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WO2018220137A1 - Ensemble pour la production d'implants tissulaires - Google Patents

Ensemble pour la production d'implants tissulaires Download PDF

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Publication number
WO2018220137A1
WO2018220137A1 PCT/EP2018/064371 EP2018064371W WO2018220137A1 WO 2018220137 A1 WO2018220137 A1 WO 2018220137A1 EP 2018064371 W EP2018064371 W EP 2018064371W WO 2018220137 A1 WO2018220137 A1 WO 2018220137A1
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WO
WIPO (PCT)
Prior art keywords
mould element
cavity
mould
tissue
membrane
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/EP2018/064371
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English (en)
Inventor
Stephan Fox
Jimmy Hu
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Eidgenoessische Technische Hochschule Zurich ETHZ
Original Assignee
Eidgenoessische Technische Hochschule Zurich ETHZ
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Filing date
Publication date
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Publication of WO2018220137A1 publication Critical patent/WO2018220137A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
    • A61F2/02—Prostheses implantable into the body
    • A61F2/10—Hair or skin implants
    • A61F2/105—Skin implants, e.g. artificial skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/24—Collagen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A61L27/3813—Epithelial cells, e.g. keratinocytes, urothelial cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/52—Hydrogels or hydrocolloids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/60—Materials for use in artificial skin
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B29—WORKING OF PLASTICS; WORKING OF SUBSTANCES IN A PLASTIC STATE IN GENERAL
    • B29C—SHAPING OR JOINING OF PLASTICS; SHAPING OF MATERIAL IN A PLASTIC STATE, NOT OTHERWISE PROVIDED FOR; AFTER-TREATMENT OF THE SHAPED PRODUCTS, e.g. REPAIRING
    • B29C33/00—Moulds or cores; Details thereof or accessories therefor
    • B29C33/0011—Moulds or cores; Details thereof or accessories therefor thin-walled moulds
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B29—WORKING OF PLASTICS; WORKING OF SUBSTANCES IN A PLASTIC STATE IN GENERAL
    • B29C—SHAPING OR JOINING OF PLASTICS; SHAPING OF MATERIAL IN A PLASTIC STATE, NOT OTHERWISE PROVIDED FOR; AFTER-TREATMENT OF THE SHAPED PRODUCTS, e.g. REPAIRING
    • B29C33/00—Moulds or cores; Details thereof or accessories therefor
    • B29C33/0077—Moulds or cores; Details thereof or accessories therefor characterised by the configuration of the mould filling gate ; accessories for connecting the mould filling gate with the filling spout
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B29—WORKING OF PLASTICS; WORKING OF SUBSTANCES IN A PLASTIC STATE IN GENERAL
    • B29C—SHAPING OR JOINING OF PLASTICS; SHAPING OF MATERIAL IN A PLASTIC STATE, NOT OTHERWISE PROVIDED FOR; AFTER-TREATMENT OF THE SHAPED PRODUCTS, e.g. REPAIRING
    • B29C33/00—Moulds or cores; Details thereof or accessories therefor
    • B29C33/38—Moulds or cores; Details thereof or accessories therefor characterised by the material or the manufacturing process
    • B29C33/40—Plastics, e.g. foam or rubber
    • B29C33/405—Elastomers, e.g. rubber
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2240/00—Manufacturing or designing of prostheses classified in groups A61F2/00 - A61F2/26 or A61F2/82 or A61F9/00 or A61F11/00 or subgroups thereof
    • A61F2240/001—Designing or manufacturing processes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2240/00—Manufacturing or designing of prostheses classified in groups A61F2/00 - A61F2/26 or A61F2/82 or A61F9/00 or A61F11/00 or subgroups thereof
    • A61F2240/001—Designing or manufacturing processes
    • A61F2240/002—Designing or making customized prostheses
    • A61F2240/004—Using a positive or negative model, e.g. moulds
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B29—WORKING OF PLASTICS; WORKING OF SUBSTANCES IN A PLASTIC STATE IN GENERAL
    • B29C—SHAPING OR JOINING OF PLASTICS; SHAPING OF MATERIAL IN A PLASTIC STATE, NOT OTHERWISE PROVIDED FOR; AFTER-TREATMENT OF THE SHAPED PRODUCTS, e.g. REPAIRING
    • B29C33/00—Moulds or cores; Details thereof or accessories therefor
    • B29C33/30—Mounting, exchanging or centering
    • B29C33/306—Exchangeable mould parts, e.g. cassette moulds, mould inserts
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B29—WORKING OF PLASTICS; WORKING OF SUBSTANCES IN A PLASTIC STATE IN GENERAL
    • B29K—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES B29B, B29C OR B29D, RELATING TO MOULDING MATERIALS OR TO MATERIALS FOR MOULDS, REINFORCEMENTS, FILLERS OR PREFORMED PARTS, e.g. INSERTS
    • B29K2995/00—Properties of moulding materials, reinforcements, fillers, preformed parts or moulds
    • B29K2995/0037—Other properties
    • B29K2995/0056—Biocompatible, e.g. biopolymers or bioelastomers

Definitions

  • the present invention relates to an assembly for the production of tissue implants via an injection moulding process.
