EP3882385A1 - Automatisierte herstellung von dreidimensionalen zellmatrizen mit nanofasern mit kontrollierter ausrichtung und gleichmässiger zellverteilung - Google Patents

Automatisierte herstellung von dreidimensionalen zellmatrizen mit nanofasern mit kontrollierter ausrichtung und gleichmässiger zellverteilung Download PDF

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Publication number
EP3882385A1
EP3882385A1 EP21160776.7A EP21160776A EP3882385A1 EP 3882385 A1 EP3882385 A1 EP 3882385A1 EP 21160776 A EP21160776 A EP 21160776A EP 3882385 A1 EP3882385 A1 EP 3882385A1
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Prior art keywords
nanofibres
deposition table
dimensional
nanofibre
deposition
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EP21160776.7A
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English (en)
French (fr)
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EP3882385C0 (de
EP3882385B1 (de
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António Manuel GODINHO COMPLETO
Paula Alexandina DE AGUIAR PEREIRA MARQUES
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Universidade de Aveiro
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Universidade de Aveiro
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    • D—TEXTILES; PAPER
    • D04—BRAIDING; LACE-MAKING; KNITTING; TRIMMINGS; NON-WOVEN FABRICS
    • D04H—MAKING TEXTILE FABRICS, e.g. FROM FIBRES OR FILAMENTARY MATERIAL; FABRICS MADE BY SUCH PROCESSES OR APPARATUS, e.g. FELTS, NON-WOVEN FABRICS; COTTON-WOOL; WADDING ; NON-WOVEN FABRICS FROM STAPLE FIBRES, FILAMENTS OR YARNS, BONDED WITH AT LEAST ONE WEB-LIKE MATERIAL DURING THEIR CONSOLIDATION
    • D04H1/00—Non-woven fabrics formed wholly or mainly of staple fibres or like relatively short fibres
    • D04H1/70—Non-woven fabrics formed wholly or mainly of staple fibres or like relatively short fibres characterised by the method of forming fleeces or layers, e.g. reorientation of fibres
    • D04H1/72—Non-woven fabrics formed wholly or mainly of staple fibres or like relatively short fibres characterised by the method of forming fleeces or layers, e.g. reorientation of fibres the fibres being randomly arranged
    • D04H1/728—Non-woven fabrics formed wholly or mainly of staple fibres or like relatively short fibres characterised by the method of forming fleeces or layers, e.g. reorientation of fibres the fibres being randomly arranged by electro-spinning
    • D—TEXTILES; PAPER
    • D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
    • D01D—MECHANICAL METHODS OR APPARATUS IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS
    • D01D5/00—Formation of filaments, threads, or the like
    • D01D5/0007—Electro-spinning
    • D01D5/0061—Electro-spinning characterised by the electro-spinning apparatus
    • D01D5/0076—Electro-spinning characterised by the electro-spinning apparatus characterised by the collecting device, e.g. drum, wheel, endless belt, plate or grid
    • D—TEXTILES; PAPER
    • D04—BRAIDING; LACE-MAKING; KNITTING; TRIMMINGS; NON-WOVEN FABRICS
    • D04H—MAKING TEXTILE FABRICS, e.g. FROM FIBRES OR FILAMENTARY MATERIAL; FABRICS MADE BY SUCH PROCESSES OR APPARATUS, e.g. FELTS, NON-WOVEN FABRICS; COTTON-WOOL; WADDING ; NON-WOVEN FABRICS FROM STAPLE FIBRES, FILAMENTS OR YARNS, BONDED WITH AT LEAST ONE WEB-LIKE MATERIAL DURING THEIR CONSOLIDATION
    • D04H3/00—Non-woven fabrics formed wholly or mainly of yarns or like filamentary material of substantial length
    • D04H3/016—Non-woven fabrics formed wholly or mainly of yarns or like filamentary material of substantial length characterised by the fineness
    • D—TEXTILES; PAPER
    • D04—BRAIDING; LACE-MAKING; KNITTING; TRIMMINGS; NON-WOVEN FABRICS
    • D04H—MAKING TEXTILE FABRICS, e.g. FROM FIBRES OR FILAMENTARY MATERIAL; FABRICS MADE BY SUCH PROCESSES OR APPARATUS, e.g. FELTS, NON-WOVEN FABRICS; COTTON-WOOL; WADDING ; NON-WOVEN FABRICS FROM STAPLE FIBRES, FILAMENTS OR YARNS, BONDED WITH AT LEAST ONE WEB-LIKE MATERIAL DURING THEIR CONSOLIDATION
    • D04H3/00—Non-woven fabrics formed wholly or mainly of yarns or like filamentary material of substantial length
    • D04H3/02—Non-woven fabrics formed wholly or mainly of yarns or like filamentary material of substantial length characterised by the method of forming fleeces or layers, e.g. reorientation of yarns or filaments
    • D04H3/04—Non-woven fabrics formed wholly or mainly of yarns or like filamentary material of substantial length characterised by the method of forming fleeces or layers, e.g. reorientation of yarns or filaments in rectilinear paths, e.g. crossing at right angles
    • D04H3/045—Non-woven fabrics formed wholly or mainly of yarns or like filamentary material of substantial length characterised by the method of forming fleeces or layers, e.g. reorientation of yarns or filaments in rectilinear paths, e.g. crossing at right angles for net manufacturing

Definitions

  • This invention relates to a system and a process for the automated manufacture of three-dimensional cell matrices by electrospinning from nanofibres of controlled alignment and uniform cell distribution throughout their thickness.
