WO2013093921A1 - Fibres de polymère synthétique enrobées de collagène - Google Patents

Fibres de polymère synthétique enrobées de collagène Download PDF

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Publication number
WO2013093921A1
WO2013093921A1 PCT/IL2012/050543 IL2012050543W WO2013093921A1 WO 2013093921 A1 WO2013093921 A1 WO 2013093921A1 IL 2012050543 W IL2012050543 W IL 2012050543W WO 2013093921 A1 WO2013093921 A1 WO 2013093921A1
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collagen
fiber
fibers
synthetic polymer
isolated
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PCT/IL2012/050543
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English (en)
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Oded Shoseyov
Sigal SHARON
Shaul Lapidot
Yuval Nevo
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Collplant Ltd.
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Publication of WO2013093921A1 publication Critical patent/WO2013093921A1/fr

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    • D—TEXTILES; PAPER
    • D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
    • D06M—TREATMENT, NOT PROVIDED FOR ELSEWHERE IN CLASS D06, OF FIBRES, THREADS, YARNS, FABRICS, FEATHERS OR FIBROUS GOODS MADE FROM SUCH MATERIALS
    • D06M15/00—Treating fibres, threads, yarns, fabrics, or fibrous goods made from such materials, with macromolecular compounds; Such treatment combined with mechanical treatment
    • D06M15/01—Treating fibres, threads, yarns, fabrics, or fibrous goods made from such materials, with macromolecular compounds; Such treatment combined with mechanical treatment with natural macromolecular compounds or derivatives thereof
    • D06M15/03—Polysaccharides or derivatives thereof
    • D06M15/05—Cellulose or derivatives thereof
    • 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
    • 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
    • A61L27/28—Materials for coating prostheses
    • A61L27/34—Macromolecular materials
    • 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
    • A61L27/3834—Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem 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
    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
    • A61L31/04—Macromolecular materials
    • 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
    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
    • A61L31/08—Materials for coatings
    • A61L31/10—Macromolecular materials
    • D—TEXTILES; PAPER
    • D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
    • D01F—CHEMICAL FEATURES IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS; APPARATUS SPECIALLY ADAPTED FOR THE MANUFACTURE OF CARBON FILAMENTS
    • D01F8/00—Conjugated, i.e. bi- or multicomponent, artificial filaments or the like; Manufacture thereof
    • D—TEXTILES; PAPER
    • D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
    • D06M—TREATMENT, NOT PROVIDED FOR ELSEWHERE IN CLASS D06, OF FIBRES, THREADS, YARNS, FABRICS, FEATHERS OR FIBROUS GOODS MADE FROM SUCH MATERIALS
    • D06M15/00—Treating fibres, threads, yarns, fabrics, or fibrous goods made from such materials, with macromolecular compounds; Such treatment combined with mechanical treatment
    • D06M15/01—Treating fibres, threads, yarns, fabrics, or fibrous goods made from such materials, with macromolecular compounds; Such treatment combined with mechanical treatment with natural macromolecular compounds or derivatives thereof
    • D06M15/15—Proteins or derivatives thereof

Definitions

  • the present invention in some embodiments thereof, relates to collagen coated synthetic polymers for tissue repair and regenerative medicine and fabrics comprising same.
  • Synthetic biomaterials are generally more biologically inert than natural biomaterials. They have more predictable properties and batch-batch uniformity as well as having the unique advantage of having tailored property profiles for specific applications.
  • Polymeric fibers are considered attractive materials for the fabrication of biomedical devices such as temporary prostheses, sensors and drug and enzymes carriers due to their flexibility and reliability. It is believed that it is the combination of the interconnected pores of the polymeric fibers, the high versatility of the fiber surface and the ability to control fiber and textile processing to a high degree, which enables the tailoring of specific textile engineering design to provide the desired mechanical properties.
  • PLA Poly(lactic acid)
  • PLA is a relevant bio-absorbable polymer that has been used as a scaffold material due to its biocompatibility and thermoplastic characteristics.
  • PLA has good tensile strength, low extension and a high modulus (approximately 4.8 GPa). Accordingly, it has been considered as an ideal biomaterial for load bearing applications, such as orthopaedic fixation devices.
  • PLA polymers may form a range of different desired shapes by various techniques including molding, fiber extrusion, and solvent casting depending on the application. Compared with other popular bio- absorbable polymers such as Poly(glycolic acid) (PGA), it is relatively more hydrophobic with a longer degradation period (more than 24 months).
  • PGA Poly(glycolic acid)
  • Poly(glycolic acid) was one of the first biodegradable synthetic polymers investigated for biomedical applications.
  • This polymer is a highly crystalline polymer and therefore exhibits a high tensile modulus with very low solubility in organic solvents.
  • PGA has been fabricated into a variety of forms and structures. Extrusion, injection and compression molding as well as particulate leaching and solvent casting, are some of the techniques used to develop PGA-based structures for biomedical applications. Due to its high rate of degradation, acidic degradation products and low solubility, several copolymers of lactides and glycolides have been developed so as to form polymers with increased property modulation.
  • PCL Polycaprolactone
  • a drawback of fabrication of biomedical devices from synthetic polymers is their lack of necessary specific bioactive abilities to accelerate extra cellular matrix (ECM) secretion and regeneration of cultured cells.
  • ECM extra cellular matrix
  • natural biomaterials such as collagen, allow rapid cell expansion.
