WO2018122555A1 - Membrane tissulaire - Google Patents

Membrane tissulaire Download PDF

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Publication number
WO2018122555A1
WO2018122555A1 PCT/GB2017/053892 GB2017053892W WO2018122555A1 WO 2018122555 A1 WO2018122555 A1 WO 2018122555A1 GB 2017053892 W GB2017053892 W GB 2017053892W WO 2018122555 A1 WO2018122555 A1 WO 2018122555A1
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WO
WIPO (PCT)
Prior art keywords
face
tissue
membrane
scaffold
substrate
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PCT/GB2017/053892
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English (en)
Inventor
Lobat TAYEBI
Hua Ye
Zhanfeng Cui
Original Assignee
Oxford University Innovation Limited
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Publication of WO2018122555A1 publication Critical patent/WO2018122555A1/fr

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    • 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
    • A61L27/56—Porous materials, e.g. foams or sponges
    • 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
    • A61L27/20—Polysaccharides
    • 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
    • A61L27/222—Gelatin
    • 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
    • A61L27/227—Other specific proteins or polypeptides not covered by A61L27/222, A61L27/225 or A61L27/24
    • 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
    • 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

Definitions

  • the present disclosure relates to an epithelial tissue scaffold membrane.
  • the present disclosure also relates to a method of producing such a scaffold membrane, as well as to a method of guided tissue regeneration that comprises the use of such a scaffold membrane.
  • Oral mucosal defects may be reconstructed by autografts from the oral mucosa, or using split thickness skin grafts. These techniques, however, can cause donor site morbidity. Furthermore, because such grafts do not have an intact blood supply, the supply of blood to such grafts may be sub-optimal. Blood supply can be improved with vascularised skin flaps, which have an intact blood supply. However, such flaps express a different keratinisation pattern and, hence, can be more susceptible to scar formation. They may also be more susceptible to infection in a wet oral environment.
  • tissue scaffolds have been used to aid tissue regeneration.
  • Scaffolds are structures that have been engineered to cause desirable cellular interactions to aid the formation of new functional tissues. Cells may be seeded into these structures to support three-dimensional tissue formation.
  • Figure 1 is a perspective view of a scaffold membrane according to a first embodiment of the present disclosure
  • Figure 2 is a top down view of the scaffold membrane shown in Figure 1 ;
  • Figure 3 is a perspective view from the bottom of the tissue scaffold shown in Figure 1 ;
  • Figures 4 and 5 are SEM images of tissues scaffolds according to a second and third embodiment of the present disclosure.
  • Figure 6 to 9 show various histological images of tissue grown using a scaffold membrane according to a fourth embodiment of the present disclosure as described in Example 5; DESCRIPTION
  • an epithelial tissue scaffold membrane comprising a porous substrate comprising a biocompatible (e.g. hydrogel) polymer, said substrate having a first face for supporting the growth of epithelial tissue, and a second face, opposing the first face, for supporting the growth of underlying tissue, wherein the pore structure at the first face is different from the pore structure at the second face.
  • a biocompatible e.g. hydrogel
  • the pores at the first face of the porous substrate have a different structure from the pores at the second face of the substrate.
  • the average pore size at the first face may be less than the average pore size at the second face.
  • the porosity at the first face may be less than the porosity at the second face.
  • the average pore size at the first face may be the same as the average pore size at the second face but there may be fewer pores per unit area at the first face than the second face.
  • the first face of the porous substrate is configured to support the growth of epithelial tissue.
  • the first face of the porous substrate may provide a pore structure for
  • the second face of the porous substrate is configured to support the growth of underlying (e.g. connective tissue.
  • the second face of the porous substrate may provide a pore structure for accommodating cells for underlying tissue, for example, fibroblasts for the generation of connective tissue.
  • the pores of the first face may be structured (e.g. sized) to accommodate and retain e.g. keratinocytes in the region of the first face of the porous substrate.
  • keratinocytes By retaining the keratinocytes in this manner, the risk of keratinocyte cells penetrating through to the second face of the substrate is reduced. This can help to improve definition between layers of epithelial and underlying e.g. connective tissue in the (re)generated tissue by reducing the risk of keratinocytes interfering the growth of connective tissue.
  • the pores of the second face may be structured (e.g. sized) to accommodate e.g. fibroblasts.
  • the pore structure at the second face may also allow fibroblasts to penetrate through the thickness of the porous substrate to engage with the (re)generated epithelial tissue. This penetration may facilitate a desirable interface between the (re)generated epithelial and connective tissue, thereby ensuring an adequate blood supply to the
  • the average pore size at the first face of the porous substrate may be 0.5 to 200 microns, for example, 1 to 200 microns. In one example, the average pore size at the first face may be 5 to 180 microns, preferably 10 to 100 microns. In a preferred example, the average pore size at the first face may be 0.5 to 100 microns, preferably 1 to 90 microns, for instance, 10 to 75 microns.
  • the average pore size at the second face of the porous substrate may be 100 to 1000 microns, for example, 250 to 700 microns. In one example, the average pore size at the second face may be 350 to 600 microns, preferably 400 to 500 microns. In a preferred example, the average pore size at the first face may be 400 to 450 microns.
  • Pore size may be determined by any suitable method.
  • the pore size may be determined by imaging the substrate, for example, using SEM and determining the average pore size with the help of image processing and analytical software, for example, ImageJTM
  • the pore size may reflect the largest dimension across the size of the pore.
  • a circle may be "fitted" into the size of the pore and the diameter of the largest circle that can be fitted into the pore determined.
  • the process may be repeated across a number of pores across the SEM image and averaged to provide an average pore size.
  • the process may be repeated across at least 10 pores, preferably at least 20 pores, more preferably across 30 pores to provide an average pore size.
  • the pores at the first face and the pores at the second face may be substantially uniform in size.