  • the present invention has many applications in various areas, in the manufacture of products or structures, on a nanometric scale, which depend on high surface area, such as in biotechnology, in the pharmaceutical and in tissue engineering areas, in particular in regenerative medicine.
  • nanomaterials associated with the different possibilities of morphologies and functionalities reveal a series of possibilities for new fields of application and drive the progress in the processing of these nanostructures.
  • the electrospinning or electrostatic spinning method is very advantageous, since the fibres obtained with this technique have a high surface area, combined with a low production cost and the possibility of being formed from a wide variety of polymers or composites.
  • This technique is based on the application of high voltage (5-50 KV) and low current (0.5-1 ⁇ A) electric fields for the production of very small diameter fibres. In this process, the electrostatic forces control the formation and deposition of these fibres.
  • the key configuration of a generic electrospinning process consists of a syringe, where the molten polymer or polymeric solution is introduced, which is connected to a capillary tube, a diffuser pump, which controls the flow of the polymeric solution to be supplied, so that a drop of solution is always maintained at the tip of the capillary tube, a metal collector, maintained at zero potential (grounded), where the fibres produced will be collected, a high voltage source, responsible for producing a difference in potential between the tip of the capillary tube and the collector.
  • a high voltage source responsible for producing a difference in potential between the tip of the capillary tube and the collector.
  • the balance of electrostatic charges to which the droplet is subjected namely the surface tension force of the solution and the force exerted by the applied electric field, begins to suffer an imbalance and, from a certain critical value of electric field, a jet of polymeric material from the capillary tube is projected and accelerated towards the collector.
  • the jet with the polymeric solution suffers evaporation of a large part of its solvent, thus ensuring that the fibres formed have enough rigidity to support their own weight.
  • the solvent that remains in the solution such as moisture, allows the adhesion of one fibre to another, as they are deposited in layers, forming a non-woven web.
  • the electrospun fibres form a two-dimensional, randomly oriented blanket or fabric due to the instability of the jet path.
  • Oriented fibre networks have the possibility of developing anisotropic properties in materials. These relationships are quite obvious in the field of tissue engineering.
  • Typical examples include the production of polymeric meshes, containing aligned fibres, used as substrates for culture and regeneration of neural cells, due to the inherently anisotropic nature of nerves and their regenerative mechanisms.
  • the scaffold material has a three-dimensional structure of controlled porosity, in order to allow the development of the three-dimensional cell matrix throughout the depth of the matrix.
  • document US20110018174A1 discloses the production of aligned electrospun fibres, with location and orientation control of the fibres, using for this purpose a device that provides a voltage depending on the selected time, whereby that voltage is applied to a collector with multiple electrodes.
  • said document does not disclose a process capable of forming a three-dimensional matrix of aligned fibres in any desired thickness.
  • the limitations related to the current processes of electrospinning the aligned fibres are mainly related to the fact that, as the aligned and electrically charged fibres are deposited one over the other, the increasing electric charge tends to repel the new fibres from being deposited, preventing their correct alignment and limiting their thickness to a few tenths of a millimetre of the matrix of the formed fibres.
  • cell matrices of nanofibres with a high thickness, i.e. in the order of several millimetres, with control of the alignment of the fibres throughout the thickness, with a uniform distribution of cells throughout the volume of the three-dimensional nanofibre matrix, with control of the distance between aligned nanofibres (inplane porosity) in the deposition plane forming two-dimensional nanofibre meshes and control of the degree of compaction (porosity throughout the thickness) between layers of the deposited two-dimensional nanofibre meshes.
  • the porosity of these nanofibre matrices is of utmost relevance in enhancing cell migration and multiplication, as well as the delivery of nutrients to the cells, throughout the volume of the three-dimensional nanofibre matrix.