  • Collagen is the most widely utilized natural polymer for biomedical applications and tissue engineering due to its excellent biocompatibility, biodegradability and safety.
  • animal-derived collagen is problematic due to the possible risks of contamination by non-conventional infectious agents. While the risks raised by bacterial or viral contamination can be fully controlled, prions are less containable and present considerable health risks. These infectious agents which appear to have a protein-like nature, are involved in the development of degenerative animal encephalopathy (sheep trembling disease, bovine spongiform encephalopathy) and human encephalopathy (Creutzfeld-Jacob disease, Gerstmann-Straussler syndrome, and kuru disease). Due to the lengthy time before onset of the disease, formal controls are difficult to conduct.
  • Cellulose is one of the most abundant polymers on earth. It can be found in all plants and it can also be produced by certain bacteria and sea animals. Cellulose is a polysaccharide mainly composed of cellobiose units linked together by ⁇ - 1,4- glycosidic linkages. Various models have been proposed to explain the structure of cellulose in the plant cell wall, but the most accepted explanation is that due to the linearity of the cellulose backbone, chains form a framework of elementary microfibrils with crystalline and amorphous regions.
  • cellulose nanocrystals Due to its high modulus of elasticity (MOE), calculated as 138 GPa, crystalline cellulose has been exploited as a reinforcement agent for a variety of composites during acid hydrolysis, the amorphous domains of the microfibrils are degraded, resulting in the preservation of crystallites which are called cellulose nanocrystals (CNs).
  • the size of these nanocrystals varies, depending upon the source from which they were obtained; but usually they are in the size range of 100- 1000 nm in length and 3-50 nm in width.
  • the reinforcing ability of CNs lies in their high surface area and good mechanical properties.
  • Cellulose nanocrystals have a broad variety of applications such as reinforcement agents for plastic composites, use in sensors, smart materials, membranes, textiles, electro-optic devices as well as biomedical purposes.
  • U.S. Patent application No. 20020131989 teaches implanted degradable devices fabricated from a polymeric fibrous matrix.
  • the matrix may be coated with a number of alternative adhesive biological factors including collagen.
  • an internal core which comprises a synthetic polymer
  • a method for treating a hernia or uterovaginal prolapse in a subject in need thereof comprising making an incision into an affected area of the subject, placing the implantable device of the present invention onto the affected area, and securing the device to the affected area, thereby treating the hernia or uterovaginal prolapse.
  • a method of generating the fiber the present invention comprising:
  • a fabric comprising the isolated fibers described herein.
  • a scaffold comprising the isolated fibers described herein.
  • an electrospun element comprising the isolated fiber described herein.
  • an implantable device comprising the fiber described herein.
  • the collagen comprises recombinant collagen.
  • the isolated fiber further comprises an adhesive layer situated between the internal core and the intermediate layer.
  • the adhesive layer comprises gelatin.
  • a diameter of the internal core is between 50-150 ⁇ .
  • the thickness of the intermediate layer is between 0.1-10 ⁇ .
  • the thickness of the outer layer is between 1-11 ⁇ .
  • the recombinant collagen is human collagen.
  • the recombinant collagen is generated in plants.
  • the synthetic polymer is biodegradable.
  • the biodegradable synthetic polymer is selected from the group consisting of polylactic acid (PLA), polyglycolic acid (PGA), polycaprolactone (PCL), poly(Lactide-co-Glycolide) (PLGA), polydioxanone (PDO), trimethylene carbonate (TMC), polyethyleneglycol (PEG) and a combination of same.
  • the synthetic polymer is nonbiodegradable.
  • the non-biodegradable synthetic polymer is selected from the group consisting of polyurethane, polycarbonate, polyacrylonitrile, polyethyleneoxide, polyaniline, polyvinyl carbazole, polyvinyl chloride, polyvinyl fluoride, polyvinyl imidazole, polyvinyl alcohol, polystyrene and poly(vinyl phenol), aliphatic polyesters, polyacrylates, polymethacrylate, acyl- sutostituted cellulose acetates, non-biodegradable polyurethanes, polystyrenes, chlorosulphonated polyolifins, polyethylene oxide and polytetrafluoroethylene.
  • the collagen comprises liquid crystal collagen.
  • the collagen comprises recombinant collagen.
  • the method further comprises crosslinking the collagen following step (b).
  • the method further comprises fibrillating the collagen following step (b). According to some embodiments of the invention, the method further comprises contacting the synthetic polymer fiber with gelatin prior to step (a).
  • the method further comprises plasma treating the outer surface of the synthetic polymer fiber prior to step (a).
  • the plasma treating comprises oxygen plasma treating or ammonia plasma treating.
  • the scaffold is seeded with cells.
  • the cells comprise stem cells.
  • the stem cells comprise mesenchymal stem cells.
  • the implantable device is a surgical mesh.
  • the implantable device is configured for pelvic floor repair.
  • the implantable device is configured for hernia repair.
  • the implantable device is configured for plastic surgery.
  • the implantable device is configured for breast reconstruction.
  • the implantable device is configured for urinary or fecal incontinence repair.
  • the implantable device is configured for cardiovascular procedures.
