to the bio-implantable microfiber scaffold and the manufacturing method thereof according to the present invention, a three-dimensional tissue fiber frame can be created by laminating oriented and non-oriented fiber membranes and then rolling or twisting them, and in the case of vascular tissue fibers, an axially aligned surface that does not impede blood flow and a random structure can be implemented simultaneously. In this way, by utilizing hard and soft tissue fibers together to form a structure, the two mechanical properties can be mixed, and mechanical strength and flexibility can be adjusted according to the required properties of each site.

[0251] As a result, the bio-implantable microfiber scaffold according to the present invention and the method for manufacturing the same can be utilized in various medical fields such as artificial organs, tissue regeneration, and surgical sutures, and has the advantage of enabling customized treatment and transplantation applications by precisely imitating actual human tissue.

[0252]

[0253] Although the present invention has been described with reference to the drawings and embodiments, it does not mean that the scope of protection of the present invention is limited by the drawings or embodiments, and it will be understood that a person skilled in the art can modify and change the present invention in various ways without departing from the spirit and scope of the present invention as described in the following claims.

[0254] Although the present invention described above is described based on a series of functional blocks, it is not limited to the above-described embodiments and the attached drawings, and it will be apparent to those skilled in the art to which the present invention pertains that various substitutions, modifications, and changes are possible within a scope that does not depart from the technical spirit of the present invention.

[0255] The combination of the above-described embodiments is not limited to the above-described embodiments, and various combinations may be provided in addition to the above-described embodiments depending on implementation and / or needs.

[0256] In the above-described embodiments, the methods are described based on a flowchart as a series of steps or blocks. However, the present invention is not limited to the order of the steps, and some steps may occur in a different order or simultaneously with other steps described above. Furthermore, those skilled in the art will understand that the steps depicted in the flowchart are not exclusive, and other steps may be included, or one or more steps in the flowchart may be deleted without affecting the scope of the present invention.

[0257] The above-described embodiments include examples of various aspects. While not all possible combinations to illustrate the various aspects can be described, those skilled in the art will recognize that other combinations are possible. Accordingly, the present invention is intended to encompass all other alterations, modifications, and variations within the scope of the following claims.

Claims

1. A method for manufacturing a bio-insertable microfiber scaffold, A first step of manufacturing a two-dimensional microfiber membrane by electrospinning a polymer solution; and A second step of manufacturing one-dimensional microfibers by rolling or twisting the two-dimensional microfiber membrane; A method for manufacturing a bio-implantable microfiber scaffold, comprising:

2. In paragraph 1, A method for manufacturing a bio-implantable microfiber scaffold, further comprising a step of coating the two-dimensional microfiber membrane with parylene between the first and second steps.

3. In paragraph 2, A method for manufacturing a bio-insertable microfiber scaffold, further comprising a step of tensioning the two-dimensional microfiber membrane after coating it with perylene.

4. In paragraph 1, A method for manufacturing a bio-implantable microfiber scaffold, wherein rolling or twisting of the two-dimensional microfiber membrane in the second step is performed by immersing it in water or spraying water using a sprayer.

5. In paragraph 1, A method for manufacturing a bio-implantable microfiber scaffold, wherein in the second step, the entire two-dimensional microfiber membrane is twisted by hand or machine to manufacture one-dimensional microfibers.

6. In paragraph 1, A method for manufacturing a bio-implantable microfiber scaffold, further comprising, after the second step, a step of performing a multi-array process using the one-dimensional microfibers manufactured in the second step.

7. In paragraph 6, A method for manufacturing a bio-implantable microfiber scaffold, wherein the above multi-array process is a braiding, weaving or knitting process.

8. In paragraph 1, A method for manufacturing a bio-implantable microfiber scaffold, further comprising, in the first step, a step of manufacturing and laminating two or more two-dimensional microfiber membranes.

9. In paragraph 8, A method for manufacturing a bio-implantable microfiber scaffold, wherein at least one of the two or more two-dimensional microfiber membranes is an oriented microfiber membrane and at least one is an unoriented microfiber membrane.

10. In paragraph 1, A method for manufacturing a bio-implantable microfiber scaffold, further comprising a step of loading cells onto the two-dimensional microfiber membrane between the first and second steps.

11. In paragraph 10, A method for manufacturing a bio-implantable microfiber scaffold, further comprising the step of preparing at least three of the above two-dimensional microfiber membranes and loading hard tissue cells including osteoblasts, soft tissue cells including fibroblasts, or vascular tissue cells including vascular endothelial cells and angiogenesis-inducing substances onto each of the two-dimensional microfiber membranes.

12. In paragraph 11, In the second step, the two-dimensional microfiber membrane loaded with the hard tissue cells, the two-dimensional microfiber membrane loaded with the soft tissue cells, and the two-dimensional microfiber membrane loaded with the vascular tissue cells are each rolled or twisted to manufacture one-dimensional hard tissue microfibers, one-dimensional soft tissue microfibers, and one-dimensional vascular tissue microfibers. A method for manufacturing a bio-implantable microfiber scaffold, further comprising the step of manufacturing a hard tissue and soft tissue scaffold by structuring one-dimensional hard tissue microfibers, one-dimensional soft tissue microfibers, and one-dimensional vascular tissue microfibers.

13. In paragraph 12, A method for manufacturing a bio-implantable microfiber scaffold, wherein the ratio of the one-dimensional hard tissue microfibers, one-dimensional soft tissue microfibers, and one-dimensional vascular tissue microfibers is controlled during the structuring of the one-dimensional hard tissue microfibers, one-dimensional soft tissue microfibers, and one-dimensional vascular tissue microfibers to form a gradient between the hard tissue and the soft tissue.

14. In paragraph 12, A method for manufacturing a bio-implantable microfiber scaffold, further comprising a step of coating the surface of the manufactured hard and soft tissue scaffolds with a decellularized hydrogel.

15. In paragraph 10, A method for manufacturing a bio-insertable microfiber scaffold, wherein the step of loading cells onto the above two-dimensional microfiber membrane is performed by a bioink printing method.

16. A bio-implantable microfiber scaffold manufactured by a method for manufacturing a bio-implantable microfiber scaffold according to any one of claims 1 to 15.

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