  • the present invention proposes to solve the problems of the prior art, described above, through the implementation of a system and a process for the automated manufacture of three-dimensional cell matrices, which can present various patterns of fibre alignment, throughout the thickness of the matrix and with a uniform distribution of cells throughout the thickness of the matrix, this thickness being dependent on the number of layers of fibres deposited, the thickness of the fibres and the degree of compaction between layers.
  • the present invention relates to a system and process for the automated manufacture of three-dimensional cell matrices, by electrospinning, from nanofibres with controlled alignment and uniform cell distribution throughout their thickness.
  • the system of the present invention comprises a module (A) for forming nanofibres by electrospinning, a module (B) for collecting the formed nanofibres, a module (C) for deposition of the collected nanofibres, a module (D) for electropulverisation of cells, a vacuum pump and a computational unit according to claim 1.
  • the module (B), nanofibre collector comprises two collecting cylinders, with coaxial axes and perpendicular to the axis of the electrospinning capillary tube, where each cylinder is provided with continuous rotation movement by an electric motor controlled by a computerised unit, in which the electrospun nanofibres are collected on the surfaces and between the surfaces of the collecting cylinders, also comprising this module (B), brushes for removing the nanofibres, which remain on the cylindrical surfaces, and where the module (C), of fibre deposition, comprises a deposition table, able to be moved linearly, in the direction parallel to its surface and in the direction of the axis of the electrospinning capillary tube, and rotationally, around its longitudinal axis, assures that the electrospinning process occurs in a continuous and automatic way, with formation of three-dimensional matrices with uniform distribution of the cells throughout the thickness of the three-dimensional cell matrix and with alignment and distance between nanofibres controlled according to the
  • module (D) for electropulverisation of cells, makes it possible for the tissue engineering process, by electrospinning, to take place in a continuous and automated manner, capable of producing three-dimensional cell matrices suitable for use in medicine, regenerative medicine, cartilage engineering, etc., according to claim 15.
  • the present invention relates to a continuous and automated process for forming three-dimensional cell matrices with nanofibres of controlled alignment and uniform cell distribution throughout their thickness, according to claim 3.
  • the process of the present invention allows to obtain, in a totally automatic way and without manual intervention, three-dimensional cell matrices of aligned polymeric fibres, with a uniform cell distribution throughout the thickness of the matrix, which can present several alignment patterns, of the nanofibres that compose them, throughout their thickness, being the thickness of the matrix dependent on the number of fibre layers deposited, the thickness of the fibres and the degree of compaction between layers.
  • This process has the additional advantage of being versatile, simple, and operating in automatic and continuous mode, so it is not necessary to produce a series of layers of nanofibre meshes with a certain alignment, to manually add other layers with different alignments and to avoid the laborious manual process of cell seeding of the three-dimensional matrices of nanofibres previously manufactured, overcoming the limited capacity of this manual process to ensure the uniformity/control of the cells distribution in the three-dimensional matrices of nanofibres throughout the thickness.
  • This invention relates to a system and a process for the automated manufacture of three-dimensional cell matrices, obtaining nanofibres with controlled alignment and uniform cell distribution throughout their thickness, with the possibility of producing various fibre alignment patterns throughout the matrix thickness with controlled thickness and uniform cell distribution throughout the matrix thickness, this thickness being dependent on the number of layers of deposited nanofibre meshes, the thickness of the nanofibres and the degree of compaction between layers.
  • the system of the present invention comprises a module (A) for forming nanofibres by electrospinning, a module (B) for collecting the produced nanofibres, a module (C) for deposition of the collected nanofibres, and a module (D) for electropulverisation of cells.
  • Module (A) comprises a syringe to contain a polymer solution, connected to an injector pump, connected to an electrospinning capillary tube, which is connected to a voltage source, configured to provide positive polarity.
  • Module (A) is aligned perpendicularly with the axes of the two collector cylinders of module (B), the nanofibre collector.
  • Module (B) comprises two collector cylinders, each equipped with a continuous rotation movement, through the action of an electric motor, which is controlled by a computerised unit.
  • the distance between the top faces of the two cylinders is equal to the diameter of the nanofibre deposition table of module (C), and the cylindrical surfaces of the collector cylinders are made of conductive material, and may have negative or neutral polarity.
  • the nanofibre of polymeric material formed by electrospinning from the capillary tube of the module (A), with positive polarity, moves by the action of an electric field towards a collector module (C), and these are collected on the cylindrical surfaces and between the cylindrical surfaces of the collector cylinders, which are in continuous rotation.
  • the collector module (C) has brushes to remove the nanofibres that remain on the cylindrical surfaces at the end of each fibre production cycle, thus ensuring the continuous electrospinning of the nanofibres on and between the rotating cylindrical surfaces of the collector cylinders.