  • the implantable device further comprises a bioactive agent selected from the group consisting of antimicrobials, antibacterials, anti-fungals, antibiotics, anti-viral agents, analgesics, antiadhesives, anesthetics, anti-inflammatories, antispasmodics, hormones, growth factors, muscle relaxants, antineoplastics, immunogenic agents, immunosuppressants, steroids, lipids, narcotics, lipopolysaccharides, polysaccharides, polypeptides, enzymes, and combinations thereof.
  • a bioactive agent selected from the group consisting of antimicrobials, antibacterials, anti-fungals, antibiotics, anti-viral agents, analgesics, antiadhesives, anesthetics, anti-inflammatories, antispasmodics, hormones, growth factors, muscle relaxants, antineoplastics, immunogenic agents, immunosuppressants, steroids, lipids, narcotics, lipopolysacchari
  • FIGs. 1A-E are scanning electron microscopy (SEM) micrographs of PLA fibers (A) poly(lactic acid) (PLA) fiber with nanocellulose crystal (NCC) coating without gelatin coating (B), gelatin-coated PLA fiber (C), gelatin/NCC-coated PLA fiber (D) and gelatin/NCC/rhcollagen -coated PLA fiber (E).
  • SEM scanning electron microscopy
  • the present invention in some embodiments thereof, relates to collagen coated synthetic polymers for tissue repair and regenerative medicine and fabrics comprising same.
  • Biodegradable synthetic polymers offer a number of advantages over other materials for developing scaffolds in tissue engineering.
  • the key advantages include the ability to tailor mechanical properties and degradation kinetics to suit various applications.
  • Synthetic polymers are also attractive because they can be fabricated into various shapes with desired pore morphologic features conducive to tissue in-growth.
  • polymers can be designed with chemical functional groups that can induce tissue in-growth.
  • the present inventors propose coating synthetic polymers with nano cellulose crystals (NCC) followed by coating with collagen.
  • NCC nano cellulose crystals
  • an isolated fiber comprising:
  • fiber refers to an elongated, thread-like structure having a characteristic longitudinal dimension, typically a "length”, and a characteristic transverse dimension, typically a “diameter” or a “width”, wherein the ratio of the characteristic longitudinal dimension to the characteristic transverse dimension is greater than or equal to about 50, more typically greater than or equal to about 100.
  • synthetic polymer fibers are initially coated in cellulose nanocrystals and subsequently in collagen.
  • the synthetic fibers are composed of polymers which may be a biodegradable or non-biodegradable or a mixture of both.
  • biodegradable synthetic polymers include, but are not limited to polylactic acid (PLA), polyglycolic acid (PGA), polycaprolactone (PCL), poly(Lactide-co-Glycolide) (PLGA), polydioxanone (PDO), trimethylene carbonate (TMC), polyethyleneglycol (PEG) and a combination of same.
  • non-biodegradable polymer examples include, but are not limited to polycarbonate, polyacrylonitrile, polyethyleneoxide, polyaniline, polyvinyl carbazole, polyvinyl chloride, polyvinyl fluoride, polyvinyl imidazole, polyvinyl alcohol, polystyrene and poly(vinyl phenol), aliphatic polyesters, polyacrylates, polymethacrylate, acyl- sutostituted cellulose acetates, non-biodegradable polyurethanes, polystyrenes, chlorosulphonated polyolifins, polyethylene oxide and polytetrafluoroethylene.
  • Fibers are created by forcing, usually through extrusion, synthetic polymers through holes (i.e. spinnerets) into the air, forming a thread.
  • the elongate fibers for use in the present invention can be formed by a number of methods well known in the art, including, but not limited to, melt- spinning, wet- spinning, dry-spinning, dry-jet wet spinning, electro spinning, or extrusion (Ziabicki, A. "Fundamentals of Fiber Formation,” Wiley, New York (1976); Kroschwitz, J. I., “Encyclopedia of Polymer Science and Engineering. Second Edition, Vol. 6. John Wiley & Sons. New York (1986), which are hereby incorporated by reference in their entirety).
  • melt- spinning the fiber material is usually melted and pumped through a spinneret (die) with numerous holes (one to thousands).
  • the molten fibers are cooled, solidified, and can be collected on a stick or on a take-up wheel.
  • a classic article which discusses structure development during melt spinning is: Dees et al., J. Appl. Polym. Sci., 18: 1053-1078 (1974).
  • Dry spinning also can be used to form fibers from a solution.
  • the fiber material is dissolved in a volatile solvent and the solution is pumped through a spinneret (die) with numerous holes (one to thousands).
  • a spinneret die
  • numerous holes one to thousands.
  • air is used to evaporate the solvent so that the fibers solidify and can be collected on a take-up wheel.
  • Stretching of the fibers provides for orientation of the polymer chains along the fiber axis.
  • a more detailed study of dry spinning is provided in Ohzawa et al. J. Appl. Polym. Sci., 13, pp. 257-283 (1969).
  • Fibers for the purposes of the present invention can also be produced by wet spinning.
  • Wet spinning is the one of the earliest process of producing fibers and can be used to make the fibers of the present invention. It is used for fiber-forming substances that have been dissolved in a solvent. The spinnerets are submerged in a chemical bath, and, as the filaments emerge, they precipitate from solution and solidify. Because the solution is extruded directly into the precipitating liquid, this process for making fibers is called wet spinning.
  • dry-wet spinning is a special process which can be used to obtain high strength or other special fiber properties.