  • at least 70%, preferably, at least 80%, more preferably at least 90 %, for instance, up to 100% of the pores at the first face have a pore size within 20% of the average pore size.
  • the average pore size at the first face is 50 microns
  • at least 70%, preferably, at least 80%, more preferably at least 90 %, for instance, up to 100% of the pores at the first face have a pore size within 20% of the 40 to 60 microns.
  • at least 70%, preferably, at least 80%, more preferably at least 90 %, for instance, up to 100% of the pores at the first face have a pore size within 5 to 15%, for instance, 10% of the average pore size.
  • At least 70%, preferably, at least 80%, more preferably at least 90 %, for instance, up to 100% of the pores at the second face have a pore size within 20% of the average pore size.
  • at least 70%, preferably, at least 80%, more preferably at least 90 %, for instance, up to 100% of the pores at the second face have a pore size of 320 to 480 microns.
  • at least 70%, preferably, at least 80%, more preferably at least 90 %, for instance, up to 100% of the pores at the second face have a pore size within 50 to 15%, for instance, 10% of the average pore size.
  • the porous substrate may be formed by 3-D printing strands of polymer criss-crossed in a lattice pattern (as discussed below). This can result in a substrate having a regular pore structure with substantially little deviation between the sizes of the pores.
  • the porous substrate may have a thickness of 50 to 3000 microns, preferably 150 to 500 microns, more preferably 200 to 400 microns. In one example, the porous substrate may have a thickness of 150 to 200 microns. In another example, the porous substrate may have a thickness of 300 to 400 microns. The precise thickness may depend on the intended application of the porous substrate. For example, where the porous substrate is used as a scaffold for regenerating oral mucosal tissue, the thickness may be in the range of 300 to 400 microns. On the other hand, for ocular applications, the thickness may be in the range of 10 to 200 microns. The thickness of the substrate may be determined by any suitable technique. Examples include using laser microscope, profilometer and micrometer.
  • the porous substrate may allow molecules to pass from the second face of the membrane to the first face of the membrane.
  • the porous substrate may be permeable to molecules having hydrodynamic diameter of up to 5 microns.
  • the porous substrate may be permeable to molecules having a diameter of up to 3 microns.
  • the porous substrate may be permeable to, for example, the flow of nutrients to support the growth of, for example, keratinocyte and fibroblast cells.
  • Hydrodynamic diameter may be determined by any suitable technique, for example, dynamic light scattering. Examples of nutrients that may pass through from the second face of the membrane to the first face of the membrane include glucose.
  • the porous substrate may comprise a three-dimensional framework.
  • the framework comprises a pore structure, whereby the average pore size at the first face is smaller than the average pore size at the second face.
  • the framework comprises a three-dimensional network of pores e.g. through-pores that extend from one face of the substrate to the other.
  • the average pore size of the through-pores may increase from the first face of the substrate to the second. This change of pore size may be tailored to retain cells e.g. keratinocytes at or near the first face, while allowing other cells e.g.
  • fibroblasts to penetrate the substrate from the second face. This penetration allows e.g. fibroblasts to propagate through the second face and form connective tissue that engages the epithelial tissue supported at the first face. This engagement may allow nutrients to be transported through the connective tissue to the epithelial tissue at the first face.
  • This change of pore size may also be tailored to facilitate the formation of a desirable interface between e.g. the (re)generated epithelial and connective tissue.
  • the framework may comprise a first region that extends from the first face of the substrate to a first predetermined depth within the substrate.
  • This first region may accommodate keratinocytes, such that growth of e.g. epithelial tissue can be supported within the first region.
  • the average pore size of the first region may be less than 200 microns, for example, 10 to 100 microns. In one example, the average pore size in the first region may be 20 to 100 microns, preferably 20 to 50 microns.
  • the average pore size of the first region may be substantially the same throughout the first region. Alternatively, the average pore size of the first region may vary. For example, the average pore size at the first face of the first region may increase towards the first predetermined depth within the substrate.
  • the framework may also comprise a second region that extends from the second face of the substrate to a second predetermined depth within the substrate.
  • This second region may accommodate e.g. fibroblasts, such that growth of connective tissue can be supported within the second region.
  • the average pore size of the second region may be greater than 250 microns, for example, 300 to 700 microns. In one example, the average pore size in the second region may be 350 to 600 microns, for example, 400 to 500 microns.
  • the average pore size of the second region may be substantially the same throughout the second region. Alternatively, the average pore size of the second region may vary. For example, the average pore size at the second face of the second region may decrease towards the second predetermined depth within the substrate.
  • the first region and second region may be adjacent (e.g. in contact) with one another. Alternatively, the first region and second region may be separated by one or more intermediate regions.
  • the intermediate region(s) may have an average pore size that is intermediate the average pore size of the first region and second region.
  • the first region may have a thickness of 50 to 300 microns, preferably 100 to 200 microns.
  • the second region may have a thickness of 50 to 300 microns, preferably 100 to 300 microns.
  • the porous substrate may comprise a plurality of layers.
  • the pore structure of the layers may increase from the first face to the second face of the substrate.
  • the pore structure may increase in a gradual manner from the first face of the substrate to the second.
  • the pore structure may change in a stepped manner.
  • the porous substrate may comprise a three-dimensional framework formed from overlapping polymer layers. Each polymer layer may itself be a porous substrate.
  • the pores of the framework may be defined by the juxtaposition of two or more polymer layers.
  • each polymer layer comprises an array of polymer strands.
  • the polymer strands may be arranged substantially parallel to one another.
  • the array comprises a plurality of polymers arranged so that they are substantially aligned in a first direction, x.
  • a second layer of polymer strands is disposed over the first layer, whereby the strands in the second layer are aligned in a second direction, y, which is at an angle to x.
  • the resulting structure may form a lattice.
  • the lattice may comprise a plurality of such "first" and "second" layers stacked on top of each other.
  • direction, y may be at any angle to direction, x.