  • the nanofibres electrospun between the cylindrical surfaces of the collector cylinders, in continuous rotation, are deposited on the surface of the deposition table of the deposition module.
  • the table has a substantially circular shape and is positioned between the generatrices of the two collector cylinders, with its surface perpendicular to the electrospinning capillary tube axis.
  • the deposition table has holes extending from its surface to a chamber inside the table, which is connected by a channel to a vacuum pump.
  • the suction force generated by the vacuum, in the holes of the deposition table surface attaches the deposited nanofibres to the table.
  • the linear movement of the deposition table, parallel to its surface, and the rotation movement, around its longitudinal axis, are performed by electric motors controlled by a computerised unit.
  • the nanofibres, continuously deposited and attached to the surface of the deposition table, are aligned in different directions by the rotation movement of the deposition table, and the distance between the deposited nanofibres is controlled by the linear movement of the deposition table.
  • the continuous deposition of nanofibres, by the cylindrical surfaces of the collector cylinders, allows the formation of a two-dimensional nanofibre mesh of controlled organization and distribution on the surface of the deposition table.
  • the control of the distance between the fibres deposited on the deposition table allows controlling the porosity of the mesh in its plane.
  • the controlled movement of the deposition table in the direction and opposite orientation to the electrospinning capillary tube (direction of the matrix thickness) after the formation of the two-dimensional nanofibre mesh on the surface of the deposition table allows the deposition of a new two-dimensional nanofibre mesh over the previous mesh with a new organisation of the nanofibres, the number of nanofibre mesh layers accumulated on the table is defined by the desired thickness of the three-dimensional cell matrix, the thickness of the deposited nanofibres and the magnitude of the vacuum pressure generated on the surface of the deposition table that controls the level of compaction of the nanofibre mesh layers and therefore the porosity of the three-dimensional matrix throughout the thickness.
  • the electropulverisation module of the cells can basically consist of a syringe to contain a solution with cells in suspension, connected to an injector pump, connected to an electropulverisation capillary tube, which is connected to a voltage source.
  • the voltage source is configured to provide positive polarity, being aligned with the ring-shaped collector, with internal diameter equal to the diameter of the deposition table, with negative or neutral polarity.
  • the electropulverisation of the cells starts from the capillary tube with positive polarity, in which the solution with cells moves by the action of an electric field towards the collector ring.
  • the solution with cells is seeded onto the nanofibre meshes deposited on the deposition table, which is in concentric position with the collector ring. In this position, the deposition table starts a rotation movement for a certain period of time, so that the surface of the nanofibre mesh is uniformly distributed with cells.
  • the number of nanofibre mesh layers deposited on the deposition table and the number of times the electropulverisation of the cells occurs is defined by the desired thickness of the three-dimensional cell matrix, as well as by the thickness of the deposited nanofibres, the magnitude of the vacuum pressure generated on the surface of the deposition table that controls the level of compaction of the nanofibre mesh layers and thus the porosity of the three-dimensional matrix throughout the thickness and the desired cell density of the matrix.
  • the successive layers of two-dimensional nanofibre meshes, deposited on the surface of the deposition table, are kept in position on the deposition table by the action of the vacuum generated in the holes of the upper surface of the deposition table which communicate with a chamber in its interior connected to the vacuum pump.
  • Pressure control in the vacuum pump is also intended to control the degree of compaction between the two-dimensional nanofibre mesh layers formed and thus the porosity in the direction perpendicular to the plane of the deposited nanofibre mesh layer.
  • the control of the distance between the electrospinning capillary tube with positive polarity and the collector cylinders, the control of the speed of rotation of the collector cylinders, the control of the linear and rotation movements of the deposition table, the control of the pressure of the vacuum pump, the control of the distance between the electrospinning capillary tube of the cells with positive polarity and the collector ring, the control of the voltage of the capillary tubes, of the cylindrical surfaces of the collector cylinders and the collector ring are performed by a computerised control unit that, depending on the alignment and distance between nanofibres desired for each deposited two-dimensional layer and on the thickness of the matrix desired and the cell density desired, programs the sequence of all the movements, vacuum pressure and polarity of the electrodes required, based on a computer program developed for this purpose.
  • the system of the present invention has the capacity to continuously and automatically produce three-dimensional cell matrices with nanofibres of controlled alignment and uniform cell distribution throughout their thickness.
  • matrices with various alignment patterns and with different distances between nanofibres (porosity), throughout the matrix thickness, and with a uniform distribution of tissue component cells, throughout the matrix thickness, in a controlled manner.