  • the polymer is not in a true liquid state during extrusion. Not completely separated, as they would be in a true solution, the polymer chains are bound together at various points in liquid crystal form. This produces strong inter-chain forces in the resulting filaments that can significantly increase the tensile strength of the fibers.
  • the liquid crystals are aligned along the fiber axis by the shear forces during extrusion. The filaments emerge with an unusually high degree of orientation relative to each other, further enhancing strength.
  • the process can also be described as dry-wet spinning, since the filaments first pass through air and then are cooled further in a liquid bath.
  • Electro spinning can also be used for making fibers of the present invention.
  • the high surface area and high porosity of electrospun fibers allow favorable cell interactions and hence make them potential candidates for tissue engineering applications. It uses an electrical charge to draw very fine (typically on the micro or nano scale) fibers from a liquid.
  • Electro spinning shares characteristics of both electrospraying and conventional solution dry spinning of fibers. The process is non-invasive and does not require the use of coagulation chemistry or high temperatures to produce solid threads from solution. This makes the process particularly suited to the production of fibers using large and complex molecules.
  • the diameter of the synthetic polymer fiber is between 10-500 ⁇ , more preferably between 20-200 ⁇ and more preferably between 50-100 ⁇ ,
  • the next stage in the generation of the fibers of this aspect of the invention involves coating with nano cellulose crystals.
  • cellulose refers to the polysaccharide mainly composed of cellobiose units linked together by ⁇ - 1,4- glycosidic linkages and derivatives thereof.
  • exemplary derivatives include, but are not limited to carboxymethylcellulose, methylcellulose, hydroxyethylcellulose, hydroxypropylcellulose and hydroxypropylmethylcellulose or combinations thereof.
  • the cellulose may be derived from plants, bacteria or certain sea animals. According to a preferred embodiment, the cellulose is derived from plants - e.g. cotton or wood pulp.
  • Nanocrystals are typically generated during acid hydrolysis of the cellulose, wherein the amorphous domains of the microfibrils are degraded, resulting in the preservation of crystallites.
  • Exemplary acids that can be used to generate nano cellulose crystals from cellulose include, but are not limited to sulfuric, nitric and hydrochloric acid. According to one embodiment, the acid is sulfuric acid. Acid type, acid concentration, hydrolysis time and hydrolysis temperature are factors that have been shown to govern the products of the hydrolysis process. An exemplary protocol for generating nanocrystals from cellulose is described in the Examples section herein below.
  • the nanocellulose crystals may be chemically modified and subsequently dispersed in organic solvents.
  • the nanocellulose crystals may be dispersed in polar aprotic organic solvents, such as DMF and DMSO.
  • nanocellulose crystals to the outer surface of the synthetic polymeric fibers may be effected using any method known in the art, including for example, spraying, spreading, wetting, immersing, dipping, painting, ultrasonic welding, welding, bonding or adhering.
  • a typical layer of nanocellulose crystals may be between 0.1-10 ⁇ thick.
  • the synthetic polymeric fibers may optionally be pre-treated in order to enhance the ability of the CN to coat the fibers.
  • the polymeric fibers are pre-treated by coating with gelatin.
  • Gelatin is a derivative of collagen, a principal structural and connective protein in animals.
  • Gelatin can be derived from denaturation of collagen and contains polypeptide sequences having Gly-X-Y repeats, where X and Y are most often proline and hydroxyproline residues. These sequences contribute to triple helical structure and affect the gelling ability of gelatin polypeptides.
  • Gelatin can be obtained from an animal collagen source (e.g., bovine, porcine, chicken, equine, marine) sources, e.g., bones, skin, and tendons or may be recombinantly produced, as described herein below.
  • the gelatin is derived from a recombinant human collagen, generated in plants.
  • Methods, processes, and techniques of producing gelatin from collagen include denaturing the triple helical structure of the collagen utilizing detergents, heat or denaturing agents. Additionally, these methods, processes, and techniques include, but are not limited to, treatments with strong alkali or strong acids, heat extraction in aqueous solution, ion exchange chromatography, cross-flow filtration and heat drying, and other methods that may be applied to collagen to produce the gelatin.
  • the synthetic polymer fibers are pre-treated by exposure of the surface to a plasma.
  • a plasma is a partially ionized gas generated by applying an electrical field to a gas under at least a partial vacuum.
  • the plasma is generated by introducing the gas into a vacuum chamber and electromagnetic field.
  • the resulting plasma consists of ions and free electrons, free radicals, excited state species, photons and neutrals.
  • both the ions and electrons experience the same force and are accelerated. Collisions occur between these particles which transfer kinetic energy from one to the other. Since energy transfer in two body collisions favors the lighter particle (electrons in the case of plasma), the electrons soon have much greater velocity (i.e. temperature) than the ions.
  • the plasma is selected such that it incorporates high concentrations of positive charge on the fiber surface so as to create a stable bond the synthetic fiber and the negatively charged nano cellulose crystal layer.
  • exemplary plasmas include ammonia plasmas, nitrogen plasmas, oxygen plasmas and halogen plasmas.
  • the external layer of the fibers of the present invention comprises collagen.
  • collagen refers to a polypeptide having a triple helix structure and containing a repeating Gly-X-Y triplet, where X and Y can be any amino acid but are frequently the amino acids proline and hydroxyproline. According to one embodiment, the collagen is a type I, II, III, V, XI, or biologically active fragments therefrom.