  • the quality of tissue implants is determined by the know-how and skills of the person producing such grafts. It is a first challenge to standardize the production process in order to produce tissue implants of a reproducible quality. It is a second challenge to provide an easy-to-use production process requiring a lesser degree of expertise from the operator at equivalent quality when compared to expert manual production. Finally, it is a third challenge to effectively and quickly produce tissue implants.
  • a possible approach to these challenges is the production of tissue implants using 3D bio- printing techniques which employ bio-inks that are deposited layer by layer in order to form tissue implants or parts thereof having a certain morphology and/or composition.
  • 3D bio-printing techniques offer the advantage to produce complex 3D structures in an automated fashion and are being pursued for the purposes of applications such as skin graft production.
  • the 3D bio-printing techniques inherently suffer from the drawback that they are comparatively slow, since the 3D bio-printer must produce the tissue implants layer by layer.
  • a dermal tissue equivalent i.e. an implant capable of substituting the dermal layer of the skin
  • collagen is the main component of natural skin.
  • a self-assembly method provided by Bell et al., a dilute collagen hydrogel precursor is mixed with fibroblasts and contracted by the cells themselves. As a result of contraction, excess fluid is squeezed out of the collagen hydrogel and a suitable consistency is attained.
  • a dilute hydrogel is first prepared by mixing a dilute collagen solution with a cell suspension and then mechanically compressed using an adapted device to squeeze out excessive fluid and thus arrive at a dermal equivalent which has optimal cell density and mechanical properties suitable for handling during surgical procedure on a human patient.
  • the compaction method reduces manufacturing time to minutes instead of days or weeks when compared to the self- assembly method.
  • this method suffers from the drawback that the cells are not homogenously distributed throughout the bulk, in particular in direction of the compression, of the dermal equivalent.
  • the production of more complex implants having different parts as to the type of cells becomes nearly impossible.
  • the present invention provides for an apparatus and a process by which tissue implants in general, and skin implants in general, may be provided without having to cast very large precursor collagen hydrogels, without having to rely heavily on skill of the operator and do not warp (do not deform unwantedly) after casting them.
  • the apparatus and the process according to the present invention allows for increased production speed of tissue implants while the tissue implants display excellent spatial homogeneity, i.e. the cells therein are spatially distributed in an even manner in any direction
  • It is an object of the present invention to provide an injection mould assembly for the production of tissue implants comprising an inner mould element having a mould cavity and an outer mould element having a restriction cavity, the tissue implant comprising at least a first volume of a first tissue equivalent wherein the inner mould element comprises at least one membrane and the mould cavity of the inner mould element is at least partially delimited by said at least one membrane; wherein the inner mould element comprises a gate opening allowing the injection of a first tissue equivalent precursor into the mould cavity; characterized in that the outer mould element is configured such as to receive the inner mould element within the restriction cavity and the restriction cavity comprises a rigid wall delimiting at least the part of the restriction cavity facing the membrane delimiting the mould cavity when the inner mould element is received within the restriction cavity.
  • tissue implant obtained through the above process according to the second aspect, wherein the tissue implant comprises at least a first and preferably a second volume of tissue equivalent.
  • a first tissue equivalent precursor comprised of a mixture of at least the cross-linkable hydrogel matrix precursor solution and the suspension of animal cells into a mould cavity of a mould, such as the above-mentioned mould cavity of the inner mould element,
  • the first tissue equivalent precursor to solidify in the mould cavity such as to form a tissue implant comprising a first volume of a first tissue equivalent in the mould cavity of the mould, wherein the first tissue equivalent precursor comprised of a mixture of at least the cross-linkable hydrogel matrix precursor solution and the suspension of animal cells is formed by combining at least a separate flow of cross-linkable hydrogel matrix precursor solution and at least a separate flow of suspension of animal cells in a mixing cavity located downstream of the storage compartments and upstream of the mould cavity of the mould and fluidly connected with each storage compartment and wherein the cross- linkable hydrogel matrix precursor solution is a concentrated cross-linkable hydrogel matrix precursor solution.