  • the angle may be greater than 0 to less than 180 degrees, for instance, 10 to 170 degrees, preferably 30 to 150 degrees, more preferably 40 to 120 degrees. In some examples, the angle is 60 to 100 degrees, for example 80 to 90 degrees.
  • the strands of the second layer are substantially perpendicular to the first layer.
  • the framework may comprise further layers of polymer strands. In some examples, the framework may comprise an even number of layers of polymer strands. The number of layers may be varied depending on the desired thickness substrate.
  • the distance between the polymer strands in each of the layers may be varied according to the desired pore size. For example, at the first face the distance between the polymer strands may be smaller than the distance between the polymer strands at the second face. The distance between polymer strands may be used to ensure a differentiation in porosity across the thickness of the substrate. By printing the strands to form a lattice network in this manner, pore size and/or porosity can be controlled.
  • the substrate comprises a first region comprising a first lattice portion and a second region comprising a second lattice portion.
  • the first lattice portion may be formed from a plurality (e.g. an even number) of layers of polymer strands.
  • the polymer strands in the first layer of the first lattice portion may be aligned in a first direction and the polymer strands in the second layer of the first lattice portion may be aligned at an angle to that first direction.
  • the angle may be greater than 0 to less than 180 degrees, for instance, 10 to 170 degrees, preferably 30 to 150 degrees, more preferably 40 to 120 degrees.
  • the strands in the first layer may be 60 to 100 degrees, for instance, perpendicular to strands in the second layer.
  • the second lattice portion may also be formed from a plurality of layers of polymer strands.
  • the polymer strands in the first layer of the second lattice portion may be aligned in one direction and the polymer strands in the second layer may be aligned at an angle to that direction to form a lattice.
  • the angle may be greater than 0 to less than 180 degrees, for instance, 10 to 170 degrees, preferably 30 to 150 degrees, more preferably 40 to 120 degrees.
  • the strands in the first layer may be 60 to 100 degrees, for instance, perpendicular to strands in the second layer.
  • first and second layers may be a plurality of such criss-crossed first and second layers forming the second lattice portion.
  • the number of such layers may be varied to control the thickness of the second region. This, in turn, may affect the thickness of the connective tissue layer produced.
  • the number of layers in the second lattice portion may be the same or different to the number of layers in the first lattice portion.
  • the first lattice portion may overlay the second lattice portion at an angle. Angling the lattice portions in this manner may help to vary the pore size.
  • the angle may be greater than 0 to less than 180 degrees, for instance, 10 to 170 degrees, preferably 30 to 150 degrees, more preferably 40 to 120 degrees. In some examples, the angle is 60 to 100 degrees, for example 80 to 90 degrees. For example, the angle may be about 90 degrees.
  • the distance between the polymer strands at the first face may be 1 to 250 microns, preferably 25 to 100 microns.
  • the distance between polymer strands at the second face may be 50 to 1000 microns, preferably 200 to 400 microns.
  • the distance between the polymer strands in the first lattice portion may be 1 to 400 microns, preferably 25 to 200 microns.
  • the distance between polymer strands in the second lattice portion may be 50 to 2000 microns, preferably 200 to 1000 microns.
  • the distance between polymer strands at the first face or in the first lattice portion may be the same as the distance between polymer strands at the second face or in the second lattice portion.
  • the thickness of the individual polymer strands at the first face or in the first lattice portion may be greater than the thickness of the individual polymer strands at the second face or in the second lattice portion.
  • the number of pores per unit area at the first face or in the first lattice portion may be smaller than the number of pores per unit area at the second face or in the second lattice portion.
  • the substrate may be flexible, for example, when moist or wet with water.
  • the substrate may be suturable, allowing the substrate to be sutured over a wound.
  • the porous substrate may be formed of any suitable bio-erodible polymer, preferably a hydrogel polymer. Accordingly, the polymer may erode or degrade after a period of time, for example, once tissue has been generated or regenerated over the substrate.
  • hydrogel polymer may be a crosslinked hydrogel.
  • Suitable examples include polypeptides and/or polysaccharides. Specific examples include collagen, gelatin, elastin, hyaluronate/hyaluronic acid, fibrinogen and cellulose.
  • hyaluronate and hyaluronic acid derivatives of hyaluronate/hyaluronic acid derivatives such as sodium hyaluronate may also be employed.
  • the hydrogel polymer comprises at least one of gelatin, elastin and hyaluronic acid.
  • the hydrogel polymer comprises gelatin.
  • the hydrogel polymer may additionally comprise at least one of elastin and hyaluronate and/or hyaluronic acid.
  • the hydrogel polymer comprises gelatin and elastin, and yet more preferably, the hydrogel polymer comprises gelatin, elastin and hyaluronate/hyaluronic acid.
  • the relative amounts of gelatin, elastin and hyaluronate/ hyaluronic acid may be varied to provide the polymer with desired properties, for example, gel point, viscosity, mechanical strength and flexibility.
  • the hydrogel polymer comprises gelatin, elastin and sodium hyaluronate with the weight ratio of 8: 2: 0.5 (gelatin : elastin : sodium hyaluronate). All figures are presented as % w/v.
  • the polymer may have a gel point of 15-45 C, preferably 20-30.
  • the gel point of the polymer may be important.
  • the gel point may be sufficiently high for the polymer to be ejected from a print nozzle in liquid form.
  • the gel point may advantageously be sufficiently low to allow the polymer to harden once printed.
  • the polymer may be crosslinked. Crosslinking may improve the mechanical strength and risk of premature degradation of the scaffolds. Any crosslinking agent may be employed. Examples include 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N- hydroxysuccinimide (NHS).
  • EDC 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide
  • NHS N- hydroxysuccinimide
  • the polymer is a hydrogel that is crosslinked.
  • the polymer may be a crosslinked gelatin-containing polymer.
  • the polymer may be a crosslinked polymer comprising gelatin, hyaluronic acid and elastin.