  • a collagen of the present invention also refers to homologs (e.g., polypeptides which are at least 50 , at least 55 , at least 60 , at least 65 , at least 70 , at least 75 %, at least 80 %, at least 85 %, at least 87 %, at least 89 %, at least 91 %, at least 93 , at least 95 % or more say 100 % homologous to collagen sequences listed in Table 1 as determined using BlastP software of the National Center of Biotechnology Information (NCBI) using default parameters).
  • the homolog may also refer to a deletion, insertion, or substitution variant, including an amino acid substitution, thereof and biologically active polypeptide fragments thereof
  • the collagen of the present invention comprises a sufficient portion of its telopeptides such that under suitable conditions it is capable of forming fibrils.
  • the collagen may be atelocollagen, a telocollagen or procollagen.
  • atelocollagen refers to collagen molecules lacking both the N- and C-terminal propeptides typically comprised in procollagen, but including a sufficient portion of its telopeptides such that under suitable conditions it is capable of forming fibrils.
  • procollagen refers to a collagen molecule (e.g. human) that comprises either an N-terminal propeptide, a C-terminal propeptide or both.
  • exemplary human procollagen amino acid sequences are set forth by SEQ ID NOs: 3, 4, 5 and 6.
  • telocollagen refers to collagen molecules that lack both the N- and C-terminal propeptides typically comprised in procollagen but still contain the telopeptides.
  • the telopeptides of fibrillar collagen are the remnants of the N-and C-terminal propeptides following digestion with native N/C proteinases.
  • the collagen is a mixture of the types of collagen above.
  • the collagen may be isolated from an animal (e.g. bovine or pig) or from human cadavers or may be genetically engineered using recombinant DNA technology as further described herein below. According to a specific embodiment, the collagen is devoid of animal-derived (i.e. non-human) collagen.
  • the collagen is recombinant human collagen.
  • the recombinant human collagen is generated in plants, as further described herein below.
  • collagen is typically solubilized in an acid solution where it is present in its monomeric form (i.e. non-fibrillated form) prior to coating.
  • exemplary acids for solubilizing monomeric collagen include, but are not limited to hydrochloric acid (HC1) and acetic acid.
  • collagen monomers refers to monomeric collagen that has not undergone the process of polymerization.
  • the collagen may be present in the acid solution at a concentration of about 1- 100 mg/ml.
  • An exemplary concentration of HC1 which may be used to solubilize collagen monomers and generate liquid crystal collagen is about 10 mM HC1.
  • a concentration of about 0.05 mM - 50 mM acetic acid is used to solubilize the collagen monomers.
  • An exemplary concentration of acetic acid which may be used to solubilize collagen monomers is about 0.5 M acetic acid.
  • Generating solutions of liquid crystalline collagen monomers may be effected by concentrating a liquid collagen solution.
  • the liquid collagen solution may be concentrated using any means known in the art, including but not limited to filtration, rotary evaporation and dialysis membrane.
  • Dialysis may be effected against a hygroscopic polymer, for example, PEG, a polyethylene oxide, amylose or sericin.
  • PEG is of a molecular weight of 10,000-30,000 g/mol and has a concentration of 25-50 %.
  • a slide-a-lyzer dialysis cassette (Pierce, MW CO 3500) is used.
  • the dialysis is effected in the cold (e.g. at about 4 °C).
  • the dialysis is effected for a time period sufficient to result in a final concentration of aqueous collagen solution of about 10 mg/ml or more.
  • the solution of monomeric collagen is at a concentration of about 100-200 mg/ml or between 0.7-0.3mM.
  • the solution of liquid crystalline collagen comprises high concentrations (5-30 mg/ml, depending on the collagen type) of collagen molecules in physiological buffer. It has been shown that such solutions develop long range nematic and precholesteric liquid crystal ordering extending over 100 ⁇ domains, while remaining in solution (R. Martin et al., J. Mol. Biol. 301: 11-17 (2000)).
  • the starting collagen material may be prepared by ultrasonic treatment. Brown E. M. et al. Journal of American Leather Chemists Association, 101:274-283 (2006), herein incorporated by reference by its entirety.
  • a typical thickness of the outer collagen layer is between 1 - 1 ⁇ .
  • the collagen may optionally be fibrillated so as to generate fibrils and/or crosslinked.
  • fibrillogenesis refers to the precipitation of soluble collagen in the form of fibrils
  • Fibrillogenesis is entropy driven - the loss of water molecules from monomer surfaces drives the collagen monomers out of solution and into assemblies with a circular cross-section, so as to minimize surface area. Fibrillogenesis may be performed in a variety of ways including neutralization of the pH, increasing the temperature and/or the ionic strength.
  • An exemplary alkaline solution that may be added to increase the pH of the collagen is Na 2 HP0 4 (pH-11.2).
  • the amount of alkaline solution is calculated such that the final pH of the collagen is about 7-7.5 (e.g. 7.4).
  • the present invention further contemplates crosslinking the collagen following coating of the fibers using any one of the below methods: 1. by glutaraldehyde, N-ethyl- N'-[3-dimethylaminopropyl] carbodiimide (EDC) in the presence or absence of N- hydroxy sue cinimide (NHS), genipin or other chemical crosslinking agents; 2. by glycation using different sugars; 3. by Fenton reaction using metal ions such as copper; 4. by lysine oxidase; or 5. by UV radiation (for example in the presence of a photoinitiator such as 4-(2-hydroxyethoxy)phenyl-(2-hydroxy-2-propyl)ketone - Irgacure 2959).