  • the surface of the collection table provided with holes, which are subjected to vacuum pressure, allows the attachment of the fibres to this table, also allowing the control and definition of the degree of compaction (porosity) to be presented between the different layers of deposited two-dimensional nanofibre meshes, as well as the support and deposit of the nanofibres on the deposition table.
  • the separation of the deposited fibres which occurs by the effect of nanofibres stretching on the surface of the collector cylinders, during the continuous movement of these cylinders, and the linear movement of the deposition table in the direction and opposite orientation to the electrospinning capillary tube, contributes advantageously to the deposition of successive layers of two-dimensional fibre meshes, and the linear movement of the deposition table, to the concentric position, with the collector ring of the electropulverisation module of the cells.
  • the solution with cells which is electropulverised over the nanofibre meshes deposited on the deposition table, the rotation movement of the deposition table, in this position, for a certain period of time, ensures the uniform distribution of the cells on the surface of the nanofibre mesh, while the linear movement, returning the deposition table to the fibre collector module, allows a new deposition of another layer or layers of two-dimensional nanofibre meshes.
  • the alternating and controlled movement of the deposition table between the nanofibre deposition position and the cell electropulverisation position allows to automatically manufacture a three-dimensional cell matrix with nanofibres of controlled alignment and uniform cell distribution throughout the thickness, where the number of nanofibre mesh layers deposited on the deposition table and the number of times that the electropulverisation of the cells occurs are defined by the desired thickness of the three-dimensional cell matrix, by the thickness of the deposited nanofibres, by the magnitude of the vacuum pressure generated on the surface of the deposition table that controls the level of compaction of the nanofibre mesh layers and therefore the porosity of the matrix throughout the thickness and by the cell density desired for the matrix.
  • the present invention relates to a system and a process for the automated manufacture of three-dimensional cell matrices.
  • the system of the present invention comprises:
  • the system of the present invention also comprises a computerised control unit and the electronics necessary for its proper functioning, actuators, namely the actuators of the linear and rotation movement of the deposition table, as well as all the electric wiring for the distribution of energy to the various components of the system, as well as all the devices and accessories necessary to guarantee the sterilisation, humidity and temperature conditions required to ensure the survival of the cells deposited on the nanofibre meshes during the manufacturing time of the three-dimensional cell matrix.
  • system (1) of the present invention comprises:
  • the nanofibre formation module (A) comprises:
  • the nanofibre collector module (B) comprises:
  • the nanofibre deposition module comprises:
  • the cell electropulverisation module (D) comprises:
  • the cylindrical surfaces of the collector cylinders (34, 36)) are made of conductive material, and these can have negative or neutral polarity (33).
  • the electrospun nanofibres (5) are collected on the cylindrical surfaces (34, 36) and between the cylindrical surfaces (34, 36).
  • Module (B) has brushes (32, 35) to remove the nanofibres that remain on the cylindrical surfaces (34,36) maintaining the electrical continuity of these surfaces (34,36) thus ensuring the continued electrospinning of the nanofibres on and between the cylindrical surfaces in rotation (31).
  • the nanofibres deposition module (C) comprises a deposition table (10), of circular shape, positioned between the generatrices of the collector cylinders (6, 30), with a flat surface perpendicular to the axis of the electrospinning capillary tube (4), where the nanofibres, electrified between the collector cylinders (6, 30), are deposited by rotation action (31) of them.
  • the deposition table (10) has a surface with holes (9), extending to a chamber (42), which is located inside the table (10), this chamber (42) being connected by a channel (11) to a vacuum pump (21).
  • the deposited nanofibres (8) are attached to the deposition table (10) by action of the suction force (43), generated by the vacuum in the holes of the surface of the deposition table (10).
  • the deposition table (10) moves linearly parallel to its surface (22) and has a rotation movement (24) around its longitudinal axis, the linear and rotation movements of the table are performed by electric motors (39, 40) controlled by a computerised unit (20), the control of these movements allow defining the two-dimensional organisation of the nanofibre mesh deposited on the deposition table (81, 82, 83, 84) .
  • the continuously deposited nanofibres (8) attached to the surface (9) of the deposition table (10) are aligned in different directions by the rotation movement of the deposition table (24).
  • the distance between the deposited nanofibres is controlled by the linear movement of the deposition table (22).
  • the continuous deposition of nanofibres, by the cylindrical collector surfaces (34, 36), allows the formation of a two-dimensional nanofibre mesh (81, 82, 83, 84), of controlled organisation and distribution, over the surface of the deposition table (10).
  • the control of the distance (45, 47, 49, 52) between the nanofibres deposited on the deposition table (10) allows controlling the porosity of the two-dimensional mesh on the plane thereof.
  • the linear movement of the deposition table in the direction and opposite orientation (60) to the electrospinning capillary tube (4) determines the end of an electrospinning cycle.