  • Fig. 1 shows a front view of an inner mould element (1) formed of an annular frame (2) which is delimited on both sides by a membrane (3) of square shape, which membrane is affixed to the frame by a seal (4).
  • the inner mould element further comprises a gate opening (5) through which a tissue equivalent precursor can be injected into the mould cavity.
  • Fig. 2 shows a perspective view of the inner mould element shown in Fig.1
  • Fig. 3 shows a perspective view of the inner mould element shown in Fig.l, where the membrane (3) has been partially peeled away.
  • Fig. 4 shows a perspective view of an injection mould assembly comprising the inner mould element shown in Fig. l and an outer mould element (6), in which the inner mould element (1) is received in the outer mould element (6).
  • the outer mould element (6) is formed of an upper die (8) formed by a rigid wall and an lower die (7) formed by a rigid wall, where the two dies are connected to each other through the distance adjusting means (9) which are fixed in position.
  • the upper die further comprises a gate opening (10) through which a tissue equivalent precursor can be injected into the mould cavity through the gate opening (5) of the inner mould element (1).
  • FIG. 5 shows a perspective view of an injection mould assembly comprising an inner mould element (1) and an outer mould element (6).
  • the inner mould element (1) is formed of a rectangular frame (2) which is delimited on its upper side by a membrane (3) of square shape, which membrane is affixed to the frame by a seal.
  • the inner mould element further comprises a gate opening (5) through which a tissue equivalent precursor can be injected into the mould cavity.
  • the inner mould element (1) can be inserted laterally into the outer mould element (6).
  • the outer mould element (6) further comprises a gate opening (10).
  • FIG. 1 shows a perspective view of an injection mould assembly in which the inner mould element (1) is received in the outer mould element (6).
  • FIG. 1 shows a cross-sectional view of an injection mould assembly comprising the inner mould element shown in Fig.l and an outer mould element (6), in which the inner mould element (1) is received in the outer mould element (6).
  • the outer mould element (6) is formed of an upper die (8) formed by a rigid wall and an lower die (7) fomied by a rigid wall, where the two dies are connected to each other through the distance adjusting means (9) which are fixed in a position where the upper die restricts the outward bulging of the membrane (3) during the injection of a first tissue equivalent precursor (1 1).
  • the upper die further comprises a gate opening (10) through which a tissue equivalent precursor can be injected into the mould cavity through the gate opening (5) of the inner mould element (1).
  • FIG. 1 shows a cross-sectional view of an injection mould assembly comprising the inner mould element shown in Fig.l and an outer mould element (6), in which the inner mould element (1) is received in the outer mould element (6).
  • the outer mould element (6) is formed of an upper die (8) formed by a rigid wall and an lower die (7) formed by a rigid wall, where the two dies are connected to each other through the distance adjusting means (9) which are first adjusted to a predetermined distance with respect to the membrane (3) and then fixed in a position where the upper die allows the outward bulging of the membrane (3) during the injection of a second tissue equivalent precursor (12).
  • the upper die further comprises a gate opening (10) through which a tissue equivalent precursor can be injected into the mould cavity through the gate opening (5) of the inner mould element (1).
  • Suitable membranes for use in the present invention are permeable or impermeable membranes, and in particular permeable membranes that form a selective barrier through which liquids such as aqueous solutions and/or gases such as carbon dioxide or oxygen can move.
  • Suitable permeable membranes may be porous membrane or meshed screens. Examples of such porous membranes are PET membranes or PTFE membranes, while meshed screens may for example be screens made from polyamide or polyolefins. Examples of impermeable membranes are for example aluminium foil or other barrier films.
  • the term "skin” is usually understood as referring to a construct having at least two layers and cell types.
  • the first layer comprises the dermis populated with fibroblasts in an extracellular matrix (ECM) and the second layer comprises the epidermis populated with keratinocytes.
  • ECM extracellular matrix
  • the production of the epidermis is based on seeding keratinocytes on top of a dermal equivalent, either by pipetting, robotic application (e.g. 3D bioprinting) or spraying.