  • a photoinitiator such as 4-(2-hydroxyethoxy)phenyl-(2-hydroxy-2-propyl)ketone - Irgacure 2959.
  • the fibers of the present invention may be braided, twisted, aligned, or otherwise joined to form a fabric.
  • at least two fibers may form a yarn for use in forming the fabric.
  • multiple fibers may be braided, twisted, aligned, or otherwise joined to form a multifiber yarn.
  • the fabric may be assembled from a plurality of fibers or yarns.
  • the fibers/yarns may be woven, knitted, interlaced, braided, or formed into a surgical mesh by non-woven techniques.
  • the fibers of the present invention may be used to fabricate a scaffold.
  • the term "scaffold” refers to a 3D matrix upon which cells may be cultured (i.e., survive and preferably proliferate for a predetermined time period).
  • the scaffolds of the present invention are typically sterilized, for example by oxygen plasma, following which they may be seeded with cells.
  • seeding refers to plating, placing and/or dropping the cells of the present invention into the scaffold of the present invention. It will be appreciated that the concentration of cells which are seeded on or within the scaffold depends on the type of cells used and the precise composition of the scaffold. Techniques for seeding cells onto or into a scaffold are well known in the art, and include, without being limited to, static seeding, filtration seeding and centrifugation seeding.
  • Static seeding includes incubation of a cell-medium suspension in the presence of the scaffold under static conditions and results in non-uniformity cell distribution (depending on the volume of the cell suspension); filtration seeding results in a more uniform cell distribution; and centrifugation seeding is an efficient and brief seeding method (see for example EP19980203774).
  • the cells may be seeded directly onto the scaffold, or alternatively, the cells may be mixed with a gel which is then absorbed onto the interior and exterior surfaces of the scaffold and which may fill some of the pores of the scaffold. Capillary forces will retain the gel on the scaffold before hardening, or the gel may be allowed to harden on the scaffold to become more self-supporting.
  • the cells may be combined with a cell support substrate in the form of a gel optionally including extracellular matrix components.
  • the cells may comprise a heterogeneous population of cells or alternatively the cells may comprise a homogeneous population of cells.
  • Such cells can be for example, stem cells (such as embryonic stem cells, bone marrow stem cells, cord blood cells, mesenchymal stem cells, adult tissue stem cells), progenitor cells (e.g. progenitor bone cells), or differentiated cells such as chondrocytes, osteoblasts, connective tissue cells (e.g., fibrocytes, fibroblasts and adipose cells), endothelial and epithelial cells.
  • the cells may be of autologous origin or non-autologous origin, such as postpartum-derived cells (as described in U.S. Application Nos. 10/887,012 and 10/887,446). Typically the cells are selected according to the tissue being generated.
  • stem cell refers to cells which are capable of differentiating into other cell types having a particular, specialized function (i.e., “fully differentiated” cells) or remaining in an undifferentiated state hereinafter “pluripotent stem cells”.
  • the fibers of the present invention may be used to fabricate an implantable device.
  • the fibers of the present invention may be used for pelvic floor reconstruction, urethral suspension (to prevent stress incontinence using the mesh as a sling), pericardial repair, cardiovascular patching, cardiac support (as a sock that fits over the heart to provide reinforcement), organ salvage, elevation of the small bowel during radiation of the colon in colorectal cancer patients, retentive devices for bone graft or cartilage, guided tissue regeneration, vascular grafting, dural substitution, nerve guide repair, as well as in procedures needing anti-adhesion membranes and tissue engineering scaffolds.
  • the fibers of the present invention could also find other uses, for example, in synthetic ligament and tendon devices or scaffolds. Further uses include combinations with other synthetic and natural fibers, meshes and patches.
  • the fibers and devices such as meshes and tubes derived from the fibers could be combined with autologous tissue, allogenic tissue, and/or xenogenic tissues to provide reinforcement, strengthening and/or stiffening of the tissue.
  • Such combinations could facilitate implantation of the autologous, allogenic and/or xenogenic tissues, as well as provide improved mechanical and biological properties.
  • Combination devices could be used for example in hernia repair, mastopexy/breast reconstruction, rotator cuff repair, vascular grafting/fistulae, tissue flaps, pericardial patching, tissue heart valve implants, bowel interposition, and dura patching.
  • the implantable device is a surgical mesh.
  • surgical mesh refers to a class of flexible sheets that permit the growth of tissue through openings in the mesh after the surgery has been completed to enhance attachment to surrounding tissue.
  • the fabric of the present disclosure may be incorporated (e.g. attached to, coated on, embedded or impregnated) with a bioactive agent.
  • a bioactive agent as used herein, is used in its broadest sense and includes any substance or mixture of substances that have clinical use.
  • a bioactive agent is any agent which provides a therapeutic or prophylactic effect; a compound that affects or participates in tissue growth, cell growth and/or cell differentiation; a compound that may be able to invoke or prevent a biological action such as an immune response; or a compound that could play any other role in one or more biological processes.
  • any agent which may enhance tissue repair, limit the risk of sepsis, and modulate the mechanical properties of the fabric may be added during the preparation of the mesh or may be coated on or into the major spaces or pores of the fabric.