  • the number of layers (64, 65, 66) of nanofibre meshes deposited onto the table is defined by the desired thickness (67) for each three-dimensional cell matrix (68), the thickness of the deposited nanofibres and the magnitude of the vacuum pressure generated on the surface of the deposition table (10) which controls the level of compaction of the nanofibre mesh layers and hence the porosity of the three-dimensional matrix (68) throughout the thickness (67).
  • the cell electropulverisation module (D) comprises a container (14), for containing and delivering a solution with cells in suspension, typically a syringe, and an injection pump, connected to an electropulverisation capillary tube (13), connected to a voltage source (17), configured to provide positive polarity (16), an adjustable length support (15), a ring-shaped collector (12) with an internal diameter identical to the diameter of the deposition table (10), this ring having negative or neutral polarity (18).
  • the deposition table (10) After deposition of one or more mesh layers (81,82,83,84) of nanofibres over the deposition table (10), it moves linearly (55) towards the cell electropulverisation module (D), until it is centred with the ring-shaped collector (12), which presents negative or neutral polarity (18). In this position, the deposition table (10) starts a rotation movement (57) on its axis and the vacuum system is turned off, initiating the seeding of the cells (56) from the electropulverisation capillary tube (13), with positive polarity (16), on the nanofibre meshes (81,82,83,84), during a certain period of time, thus leaving the surface of the nanofibre mesh with cells (58) uniformly distributed.
  • the deposition table (10) moves (59) again to the fibre collector module, in order to proceed to a new deposition cycle of another layer or layers of two-dimensional nanofibre meshes with controlled orientation and distance between nanofibres.
  • the deposition table (10) is alternately moved (55, 59) between the position of the nanofibre collector module, where the nanofibres are deposited, and the electropulverisation module, where the cells (56) are seeded in a controlled manner by the computerised unit (20), thus obtaining a three-dimensional cell matrix (68) with nanofibres of controlled alignment and uniform cell distribution throughout the thickness (67).
  • the number of nanofibre mesh layers deposited (64,65,66) over the deposition table (10) and the number of times the electropulverisation of the cells (56) occurs over these is defined by the thickness (67) desired for the three-dimensional cell matrix (68), by the thickness of the deposited nanofibres, the magnitude of the vacuum pressure generated at the surface of the deposition table that controls the level of compaction of the nanofibre mesh layers and thus the porosity of the three-dimensional matrix throughout the thickness and the cell density desired for the three-dimensional cell matrix (68).
  • the successive two-dimensional layers of nanofibre meshes, deposited (81,82,83,84) onto the deposition table (10), are held in position by the action of a suction force (43) generated by the vacuum pressure in the holes of the surface (9) of the deposition table (10), which communicate with a chamber (42), in its interior, connected by a channel (11) to the vacuum pump (21).
  • control of the pressure in the vacuum pump (21) is also intended to control the suction forces (43) on the fibres and the degree of compaction between the formed two-dimensional nanofibre mesh layers (81,82,83,84) and, consequently, the porosity in the direction perpendicular to the plane of the deposited fibre layer.
  • the control of the distance between the electropulverisation capillary tube (4) with positive polarity and the generatrix of the collector cylinders (6, 30), the control of the rotation speed (31) of the collector cylinders (6, 30), the control of the linear (22) and rotation movements (24) of the deposition table, control of the pressure of the vacuum pump (21), control of the distance between the electropulverisation capillary tube (13) of the cells with positive polarity and the collector ring (12), control of the voltage on the capillary tubes (38) and the cylindrical surfaces (34, 36) of the collector cylinders (6, 30) and the collector ring (12) is performed by a computerised control unit (20), which, depending on the desired alignment and distance of the nanofibres for each deposited two-dimensional nanofibre mesh layer (81, 82, 83, 84), the desired thickness (67) of the matrix (68) and the desired cell density, programmes the sequence of all the necessary movements, vacuum pressure and polarity of the electrodes on the basis of a computer programme specifically developed for this
  • the process of the present invention is carried out in several steps using the manufacturing system (1), as described in the previous section.
  • the production of three-dimensional cell matrices with nanofibres of controlled alignment and uniform cell distribution throughout the thickness occurs from the alternating linear movement (55, 59) of the deposition table (10) between the position of the nanofibre collector module, where the aligned nanofibres (8) are deposited, and the electropulverisation module, where the cells (56) are seeded onto the two-dimensional nanofibre mesh layers (81,82,83,84).