  • the dermal equivalent can vary in the composition and fabrication method of the ECM and the integration of the fibroblasts. In dermal equivalents for clinical application, it is important to have a homogenous distribution of vital fibroblasts throughout the bulk of epidermal layer and to have sufficient mechanical stability for handling during surgery.
  • It is an object of the present invention to provide an injection mould assembly for the production of tissue implants comprising an inner mould element having a mould cavity and an outer mould element having a restriction cavity, the tissue implant comprising at least a first volume of a first tissue equivalent wherein the inner mould element comprises at least one membrane and the mould cavity of the inner mould element is at least partially delimited by said at least one membrane; wherein the inner mould element comprises a gate opening allowing the injection of a first tissue equivalent precursor into the mould cavity; characterized in that the outer mould element is configured such as to receive the inner mould element within the restriction cavity and the restriction cavity comprises a rigid wall delimiting at least the part of the restriction cavity facing the membrane delimiting the mould cavity when the inner mould element is received within the restriction cavity.
  • the rigid wall delimiting at least the part of the restriction cavity facing the membrane delimiting the mould cavity when the inner mould element is received within the restriction cavity essentially abuts against or rests on the outer side of the membrane delimiting the mould cavity. This way, a deformation and in particular bulging of the membrane during the injection of a first volume of a first tissue equivalent precursor is prevented.
  • the rigid wall of the restriction ca ity is configured such as to be adjustable with respect to the distance between at least one membrane of the inner mould element and the rigid wall.
  • the rigid wall may be connected to distance adjusting means such as actuators that allow controlling the position of the rigid wall. This allows, at least in the region of the membrane delimiting the mould cavity, to adjust the extent to which the membrane bulges outward in response to the pressure of the tissue equivalent precursor being injected.
  • the rigid wall in a first step, may be in a resting position in which the rigid wall essentially rests on the outer side of the membrane during the injection of a first volume of a first tissue equivalent precursor such as to restrict any outward bulging of the membrane due to injection pressure.
  • the rigid wall may then be in a position in which the rigid wall is at an increased distance from the membrane during the injection of a further volume of a further tissue equivalent precursor such as to not restrict any outward bulging of the membrane due to injection pressure.
  • This allows the further tissue equivalent precursor to enter between the membrane and the first tissue equivalent precursor and thereby expand the membrane such as to form a second volume of further tissue equivalent on top of, or adjacent to, the first volume of first tissue equivalent.
  • the at least one membrane of the inner mould element is an elastic membrane, preferably having an elasticity such as to be allow the expansion thereof when an injection pressure is applied and to allow the contraction thereof when the injection pressure is removed.
  • the at least one membrane of the inner mould element is sealed to the inner mould, and in particular to the closed frame.
  • the at least one membrane of the inner mould element is sealed to the inner mould, and in particular to the closed frame, such as to provide a peelable seal.
  • a peelable seal allows to easily peel away the membrane in order to isolate the tissue implant from the inner mould element, much like a lid of a receptacle. This may be achieved according to techniques known to the person of skill in the art, such as for example by providing the membrane with a heat seal layer which can be heat sealed to the inner mould element.
  • the heat seal may for example comprise ethylene-acid copolymers, ionomers or LDPE.
  • Alternative methods to form seals are HF, ultrasound or laser welding.
  • a seal may also be achieved by depositing an adhesive material where a seal is to be formed.
  • the membrane is provided with one or more pull tabs in order to facilitate the peeling away of the membrane.
  • One or more pull tabs may be formed, for example, by providing a rectangular membrane delimiting a circular part of the mould cavity through a circular seal, wherein the rectangular membrane, at least in one direction, extends beyond the circular seal.
  • the elements of the injection mould assembly for the production of tissue implants may be from any suitable material and in particular may be formed from polymer material.
  • the inner mould element is made from a biocompatible material such as for example a biocompatible polymer.
  • a biocompatible polymer can be polyolefins, polyester, PEEK, styrene copolymers and such.
  • the advantage of using such an inner mould element is that, after solidification of the tissue equivalent precursor and before implantation of the tissue implant, the inner mould element incorporating the solidified tissue equivalent precursor therein may be placed integrally into a cell culturing medium for further cultivation. The membrane of the inner mould element then allows the exchange of nutrients and gases between the cell culturing medium and the cells of the tissue implant.