  • the scaffold membrane may be produced using any suitable technique.
  • the scaffold membrane may be produced by 3-D printing.
  • a 3-D printer may be used to print an ink comprising a biocompatible polymer to form a porous substrate having a first face and a second face opposing the first face, wherein the pores at the first face have an average pore size that is smaller than the pores at the second face.
  • the ink comprising the biocompatible polymer may comprise molten biocompatible polymer, or a solution or suspension of the biocompatible polymer.
  • the solution or suspension may comprise a solvent, for example, water (e.g. deionised water).
  • Suitable biocompatible polymers are described above.
  • the ink may comprise a solution or suspension of the biocompatible polymer in a concentration of 1 to 40 % (w/v), preferably 5 to 30% (w/v), more preferably 10 to 20% (w/v).
  • the ink may comprise at least one of gelatin, elastin and
  • the ink may comprise gelatin.
  • the ink may additionally include at least one of elastin and hyaluronate.
  • the ink comprises gelatin, elastin and hyaluronate.
  • hyaluronate and hyaluronic acid have been used to include derivatives of such compounds, for example, sodium hyaluronate.
  • the ink may comprise gelatin in an amount of 1 to 30 % (w/v), preferably 5 to 20 % (w/v), more preferably 8 to 16 % (w/v), for example, about 8 % (w/v).
  • the ink may additionally comprise elastin.
  • the elastin may be present in an amount of 1 to 5 % (w/v), more preferably, 2 to 4 % (w/v), for example 2 % (w/v).
  • the ink may additionally comprise hyaluronate or hyaluronic acid.
  • the hyaluronate or hyaluronic acid may be present in addition to gelatin and elastin.
  • Suitable amounts of hyaluronate or hyaluronic acid may range from 0 to 5 % (w/v), preferably 0.1 to 3 % (w/v), for example, 0.2 to 1 % (w/v) (e.g. 0.5 % w/v).
  • the ink comprises 8 to 16 % (w/v) gelatin, 2 to 4 % (w/v) elastin and 0.2 to 1 % (w/v) hyaluronate/hyaluronic acid.
  • the ink may be printed to form the substrate using any suitable technique.
  • the ink may be printed as polymer strands using a needle.
  • the needle may have a diameter of 100 to 450 microns, for example, 250 microns.
  • the substrate may be printed by printing overlaying layers of polymer.
  • Each polymer layer may comprise a plurality of polymer strands.
  • the distance between the polymer strands and/or angle of the polymer strands may be varied to achieve the desired pore structure in the substrate.
  • the thickness of the polymer strands may also be varied.
  • the hydrogel polymer may be allowed to solidify or set.
  • the printed polymer may also be crosslinked, for example, using a crosslinking agent.
  • the polymer is a hydrogel that is crosslinked.
  • the polymer may comprise gelatin and, optionally, at least one of elastin and
  • the gelatin may be crosslinked.
  • Suitable crosslinking agents include 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS).
  • the substrate may be washed, for example, with deionised water.
  • the substrate may be stored in any suitable manner.
  • the substrate may be dehydrated and/or stored at low temperatures.
  • the substrate is stored in alcohol, for example, ethanol.
  • the alcohol may be a solution comprising at least 70% (v/v) alcohol, for example, 80 to up to 100% alcohol.
  • the substrate may be rehydrated in water prior to use. Once rehydrated, the substrate may regain its original flexibility.
  • the scaffold membrane described in this disclosure may be used in a method of guided tissue generation or regeneration of tissue.
  • tissue it is meant any human or animal tissue.
  • the scaffold membrane is used to generate epithelial tissue and underlying tissue adjacent the epithelial tissue.
  • the scaffold membrane may be used to support the growth of epithelial tissue at its first face and underlying (e.g. connective tissue) at its second face, while providing desirable cell differentiation at the interface between these two tissue types.
  • an in-vivo method may comprise placing the second face of the tissue scaffold over an exposed area of e.g. connective tissue.
  • the first face of the tissue scaffold may be in contact with keratinocyte cells.
  • the second face of the tissue scaffold may be in contact with fibroblasts.
  • the tissue scaffold may be secured by suturing.
  • cells e.g. keratinocytes may grow over the first face of the substrate to form epithelial tissue.
  • Different cells e.g. fibroblasts may be grown over the second face of the substrate to form connective tissue.
  • This change of pore size through the thickness of the substrate may be tailored to prevent fibroblast immigration to the first face. This may facilitate the formation of a desirable interface between the (re)generated epithelial and connective tissue.
  • the scaffold is bio-erodible.
  • the scaffold may dissolve or otherwise disintegrate e.g. once the tissue is regenerated.
  • the scaffold may be used as barrier membrane for applications such as Guided Tissue Regeneration (GTR) (including Guided Bone Regeneration (GBR).)
  • GTR Guided Tissue Regeneration
  • the scaffold can also be used to regenerate any soft tissue, for example, oral mucosal tissue (e.g. full thickness oral mucosa).
  • the scaffold may also be used to regenerate bone tissue.
  • the tissue scaffold may be used to generate tissue for burns treatment, intraoral grafting, substitution urethroplasty and ocular surface reconstruction.
  • the scaffold membrane may be used as a cell-free construct for reconstruction of oral mucosa, ocular surface/conjunctival defect, cartilage defects, skin or ear tympanic membrane.
  • the scaffold is used to generate synthetic tissue in-vitro. This may be achieved by seeding the first face of the scaffold with cells and the second face of the scaffold with cells.
  • the cells may be the same or different.
  • the first face is seeded with keratinocytes, and the second face of the scaffold with fibroblasts.
  • the synthetic tissue produced in this way may comprise a tissue scaffold as supporting a layer of epithelial tissue at its first face and a layer of connective tissue at its second face. Over time, the scaffold may dissolve, leaving a sample of synthetic tissue.