  • the continuously deposited nanofibres (8) attached to the surface of the deposition table (10) are aligned in different directions by the rotation movement of the deposition table (24), the distance between the deposited nanofibres is controlled by the linear movement (22) of the deposition table (10), the continuous deposition of nanofibres by the cylindrical surfaces (34, 36) of the collector cylinders (6, 30) allows the formation of two-dimensional nanofibre meshes (81,82,83,84) of controlled organization and distribution on the surface (9) of the deposition table (10), the control of the distance (45, 47, 49, 52) between the fibres deposited on the deposition table (10) allows controlling the porosity of the two-dimensional mesh in the plane thereof;
  • the controlled linear movement of the deposition table in the direction and opposite orientation (60) to the electrospinning capillary tube (4) allows the deposition of a new two-dimensional nanofibre mesh over the previous mesh with a new organisation of the nanofibres, being the number of layers (64,65, 66) of nanofibre meshes deposited on the table (10) defined by the desired thickness (67) of the three-dimensional cell matrix (68), the thickness of the deposited nanofibres and the magnitude of the vacuum pressure generated on the surface of the deposition table (9) which controls the level of compaction of the nanofibre mesh layers and thus the porosity of the three-dimensional matrix throughout the thickness (67).
  • the process for manufacturing three-dimensional cell matrices with nanofibres of controlled alignment and uniform cell distribution throughout the thickness of the present invention comprises the following steps:
  • the controlled movement (27) of the deposition table (10) in the direction and opposite orientation (60) to the electrospinning capillary tube (4) after a set of layers of deposited fibre meshes followed by electropulverisation of the cells (56) allows the accumulation of successive layers of two-dimensional nanofibre meshes (81,82,83, 84) with cells (58) and the formation of a three-dimensional cell matrix (68) with nanofibres of controlled alignment and uniform cell distribution with thickness (67) dependent on the number of layers (64,65,66) of deposited fibres, the thickness of the nanofibres and the degree of compaction between layers which is controlled by vacuum pressure and the vacuum pressure attaches the fibres to the table.
  • the obtained matrix thickness varies according to the number of deposited layers of nanofibre meshes, the thickness of these nanofibres and the degree of compaction between layers, the latter controlled by the vacuum pressure exerted on the layers of fibres deposited on the surface of the deposition table.
  • the alignment and distance, between the nanofibres in each layer is controlled by the different rotation and linear movements of the deposition table in each fibre deposition step.
  • the cell density of the three-dimensional nanofibre matrix is defined by the number of deposited nanofibre layers, the thickness of the deposited nanofibres, the magnitude of the vacuum pressure generated at the surface of the deposition table that controls the level of compaction of the nanofibre mesh layers, the number of times the electropulverisation of the cells (56) over the two-dimensional nanofibre mesh occurs and the duration of the cell electropulverisation period on the meshes.
  • Example Production of a three-dimensional cell matrix with nanofibres of controlled alignment and uniform cell distribution throughout the thickness.
  • This example concerns the production of a cell matrix composed of 48 layers of aligned polymeric nanofibre meshes and cells from a chondrocyte cell line for cartilage engineering, with a total thickness of 3 mm.
  • PCL polycaprolactone
  • DCM dichloromethane
  • DMF dimethylformamide
  • the molten polymer was then electrospun using a capillary tube (4) with a flow of 2.5 mL/h, a voltage of 25 kV and a working distance of 15 cm from the cylindrical surfaces (34,36) of the collector cylinders (6,30).
  • the collector cylinders (6,30) have a diameter of 80 mm and are moved with a continuous rotational speed of 10 rpm and the cylindrical surfaces (34,36) were subjected to a negative voltage of -3 kV.
  • the deposition table has a diameter of 6 mm, and the holes on its surface (9) are subjected to a vacuum pressure of 3300 Pa.
  • a vacuum pressure 3300 Pa.
  • different combinations of speeds and duty strokes were programmed in linear movement (22) and rotation movement (24) of the deposition table (10).
  • the 48 layers of the deposited two-dimensional nanofibre meshes resulted from performing 6 consecutive cycles (6 times) of 8 layers of two-dimensional meshes with different alignments (81), (82), (83), (84), (85), (86), (87) and (88) in the following order, with the speed and stroke characteristics of the deposition table (10) for each mesh as follows:
  • chondrocyte cell line C28 / I2 was used and maintained at 37°C in a humidified atmosphere of 5% CO2 in air, in DMEM / F-12 (Sigma - Aldrich) supplemented with 10% (v / v) of FBS (Sigma -Aldrich), 1% (v / v) P / S (Sigma - Aldrich) and 0.25 ⁇ g / mL of amphotericin B.
  • Cells were harvested pre-confluence using a trypsin / EDTA (0.25%; Sigma-Aldrich) solution. 1.00 ⁇ 10 ⁇ 6 chondrocytes were suspended in 154 ⁇ L of culture medium and poured into a 5 mL plastic syringe.