  • the rigid parts of the inner mould element such as for example an annular or rectangular frame allow in general for an easier manipulation of the inner mould element, i.e. the removing of the inner mould element from the restriction cavity of the outer mould element in order to place the inner mould element into a culturing vessel and offer a hold for when the membrane is peeled away to isolate the tissue implant.
  • the inner mould element comprises two membranes facing each other and wherein each membrane is affixed to either side of a closed frame such as to form the mould cavity.
  • a closed frame such as to form the mould cavity.
  • the closed frame may for example be of annular or polygonal shape. Examples of polygonal shapes are equiangular polygonal shapes such as triangular, rectangular, square or hexagonal.
  • the at least one membrane is a porous membrane preferably having pores having a diameter of 0.25 ⁇ or more, and more preferably having a diameter in the range of 0.25 ⁇ to 7.5 ⁇ .
  • the diameters of the pores allow to exchange liquids, nutrients and gases between a cell culturing medium and a tissue implant comprised in the inner mould element.
  • the inner mould element and the restriction cavity of the outer mould element are in positive lock.
  • the outer mould element may consist of two dies such as an upper die and an lower die that can be arranged such as to lock the inner mould in place and form a restriction cavity in positive lock with the inner mould.
  • the inner mould element has a cuboid geometry and at least one face of the cuboid is formed by the at least one membrane and preferably two opposing faces of the cuboid are formed by two membranes, respectively, and the remaining faces are formed by a closed frame having a rectangular or square shape.
  • the inner mould element has a cylindrical geometry and at least one face of the cylinder is formed by the at least one membrane and preferably two opposing faces of the cylinder are formed by two membranes, respectively, and the lateral face is formed by an closed annular frame.
  • the inner mould element has a flat cylindrical geometry, i.e.
  • the inner mould element is preferably comprised of a closed annular frame on which two opposing faces of the cylinder are formed by two membranes sealed to each side of the annular frame.
  • the annular frame may for example be formed from a polymer, especially a bio-compatible polymer and the membranes may be formed from a biocompatible polymer as well, such as for example PTFE, PP, PE or PET.
  • the tissue implant can be formed in a shape that essentially corresponds to the mould cavity of the inner mould element, such as for example a flat disc shape.
  • the rigid wall of the restriction cavity is configured such as to be adjustable with respect to the distance between at least one membrane of the inner mould element and the rigid wall and wherein between steps c. and d., the process further comprises the steps cl to c3 of cl . adjusting the distance between at least one membrane of the inner mould element and the rigid wall such as to increase the distance between the at least one membrane of the inner mould element and the rigid wall,
  • the rigid wall of the outer mould element is fixed at an adjusted distance from the outer side of the membrane and thereby permits an outward bulging of the membrane during the injection of the second tissue equivalent precursor.
  • the second tissue equivalent precursor to settle or solidify in the mould cavity of the inner mould element such as to form an tissue implant comprising a second volume of a second tissue equivalent adjacent to, or on top of, the first volume of a first tissue equivalent;
  • the first and second outer mould elements are configured such as to receive the inner mould element within their respective restriction cavity and the restriction cavities each comprise a rigid wall delimiting at least the part of the restriction cavity facing the membrane delimiting the mould cavity when the inner mould element is received within the restriction cavity and wherein the rigid wall of the first outer mould element rests immovably on the outer side of the membrane, thereby restricting an outward bulging of the membrane during the injection of the first tissue equivalent precursor and wherein the rigid wall of the second outer mould element is immovably placed at an increased distance from the outer side of the membrane, thereby permitting an outward bulging of the membrane during the injection of the second tissue equivalent precursor.
  • the first tissue equivalent is a dermal equivalent comprising fibroblasts suspended in a hydrogel matrix, wherein preferably the hydrogel matrix is a collagen matrix.
  • the first tissue equivalent is a dermal equivalent comprising fibroblasts suspended in a hydrogel matrix, and in particular is a dermal equivalent comprising fibroblasts suspended in a crosslinked collagen matrix.
  • the first tissue equivalent is formed by injecting a first tissue equivalent precursor comprising fibroblasts suspended in a collagen solution into the mould cavity of the inner mould element via a gate opening of the inner mould element, wherein preferably the collagen solution has a collagen concentration of at least 10 mg/ml or of from 10 mg/ml to 25 mg/ml, preferably of at least 15 mg/ml or of from 15 mg/ml to 25 mg/nil and more preferably of at least 17.5 mg/ml or of from 17.5 mg/ml to 25 mg/ml and more preferably is a collagen solution of un-pepsinized collagen.