  • the synthetic tissue generated in this way may be used for many applications. For example, the synthetic tissue may be used in laboratory tests to gain an insight into the properties of natural tissue, or to investigate the interaction of such tissue with drugs or chemicals. Alternatively, the synthetic tissue may be used to treat wounds in a human or animal body.
  • the synthetic tissue may be barrier tissue, for example, full-thickness barrier tissue. Examples include oral mucosal and skin regeneration. [0065] Aspects of the present invention may now be described, by way of example, with reference to Figures 1 to 3.
  • Figures 1 to 3 are schematic views of an example of tissue scaffold according to an embodiment of the present disclosure. Also shown in the Figures is a Cartesian coordinate system, which is used consistently throughout.
  • the tissue scaffold comprises a substrate 10 comprising six polymer layers. Each layer comprises a plurality of polymer strands 12. The top two polymer layers define a first region or a first lattice portion 14, while the bottom four layers define a second region or second lattice portion 16.
  • the first lattice portion 14 comprises two layers of polymer strands 12.
  • the polymer strands 12 in the first (uppermost) layer are disposed at an angle of 45 degrees to the z direction.
  • the polymer strands 12 in the second layer are disposed at an angle of 135 degrees to the z direction.
  • the polymer strands 12 in the first layer are in contact with the polymer strands 12 in the second layer.
  • the distance between the polymer strands 12 (central axis to central axis) in each of these layers is approximately 0.6 microns.
  • the second lattice portion 16 comprises four layers of polymer strands 12.
  • the polymer strands 12 in the layer (third layer) in contact with the second layer of the first lattice portion are disposed at an angle of 0 degrees to the z direction.
  • the polymer strands 12 in the next (i.e. fourth) layer are disposed at an angle of 90 degrees to the z direction.
  • the polymer strands 12 in the fifth layer are disposed at an angle of 0 degrees to the z direction, while the polymer strands 12 in the sixth layer are disposed at an angle of 90 degrees to the z direction.
  • the distance between the polymer strands 12 (central axis to central axis) in the third and fourth layers is approximately 0.6 microns.
  • the distance between the polymer strands 12 (central axis to central axis) in the fifth and sixth layers is approximately 0.9 microns.
  • the varying angles and distances between the polymer strands provide the gradient in pore size across the thickness of the substrate (y direction).
  • a scaffold according to an embodiment of the invention may include a substrate having fewer or more layers.
  • the polymer strands 12 may be angled at different angles to those shown in Figures 1 to 3.
  • Gelatin Type A, from porcine skin, Bioreagent grade
  • Gelatin was purchased from Sigma- Aldrich.
  • EDC 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide
  • NHS N-Hydroxysuccinimide
  • Envisiontec 3D-Bioplotter® (Manufacturer Series, Germany) is used for the 3D printing of scaffolds.
  • the high resolution/high magnification imaging of the scaffolds was performed using JEOL-JSM Scanning Electron Microscope (SEM).
  • SEM Scanning Electron Microscope
  • the SEM microscopy was performed in vacuum condition and samples were coated with a conductive materials (i.e., gold coating in our case). Accelerating voltage of 5kV was used for the SEM imaging.
  • a 15% (w/v) solution of gelatin in deionized (Dl) water was prepared for printing.
  • Gelatin solution was printed at material container temperature of 37°C and printing platform temperature of 22°C using 250 needle. Printing pressure and speed were adjusted to 0.6 bar and 15 mm/s, respectively.
  • Figure 4 is an SEM image of the printed scaffold.
  • angles of the strand-printing in the first and second layers was altered to 45° and 135° instead of 0° and 90° resulting in different surface structure and smaller pore size.
  • Figure 5 is an SEM image of such an alternative.
  • the scaffolds were crosslinked by soaking the scaffolds in a 12mg/ml EDC and 1.5mg/ml NHS in 90% ethanol solution and kept there for 4 hours.
  • the scaffolds were washed by repeatedly soaking the scaffold in large container of Dl water (i.e., 500 ml container) and rinsing the scaffold with Dl water. It was important to remove any residual crosslinking agent as such agents can have a detrimental effect on cell- culturing.
  • the scaffolds were stored in 90% ethanol and frozen.
  • the scaffolds can be brought to room temperature and washed in deionised water prior to use.
  • Elastin with molecular weight of 60KDa (Elastin-Soluble, No. ES12) was purchased from Elastin Products Company, Inc. Sodium Hyaluronate (Research Grade, 500KDa- 749KDa).
  • Gelatin Type A, from porcine skin, Bioreagent grade
  • EDC 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide
  • NHS N-Hydroxysuccinimide
  • Table 1 Sample specification for the primary rheometry experiment.
  • the w/v refers to the weight of the materials in Dl water.
  • the gel point of the gelatin-containing solutions can be important in the printing process.
  • a sol-gel may be required to maintain the shape on the platform after printing.
  • the gel point of 15% gelatin solution was found to be around 31 C. Addition of elastin and sodium hyaluronate to 15% gelatin solution resulted in a decrease of the gel point to around 29 C. The gel point was further decreased to 26 C by reducing the concentration of gelatin to 8%, while keeping the concentration of elastin and sodium hyaluronate constant.
  • Viscosity of the ink may also an important parameter for the printing process. Very low or high viscosities may make a solution unprintable.
  • Sample 3 was found to exhibit desirable gel point and viscosity characteristics for printing.
  • an Envisiontec 3D-Bioplotter® printer (Manufacturer Series, Germany) was employed for the 3D printing of scaffolds.
  • the camera of the 3D- Bioplotter® was utilized for the imaging of each layer during the process.
  • the thickness of the substrates was measured using a Marathon Electronic Digital Micrometer. The thickness values were confirmed by scanning the edge of the substrate using the 3D Laser Measuring Microscope.
  • Dynamic mechanical analyser (DMA 8000, Perkin Elmer) was used to measure the tensile storage modulus of the printed films after crosslinking.