  • the chondrocyte suspension was subjected to electropulverisation with a flow rate of 2 mL / h at +12.5 kV through 27G capillary tube/blind needle (13) (0.4 mm diameter and 1.5 mm length) with a distance between the needle and the ring-shaped collector (12) of 70 mm, the deposition table (10) was positioned concentrically with the collector ring (12). The collector ring was subjected to a voltage of -1 kV. In this position the deposition table (10) started the rotation movement (57) at a speed of 5 rpm for 3s.
  • each four layers of two-dimensional nanofibres mesh deposited on the deposition table (10) it moved 0.0625 mm in the direction and opposite orientation (60) to the electrospinning capillary tube (4), which corresponds to the average thickness of the four layers of deposited two-dimensional nanofibres mesh.
  • the deposition table moved in the opposite orientation of the capillary tube about 3 mm, corresponding to the thickness of the matrix (67) obtained at the end of the 48 deposited layers.
  • the deposition table moves (55) to the position of the cell electropulverisation module, the vacuum on the deposition table is turned off and the cells (58) are seeded on the meshes, then the deposition table (10) moves (59) to the nanofibre collector module for new deposition of four layers of nanofibre meshes (85, 86, 87, 88), the vacuum system is turned on again.
  • This automated and alternated process between the deposition of the nanofibre meshes and the seeding of cells (58) on them was repeated until the final thickness (67) of the aligned nanofibre cell matrix was reached (65).
  • the three-dimensional matrix of aligned fibres obtained in this example exhibits, like native cartilage, a preferential alignment of the fibres in its surface area parallel to the surface, in the intermediate area it does not show any preferential alignment, and in the deeper area the fibres are aligned in a vertical manner relative to the surface.

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  • Engineering & Computer Science (AREA)
  • Textile Engineering (AREA)
  • Mechanical Engineering (AREA)
  • Nonwoven Fabrics (AREA)
  • Spinning Methods And Devices For Manufacturing Artificial Fibers (AREA)
EP21160776.7A 2020-03-04 2021-03-04 Automatisierte herstellung von dreidimensionalen zellmatrizen mit nanofasern mit kontrollierter ausrichtung und gleichmässiger zellverteilung Active EP3882385B1 (de)

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US2349950A (en) 1937-08-18 1944-05-30 Formhals Anton Method and apparatus for spinning
WO2005100654A2 (en) * 2004-04-08 2005-10-27 Research Triangle Institute Electrospinning of fibers using a rotatable spray head
WO2007090102A2 (en) * 2006-01-27 2007-08-09 The Regents Of The University Of California Biomimetic scaffolds
US20110018174A1 (en) 2009-07-22 2011-01-27 Adra Smith Baca Electrospinning Process and Apparatus for Aligned Fiber Production
US20110142806A1 (en) 2009-12-15 2011-06-16 Usa As Represented By The Administrator Of The National Aeronautics And Space Adm Electroactive Scaffold
US20120009292A1 (en) 2005-12-12 2012-01-12 University Of Washington Method and apparatus for controlled electrospinning
US8580181B1 (en) 2007-03-26 2013-11-12 Vince Beachley Fabrication of three dimensional aligned nanofiber array
WO2015138970A1 (en) * 2014-03-14 2015-09-17 Scripps Health Electrospinning of cartilage and meniscus matrix polymers
US20160004706A1 (en) 2014-07-01 2016-01-07 Microsoft Corporation Security trimming of search suggestions
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US2349950A (en) 1937-08-18 1944-05-30 Formhals Anton Method and apparatus for spinning
WO2005100654A2 (en) * 2004-04-08 2005-10-27 Research Triangle Institute Electrospinning of fibers using a rotatable spray head
US20120009292A1 (en) 2005-12-12 2012-01-12 University Of Washington Method and apparatus for controlled electrospinning
WO2007090102A2 (en) * 2006-01-27 2007-08-09 The Regents Of The University Of California Biomimetic scaffolds
US8580181B1 (en) 2007-03-26 2013-11-12 Vince Beachley Fabrication of three dimensional aligned nanofiber array
US20110018174A1 (en) 2009-07-22 2011-01-27 Adra Smith Baca Electrospinning Process and Apparatus for Aligned Fiber Production
US20110142806A1 (en) 2009-12-15 2011-06-16 Usa As Represented By The Administrator Of The National Aeronautics And Space Adm Electroactive Scaffold
WO2015138970A1 (en) * 2014-03-14 2015-09-17 Scripps Health Electrospinning of cartilage and meniscus matrix polymers
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LI ET AL., NANOLETTERS, vol. 3, no. 8, 2003, pages 1167
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