  • the second tissue equivalent is an epidermal equivalent comprising keratinocytes and preferably is a suspension of keratinocytes in cell culturing medium.
  • the second tissue equivalent is formed by injecting a second tissue equivalent precursor comprising fibroblasts, preferably suspended in a cell culturing medium, into the mould cavity of the inner mould element via a gate opening of the inner mould element.
  • the second tissue equivalent precursor then accumulates between the membrane and the first tissue equivalent because of the deformation of the membrane due to injection pressure and forms a second tissue equivalent on top of, or adjacent to, the first tissue equivalent thereby yielding a tissue implant comprising two discrete parts which differ in cell type, i.e. the first and second tissue equivalents.
  • the tissue implant is a skin implant, preferably comprising at least a dermal equivalent comprising fibroblasts in a collagen matrix and an epidermal equivalent comprising keratinocytes.
  • tissue implant obtained through the above process according to the second aspect, wherein the tissue implant comprises at least a first and preferably a second volume of tissue equivalent.
  • a first tissue equivalent precursor comprised of a mixture of at least the cross-linkable hydrogel matrix precursor solution and the suspension of animal cells into a mould cavity of a mould, such as the above-mentioned mould cavity of the inner mould element,
  • the first tissue equivalent precursor to solidify in the mould cavity such as to form a tissue implant comprising a first volume of a first tissue equivalent in the mould cavity of the mould, wherein the first tissue equivalent precursor comprised of a mixture of at least the cross-linkable hydrogel matrix precursor solution and the suspension of animal cells is formed by combining at least a separate flow of cross-linkable hydrogel matrix precursor solution and at least a separate flow of suspension of animal cells in a mixing cavity located downstream of the storage compartments and upstream of the mould cavity of the mould and fluidly connected with each storage compartment and wherein the cross- linkable hydrogel matrix precursor solution is a concentrated cross-linkable hydrogel matrix precursor solution.
  • the process for the production of an tissue implant comprising animal cells dispersed in a hydrogel matrix allows for the production of an tissue implant comprising animal cells dispersed in a hydrogel matrix, and is especially useful in the production of tissue implants such as skin grafts because on one hand, the water content of the hydrogel matrices useful in the present invention is beneficial to the tissue regeneration of skin and on the other hand, the mechanical properties conferred by the scaffold formed by the hydrogel matrices useful in the present invention render the manipulation of the engineered skin graft uncomplicated during typical surgical procedure in which the graft is applied.
  • cross-linkable hydrogel matrix precursor solution in a first storage compartment, it is possible to store the fluid solution for a prolonged time until needed for the production of the engineered tissue graft and thus to generate a fresh graft on-demand provided the animal cells are in a state where they can be combined with the cross-linkable hydrogel matrix precursor solution.
  • the cross-linkable hydrogel matrix precursor solutions are preferably aqueous solutions of cross-linkable hydrogel matrix precursor.
  • Such cross-linkable hydrogel matrix precursor includes cross-linkable hydrogel matrix precursors which are cross-linkable by addition of a cross-linking agent, change in pH, change in temperature or irradiation with for example UV.
  • the cross-linkable hydrogel matrix precursors are either cross-linkable hydrogel matrix precursors which are cross-linkable by addition of a cross-linking agent or by change of pH from for example acidic towards neutral basic.
  • Exemplary cross-linkable hydrogel matrix precursor may be selected from proteins such as gelatin, collagen, fibrinogen; from polysaccharides such as starch, agarose; from synthetic polymers such as polyacrylamide or poloxamers.
  • a preferred cross-linkable hydrogel matrix precursor solution may be for example be an aqueous solution of collagen preferably having an acidic pH.
  • the suspension of animal cells may be a suspension of animal cells in a liquid medium such as for example cell culturing medium.
  • a liquid medium such as for example cell culturing medium.
  • the cells may be suspended in DMEM (Dulbecco modified Eagle medium).
  • the concentration of animal cells within the suspension of cells may be chosen such that a animal cell target density is reached in the tissue implant. It will be apparent to a person of ordinary skill in the art that the animal cells are to be chosen depending on the type of tissue graft to be produced. As an example, in the case where the tissue graft is a skin graft, the animal cells may be chosen from fibroblasts and/or keratinocytes.