  • the selected solution (8% Gelatin/ 2% Elastin/ 0.5% Sodium Hyaluronate (w/v) in Dl water) was printed at material container temperature of 30-32°C, platform temperature of 1 1°C, printing pressure of 0.6-1.2 bar, speed of 20 mm/s using 250 needle. Pre- and post-flow delays were set to zero.
  • Each substrate contained 6 layers with the strand angels of 45, 135, 0, 90, 0, 90° for layers 1 to 6, respectively. Distances between strands were set to 0.6 ⁇ for the first 4 layers and 0.9 ⁇ for the last 2 layers.
  • the printed substrate was crosslinked by soaking it in a 6 mg/ml EDC and
  • the substrate was soaked in large amounts of Dl water (0.5-1 liter) at least three times for 15 minutes each time and then rinsed by Dl water.
  • the rinsed substrate was bendable and flexible, with easy surgical handling.
  • the thickness of the substrate based on the measurement with the Electronic Digital
  • Micrometer was approximately 100-200 ⁇ . Note that, if the crosslinking had been done immediately after the 3D printing, the thickness would be greater (toward 200 ⁇ ). By extending the time between 3D printing and crosslinking, thinner substrates could be produced (toward 100 ⁇ ). [00105] The substrate was stored in 100% ethanol in a freezer. The ethanol removed moisture from the substrate, rendering it rigid. However, once the substrate was soaked in water, it became flexible and retained its original shape and quality.
  • the average values of the roughness (Ra: Arithmetical mean deviation of the roughness profile) of the small-pore-side and large-pore-side was measured to be about 0.30 and 1.19 ⁇ , respectively.
  • the small pores had an average pore size of about 140 ⁇ .
  • the large pores had an average pore size of about 440 ⁇ .
  • the tensile modulus of the substrate (200 ⁇ thickness) was 2.3 MPa.
  • the substrate had a tensile strength of 1.38 MPa and a maximum elongation at break of 65% of its initial length.
  • DMA was used to measure the tensile storage modulus of the printed substrates after crosslinking. The frequencies in the range of 0-25 Hz were sweeped at room
  • the storage modulus did not change significantly with frequency change in the scanned range. Note that increasing the temperature to 37 °C did not have a significant impact on the storage modulus.
  • the tensile storage modulus was found to be around 300 kPa.
  • DAPI nuclei staining Frozen sections of the membranes with cells were prepared and the slides were washed with PBS 3 times and mounted using mounting medium containing 4,6-diamidino-2-phenylindole (DAPI) stain (Sigma Chemical Co.) and assessed by fluorescence microscopy to image the cells. The shiny cell nuclei were only visible on the surface of the membrane and were not detected within the membrane showing ideal GTR barrier
  • the seeded substrate was characterized using basic histology. For this purpose, the substrate was fixed in 10% (v/v) PBS-buffered formalin for 24 hours, and decalcified with formic acid for the same period.
  • the specimen was processed overnight using a bench top tissue processor (Shandon Citadel 2000, Thermo Scientific) and embedded in paraffin wax using a Leica EG1 160 embedding centre (Leica Microsystems). Then, 10 ⁇ sections were prepared, stained with haematoxylin and eosin, and examined using inverted microscope equipped with a digital camera (Olympus, Japan). The histology images showed that oral keratinocytes proliferated and formed an epithelial layer on the surface of the 3D-printed membrane with no sign of invasion into the deeper layers of the membrane showing the optimal barrier function of the membrane. Oral fibroblasts also proliferated on the other side of the membrane and formed multilayers of cells separated from the epithelial layer by the membrane.
  • Example 2 The results suggest that the 3D-printed scaffolds produced in Example 2 is able to achieve the desired barrier function of a GTR membrane in vitro by complete separation of the oral epithelial layer from the underlying tissues allowing growth and proliferation of the other cell types under the connective tissue layer.
  • the selected solution was printed at material container temperature of 32 °C, platform temperature of 12 °C, printing pressure of 0.7 bar, speed of 20 mm/s using a 250 needle. Pre- and post-flow delays were set to zero. Each membrane was composed of 6 layers with strand angles of 45, 135, 0, 90, 0 and 90°.
  • membranes were printed using the ink of Sample 3 above for use in ocular surface/conjunctival defect reconstruction.
  • the final membranes were fabricated using a 3D-bioplatter (EnvisionTEC, Germany).
  • the ink was printed at material container temperature of 32 °C, platform temperature of 12 °C, printing pressure of 0.7 bar, speed of 20 mm/s using a 250 needle.
  • Each membrane was composed of 6 layers with strand angels of 45, 135, 0, 90, 0 and 90°. Distances between strands were set to 0.6 ⁇ for the first 4 layers and 0.9 ⁇ for the last 2 layers.
  • amniotic membranes have the same pore structure throughout.
  • MTT assay was used to assess cell viability and proliferation on the proposed membrane.
  • Human limbal epithelial progenitor cells (LEPCs) at a plating density of 5 ⁇ 10 3 cells/well were seeded on membranes in 96 well plate cell cultures.
  • 20 ⁇ _ of MTT (Sigma, US) substrate (of a 2.5 mg/ml stock solution in phosphate-buffered saline) was added to each well, and then all the plates were returned to standard tissue incubator conditions for an additional 4 hours.
  • DMSO dimethyl sulfoxide
  • Defect location was first marked by 8 mm vacuum trephine, coloured with surgical ink at edge, on the supra-temporal conjunctiva of both eyes of each rabbit. Conjunctive and Tenon were excised with scissors to the level of bare sclera making sure that no Tenon is left. All defects maintained a 2-mm distance form corneal limbus. Special care was exercised so that induced defects only involve the bulbar conjunctivae, and not extend into forniceal area. The use of anchoring sutures, required in surgeries involving fornix reconstruction was not in the scope of this study.