  • bioactive agents examples include antimicrobials, analgesics, antipyretics, anesthetics, antiepileptics, antihistamines, anti-inflammatories, cardiovascular drugs, diagnostic agents, sympathomimetics, cholinomimetics, antimuscarinics, antispasmodics, hormones, growth factors, muscle relaxants, adrenergic neuron blockers, antineoplastics, immunogenic agents, immunosuppressants, gastrointestinal drugs, diuretics, steroids, lipids, lipopolysaccharides, polysaccharides, and enzymes. It is also intended that combinations of bioactive agents may be used.
  • bioactive agents which may be in the present disclosure include: local anesthetics; non-steroidal antifertility agents; parasympathomimetic agents; psychotherapeutic agents; tranquilizers; decongestants; sedative hypnotics; steroids; sulfonamides; sympathomimetic agents; vaccines; vitamins; antimalarials; anti-migraine agents; anti-parkinson agents such as L-dopa; anti-spasmodics; anticholinergic agents (e.g., oxybutynin); antitussives; bronchodilators; cardiovascular agents such as coronary vasodilators and nitroglycerin; alkaloids; analgesics; narcotics such as codeine, dihydrocodeinone, meperidine, morphine and the like; non-narcotics such as salicylates, aspirin, acetaminophen, d-propoxyphene and the like; opioid receptor antagonists such as n
  • bioactive agents include: viruses and cells; peptides, polypeptides and proteins, as well as analogs, muteins, and active fragments thereof; immunoglobulins; antibodies; cytokines (e.g., lymphokines, monokines, chemokines); blood clotting factors; hemopoietic factors; interleukins (IL-2, IL-3, IL-4, IL-6); interferons (.beta.-IFN, (.alpha.-IFN and .gamma.-IFN)); erythropoietin; nucleases; tumor necrosis factor; colony stimulating factors (e.g., GCSF, GM-CSF, MCSF); insulin; anti-tumor agents and tumor suppressors; blood proteins; gonadotropins (e.g., FSH, LH, CG, etc.); hormones and hormone analogs (e.g., growth hormone); vaccines (e.g.
  • a single bioactive agent may be utilized or, in alternate embodiments, any combination of bioactive agents may be utilized.
  • the structure of the mesh will vary depending upon the assembling technique utilized to form the mesh, as well as other factors, such as the type of fibers used, the tension at which the fibers are held, and the mechanical properties required of the mesh.
  • a mesh should have sufficient tensile strength to support a fascial wall during repair of a defect in the fascial wall causing a hernia.
  • knitting may be utilized to form a mesh of the present disclosure.
  • Knitting involves, in embodiments, the intermeshing of fibers or yarns to form loops or inter-looping of the fibers or yarns.
  • fibers and/or yarns may be warp-knitted thereby creating vertical interlocking loop chains and/or may be weft-knitted thereby creating rows of interlocking loop stitches across the mesh.
  • weaving may be utilized to a mesh of the present disclosure. Weaving may include, in embodiments, the intersection of two sets of straight yarns, warp and weft, which cross and interweave at right angles to each other, or the interlacing of two yarns at right angles to each other.
  • the strands may be arranged to form a net mesh which has isotropic or near isotropic tensile strength and elasticity.
  • the fibers/yarns may be nonwoven and formed by mechanically, chemically, or thermally bonding the fibers/yarns into a sheet or web.
  • fibers/yarns may be mechanically bound by entangling the fibers/yarns to form the mesh by means other than knitting or weaving, such as matting, pressing, stitch-bonding, needlepunching, or otherwise interlocking the fibers/yarns to form a binderless network of fibers/yarns.
  • the fibers/yarns of the mesh may be chemically bound by use of an adhesive, such as a hot melt adhesive, or thermally bound by applying a binder, such as a powder, paste, or melt, and melting the binder on the sheet or web of fibers/yarns.
  • the mesh of the present invention may comprise a backing strip which may be releasably attached to the mesh.
  • the backing strip may be formed from a range of materials, including plastics, and may be releasably attached by an adhesive.
  • the releasable attachment of a backing strip to the mesh may provide a more substantial and less flexible surgical implant, which may be more easily handled by a surgeon.
  • the backing strip can be removed from the surgical implant, the surgical implant being retained in the body and the backing material being removed by the surgeon.
  • the surgical implant can therefore benefit from reduced mass while still providing characteristics required for surgical handling.
  • a surgical mesh formed from the multi-layered fibers of the present invention may be applied during open surgery.
  • the rigidity of the surgical mesh will allow for ease of handling by the surgeon.
  • the absorbable surface material may dissolve leaving behind a sufficiently strong mesh needed to maintain the long term integrity of the hernia repair.
  • the remaining mesh will be flexible, forming to the abdominal wall.
  • the mesh may also be used, in embodiments, to prevent and/or reduce adhesions which may otherwise occur between a mesh and tissue.
  • the surgical mesh may be applied during minimally invasive surgery.
  • Laparoscopic surgical procedures are minimally invasive procedures in which operations are carried out within the body by using elongated deployment devices, inserted through small entrance openings in the body.
  • the initial opening in the body tissue to allow passage of the endoscopic or laparoscopic devices to the interior of the body may be a natural passageway of the body, or it can be created by a tissue piercing device such as a trocar.
  • narrow punctures or incisions may be made, thereby minimizing trauma to the body cavity and reducing patient recovery time.