  • the suspension of animal cells as well as the cross-linkable hydrogel matrix precursor solution are stored in their respective storage compartments before being combined in a mixing cavity to which they are fluidly connected to form a tissue equivalent precursor.
  • the storage compartments may be present as separate bodies or may be comprised in a single injection moulding apparatus, such as for example a double syringe incorporating the first and second storage compartment.
  • the double syringe may be equipped with plungers that can be independently controlled in order to adjust and maintain the flow rates from the compartments into the mixing cavity and into the mould cavity of the mould, such as the inner mould element.
  • the tissue equivalent precursor comprising the mixture of cross-linkable hydrogel matrix precursor solution and the suspension of animal cells
  • the solidification may for example be completed within 10 minutes.
  • the mixture of cross-linkable hydrogel matrix precursor solution and the suspension of animal cells is formed by combining at least a separate flow of cross-linkable hydrogel matrix precursor solution and at least a separate flow of suspension of animal cells in a mixing cavity located downstream of the storage compartments and upstream of the mould cavity of the mould such as the inner mould element and fluidly connected with each storage compartment.
  • the mixing cavity may comprise a mixing element such as a static mixing element which increases the mixing of the two streams.
  • the mixing cavity may not comprise a mixing element and rely solely on the mixing effect achieved by the turbulent flow within the mixing cavity.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Transplantation (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Veterinary Medicine (AREA)
  • Dermatology (AREA)
  • Oral & Maxillofacial Surgery (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Medicinal Chemistry (AREA)
  • Epidemiology (AREA)
  • Cell Biology (AREA)
  • Mechanical Engineering (AREA)
  • Zoology (AREA)
  • Botany (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Manufacturing & Machinery (AREA)
  • Urology & Nephrology (AREA)
  • Dispersion Chemistry (AREA)
  • Biophysics (AREA)
  • Cardiology (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Vascular Medicine (AREA)
  • Prostheses (AREA)

Abstract

L'invention concerne un ensemble de moulage par injection pour la production d'implants tissulaires comprenant un élément de moule interne (1) ayant une cavité de moule et un élément de moule externe (6) ayant une cavité de restriction, l'implant tissulaire comprenant au moins un premier volume d'un premier équivalent de tissu, l'élément de moule interne comprenant au moins une membrane (3) et la cavité de moule de l'élément de moule interne étant au moins partiellement délimitée par ladite au moins une membrane ; l'élément de moule interne comprenant une ouverture de porte (10) permettant l'injection d'un premier précurseur d'équivalent de tissu dans la cavité de moule ; l'élément de moule externe étant configuré de façon à recevoir l'élément de moule interne à l'intérieur de la cavité de restriction et la cavité de restriction comprenant une paroi rigide délimitant au moins la partie de la cavité de restriction faisant face à la membrane délimitant la cavité de moule lorsque l'élément de moule interne est reçu à l'intérieur de la cavité de restriction et la ou les membranes de l'élément de moule interne sont scellées au moule interne de façon à fournir un joint pelable, ainsi qu'un procédé pour la production d'implants tissulaires à l'aide de l'ensemble de moulage par injection.
PCT/EP2018/064371 2017-05-31 2018-05-31 Ensemble pour la production d'implants tissulaires Ceased WO2018220137A1 (fr)

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EP17173807.3 2017-05-31
EP17173807 2017-05-31

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CN113428972A (zh) * 2021-07-16 2021-09-24 南京沃谱瑞环境研究院有限公司 一种人工湿地专用填料的制备模具及其制备方法
IT202300013083A1 (it) * 2023-06-23 2024-12-23 Lp Tech S R L Stampo in silicone per la realizzazione in linea di una protesi da applicare

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US5536656A (en) * 1989-09-15 1996-07-16 Organogenesis, Inc. Preparation of tissue equivalents by contraction of a collagen gel layered on a collagen gel
US5990379A (en) * 1994-11-15 1999-11-23 Kenton W. Gregory & Sisters Of Providence Prosthetic devices including elastin or elastin-based materials
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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IT202300013083A1 (it) * 2023-06-23 2024-12-23 Lp Tech S R L Stampo in silicone per la realizzazione in linea di una protesi da applicare
WO2024261721A1 (fr) * 2023-06-23 2024-12-26 Lp Tech S.R.L Moule en silicone pour la fabrication en ligne d'une prothèse à appliquer

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