  • 3D-printed Gelatin-based membranes and AM were marked with the same vacuum trephine described above and cut into 8 mm round pieces with scissors.
  • the AM (epithelium side up) and 3D-printed gelatin-based membrane were secured to episclera with four Nylon 6.0 (Supa, Tehran, IR) sutures (one at the limbal and the other at the forniceal side of the defect) to prevent dislodging and the remaining surface was adhered to the bare sclera with fibrin glue (Beriplast ® P Combi Set, CSL Behring, USA). Both implants covered the bare area completely. The edges of the conjunctivae were slightly pulled to meet the edges of the membranes or AM.
  • the 3D-printed gelatin-based membrane studied in this investigation had transparency and (potentially) geometrical conformity.
  • the latter is, since those membranes, which were made using 3D printing in this investigation, can be fabricated in any shape (conforming to a given forniceal area shape) leveraging inherent capability of 3D printing in making complex geometries. Conformity is particularly a crucial factor in conjunctival reconstruction within forniceal area since placing anchoring sutures would be very difficult, if not impossible, if membrane does not conform to the shape of fornix.
  • Transparency is an advantage in terms of better cosmetic results; materials such as poly(lactic-co-glycolic acid)(PLGA) and Collagen-Glycosaminoglycan (CG) are not transparent, and AM has been reported to result in a blunted conjunctival appearance, and decreased clarity until complete degradation.
  • materials such as poly(lactic-co-glycolic acid)(PLGA) and Collagen-Glycosaminoglycan (CG) are not transparent, and AM has been reported to result in a blunted conjunctival appearance, and decreased clarity until complete degradation.
  • Table 1 captures clinical outcomes for the 2 nd week.
  • the mean defect diameter was 8.6 ⁇ 2.6 mm (3-10 mm), 5.8 ⁇ 2.5mm (3-10 mm) and 0.0 mm for 3D-printed gelatin-based membrane, AM and ungrafted eyes, respectively.
  • No significant difference was observed between AM and 3D-printed gelatin-based membrane groups (p 0.42) while they were both significantly different from the control group (p ⁇ 0.01).
  • Ungrafted wounds were completely epithelized. Seven out of eight eyes grafted with AM, showed epithelialization and vascularization at edges of the wound and one eye had no
  • Table 2 captures clinical outcomes for 3rd week.
  • Six out of eight eyes grafted with AM showed complete epithelialization, one had a 3-mm defect, and one eye had no epithelialization. Complete epithelialization was also observed in six out of eight eyes implanted with 3D-printed gelatin-based membrane, while two other eyes showed defects of 6 and 1 mm in size.
  • three eyes in AM group showed zero-mild inflammation and three eyes were still showing severe inflammation while six out of eight eyes grafted with 3D-printed gelatin-based membrane had zero-mild inflammation, and two eyes had severe inflammation (Table 2).
  • inflammation was mainly seen in the surrounding host conjunctivae.
  • Table 3 captures clinical outcomes for 4 th week. On day 28 th after the surgery, all grafted and ungrafted wounds had completely epithelialized. Mean inflammatory response score showed a significant difference between all groups (p ⁇ 0.01) and between treated groups (p ⁇ 0.01). The mean score was 0.0, 0.37 ⁇ 0.51 (range 0-1), and 1.5 ⁇ 0.92 (range 0-3) for the control group, eyes grafted with 3D-printed gelatin-based membranes and AM, respectively. Wounds grafted either with AM or 3D-printed gelatin-based membrane were still showing infiltration with white debris that was again confirmed to be composed of acute inflammatory cells in pathology and negative for microbial tests.
  • AM-grafted eyes were still more strongly infiltrated.
  • the 3D-printed gelatin-based membrane and AM were not totally visible under the completely epithelialized surface except in two eyes in the 3D-printed gelatin-based membrane group wherein the membranes were visible as sub-conjunctival prominences without significant inflammation of the adjacent conjunctivae.
  • the covering conjunctivae showed gross remodelling and scar tissue formation in both control eyes, three of the eight 3D-printed gelatin-based membrane grafted eyes and six of the eight AM grafted eyes.
  • the gelatin-based membrane-grafted group consistently showed milder degrees of clinically observed inflammation throughout the healing period compared to AM grated eyes.
  • H&E staining was done at the end of week 4 to assess the characteristics of the repaired conjunctivae. Complete epithelialization of the defect was seen in all groups (continuous).
  • the repaired epithelium consisted of 3-5 layers of epithelial cells in eight of the gelatin-based membrane grafted and seven of the AM grafted eyes. All eyes grafted whether with gelatin-based membrane or AM showed normal cuboidal basal cells and flattening of the epithelial cells towards the surface.
  • FIG. 6 shows the results of H&E staining.
  • Gelatin-based membrane grafted eyes are shown in (A & A').
  • 3-5 layers of epithelial cells are seen in the repaired epithelium with normal cellular morphology.
  • AM amniotic membrane
  • B & B' there are also 3-5 layers of epithelial cells (double arrow).
  • squamous metaplasia of epithelium large cells with increased nuclear to cytoplasmic ratio
  • remnant AM can be seen in subepithelial stroma (yellow arrowhead) with adjacent inflammatory reaction (B). Severe inflammatory reaction in site of transplantation ( ⁇ ').
  • ⁇ ' Severe inflammatory reaction in site of transplantation
  • Membranes showed complete degradation in six of eight of gelatin-based membrane grafted eyes and membrane remnants were seen in three eyes, while only one eye in the AM group showed complete degradation and AM remnant was seen in seven of eight eyes grafted with AM.
  • Histiocytes and giant cells suggestive of granulomatous response were present in seven of the eight AM grafted eyes, while being present in one of the gelatin-based membrane grafted eyes (p ⁇ 0.001 between all three as well as two treated groups) (table 5).
  • Figure 7 shows host's inflammatory response towards remaining 3-D printed membrane and AM. Macrophages (arrow) are shown digesting the 3-D printed membrane without an adjacent significant inflammatory response (asterisk) (A & A' & A"). Multiple giant cells (arrow) along with a severe inflammatory response (asterisk) composed of monocyte, macrophage and mast cells are seen around the remaining AM (B & B' & B").
  • Figure 8 shows the results of PAS staining. AM grafted eyes are shown in
  • A&A'&A There are fewer than 10 goblet cells (arrows) per 100 cells along the repaired epithelium. Remnant amniotic membrane (arrow head in A') is seen in the sub-epithelial area with adjacent inflammatory cells and fibrosis. 3D-printed gelatin-based membrane grafted eyes are shown in B&B'&B". 10 or more goblet cells per 100 cells are seen in the repaired epithelium. Subepithelial stoma shows no abundant inflammatory cell infiltration.
  • Figure 9 show masson trichrome staining and IHC (immunohistochemistry) for a- SMA (smooth muscle acti): 3D-printed membrane grafted eyes show random alignment of collagen fibers (asterisk) (see A&A'), and no cells staining positive for a-SMA cells (D&D'). Amniotic membrane grafted eyes show parallel deposition of collagen (arrow) along the AM remnant (arrow head) ( ⁇ & ⁇ ') and numerous elongated cells staining positive for a-SMA (E&E' ). A thick layer of densely packed collagen fibers was seen in both ungrafted eyes (C&C).
  • Immunohistochemical staining for a-SMA was done to evaluate and compare the presence of myofibroblasts (cells with contractile potentials) in study groups. Smooth muscle cells of adjacent extraocular muscles and vascular pericytes stained positive for a-SMA. Cells staining positive for a-SMA which had an elongated and fibroblast like morphology, were considered myofibroblasts (potentially contractile cells). Myofibroblast appearing cells were seen in four of 3D-printed gelatin-based membrane grafted eyes, two contained many a-SMA positive staining cells and two showed a moderate infiltration of cells in stroma.
  • the results show that effective epithelial tissue growth can be achieved using the 3- D printed membranes of Example 5.
  • the pore structure of the 3-D printed membrane is advantageous.
  • the epithelial tissue-supporting face of the membrane is smooth and has a small pore size configured to facilitate epithelial cell propagation and growth.
  • the opposite face of the membrane has a larger pores size, facilitating growth of the underlying tissue. This allows the underlying tissue to grow, providing the epithelial cells with an adequate nutrient supply, gas diffusion and adequate metabolic waste removal.
  • the small pore size in one side provides the surface required for the attachment of epithelial cells, and the large pore size on the other side facilitates the effective nutrient supply, effective gas diffusion and effective metabolic waste removal.
  • a tissue scaffold comprising a porous substrate comprising a biocompatible polymer, said substrate having a first face and a second face opposing the first face, wherein the first face is either non-porous or porous and the second face is porous, wherein, when the first face is porous, the pore structure at the first face is different from the pore structure at the second face.
  • a scaffold as defined in paragraph 1 wherein the first face is porous and the average pore size at the first face is less than the average pore size at the second face.
  • a scaffold as defined in paragraph 1 wherein the first face is porous and the porosity at the first face is less than the porosity at the second face.
  • a scaffold as defined in paragraph 1 wherein the average pore size at the first face is 0 to 200 microns and the average pore size at the second face is 250 to 700 microns.
  • scaffold as defined in paragraph 5 wherein the first face is porous and the pore size or porosity of the layers increases from the first face to the second face of the substrate.
  • a scaffold as defined in paragraph 8 wherein the first region and the second region are adjacent and in contact with one another.
  • the three-dimensional framework is formed from overlapping polymer layers, wherein each polymer layer comprises an array of polymer strands, whereby the polymer strands of one layer overlap the polymers strands of an adjacent layer at an angle so as to form a three-dimensional lattice framework.
  • a scaffold as defined in paragraph 10 or 1 1 wherein the first region is formed from a first lattice portion and the second region is formed from a second lattice portion, and wherein first lattice portion overlays the second lattice portion at an angle.
  • Engineered tissue comprising a tissue scaffold as defined in any one of the preceding paragraphs.
  • Engineered tissue as defined in paragraph 21 which is full thickness oral mucosa.
  • Engineered tissue as defined in paragraph 21 which is bone tissue.
  • a method of guided tissue regeneration comprising placing the second face of a tissue scaffold as claimed in any one of paragraphs 1 to 20 over an exposed area of tissue and optionally seeding the first face of the tissue scaffold with cells.

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Abstract

Un échafaudage tissulaire comprenant un substrat poreux comprenant un polymère biocompatible, ledit substrat ayant une première face et une seconde face opposée à la première face, la première face étant soit non poreuse, soit poreuse et la seconde face étant poreuse, lorsque la première face est poreuse, la structure de pores au niveau de la première face est différente de la structure de pores au niveau de la seconde face.
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CN108434522A (zh) * 2018-06-15 2018-08-24 天津工业大学 一种表层包埋细胞的可降解生物相容性水凝胶膜的制备方法
WO2020234167A1 (fr) * 2019-05-17 2020-11-26 ETH Zürich Hydrogel imprimable, procédé de génération d'un hydrogel imprimable, lyophilisat, produit imprimé et procédé d'impression 3d
CN110302425A (zh) * 2019-06-20 2019-10-08 温州医科大学附属第一医院 混合水凝胶生物材料的制备方法及其应用
EP4227399A1 (fr) * 2022-02-14 2023-08-16 Fundación para la Investigación Biomédica del Hospital Universitario de la Paz Constructions tissulaires artificielles pour l'ophtalmologie et procédés pour les obtenir et pour leur utilisation
WO2023152401A1 (fr) * 2022-02-14 2023-08-17 Fundación Para La Investigación Biomédica Del Hospital Universitario La Paz Constructions artificielles destinées à être utilisées en ophtalmologie, leurs procédés d'obtention et leur utilisation

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