  • Laparoscopic deployment devices may be used for transferring a mesh into a body cavity. Such devices are within the purview of those skilled in the art and include, for example, the devices disclosed in U.S. Patent Application Publication Nos. 2006/0229640, 2006/0200170, and/or 2006/0200169, the entire disclosures of each of which are incorporated herein by reference.
  • a mesh according to the present disclosure can be inserted through a small incision (e.g., from about 1 cm to about 2 cm in length) with the use of a laparoscopic deployment device, trocar, or other device.
  • the mesh may be rolled or folded so as to fit within the device for transfer into the body cavity.
  • the absorbable surface material of the bicomponent microfiber Upon exiting the transfer device, the absorbable surface material of the bicomponent microfiber provides sufficient stiffness to reopen the rolled or folded mesh into its original geometric shape.
  • the mesh can be cut to any desired size.
  • the cutting may be carried out by a surgeon or nurse under sterile conditions such that the surgeon need not have many differently sized implants on hand, but can simply cut a mesh to the desired size of the implant after assessment of the patient.
  • the implant may be supplied in a large size and be capable of being cut to a smaller size, as desired.
  • Different shapes are suitable for repairing different defects in fascial tissue, and thus by providing a surgical implant which can be cut to a range of shapes, a wide range of defects in fascial tissue can be treated.
  • the implant can have any shape that conforms with an anatomical surface of a human or animal body that may be subject to a defect to be repaired by the implant.
  • an anterior uterovaginal prolapse is elliptical in shape or a truncated ellipse
  • a posterior prolapse is circular or ovoid in shape
  • the implant shape may be any one of elliptical or truncated ellipse, round, circular, oval, ovoid or some similar shape to be used depending on the hernia or prolapse to be treated.
  • the surgical implant of the present disclosure may be useful for the repair of uterovaginal prolapse, it may also be used in a variety of surgical procedures including the repair of hernias.
  • the mesh in place once it has been suitably located in the patient.
  • the mesh can be secured in any manner known to those skilled in the art. Some examples include suturing the mesh to strong lateral tissue, gluing the mesh in place using a biocompatible glue, or using a surgical fastener, e.g., a tack, to hold the mesh securely in place.
  • the mesh may include at least one capsule containing a biocompatible glue for securing the implant in place.
  • a biocompatible glue within the purview of one skilled in the art may be used.
  • useful glues include fibrin glues and cyanoacrylate glues.
  • the mesh of the present disclosure may be secured to tissue using a surgical fastener such as a surgical tack.
  • a surgical fastener such as a surgical tack.
  • Other surgical fasteners which may be used are within the purview of one skilled in the art, including staples, clips, helical fasteners, and the like.
  • Tacks are known to resist larger removal forces compared with other fasteners.
  • tacks only create one puncture as compared to the multiple punctures created by staples.
  • Tacks can also be used from only one side of the repair site, unlike staples, clips or other fasteners which require access to both sides of the repair site. This may be especially useful in the repair of a vaginal prolapse, where accessing the prolapse is difficult enough without having to access both sides of the prolapse.
  • Suitable tacks which may be utilized to secure the mesh of the present disclosure to tissue include, but are not limited to, the tacks described in U.S. Patent Application Publication No. 2004/0204723, the entire disclosure of which is incorporated by reference herein.
  • Suitable structures for other fasteners which may be utilized in conjunction with the mesh of the present disclosure to secure same to tissue are known in the art and can include, for example, the suture anchor disclosed in U.S. Pat. No. 5,964,783 to Grafton et al., the entire disclosure of which is incorporated by reference herein. Additional fasteners which may be utilized and tools for their insertion include the helical fasteners disclosed in U.S. Pat. No. 6,562,051.
  • the surgical fasteners useful with the mesh herein may be made from bioabsorbable materials, non-bioabsorbable materials, and combinations thereof. Suitable materials which may be utilized include those described in U.S. Patent Application Publication No. 2004/0204723. Examples of absorbable materials which may be utilized include trimethylene carbonate, caprolactone, dioxanone, glycolic acid, lactic acid, glycolide, lactide, homopolymers thereof, copolymers thereof, and combinations thereof. Examples of non-absorbable materials which may be utilized include stainless steel, titanium, nickel, chrome alloys, and other biocompatible implantable metals. In embodiments, a shape memory alloy may be utilized as a fastener. Suitable shape memory materials include nitinol.
  • Surgical fasteners utilized with the mesh of the present disclosure may be made into any size or shape to enhance their use depending on the size, shape and type of tissue located at the repair site.
  • the surgical fasteners e.g., tacks
  • the mesh may be tacked and glued, or sutured and tacked, into place.
  • the surgical fasteners may be attached to the mesh in various ways.
  • the ends of the mesh may be directly attached to the fastener(s).
  • the mesh may be curled around the fastener(s) prior to implantation.
  • the fastener may be placed inside the outer edge of the mesh and implanted in a manner which pinches the mesh up against the fastener and into the site of the injury.
  • Dispersal and solubilization of native animal collagen can be achieved using various proteolytic enzymes (such as porcine mucosal pepsin, bromelain, chymopapain, chymotrypsin, collagenase, ficin, papain, peptidase, proteinase A, proteinase K, trypsin, microbial proteases, and, similar enzymes or combinations of such enzymes) which disrupt the intermolecular bonds and remove the immunogenic non-helical telopeptides without affecting the basic, rigid triple-helical structure which imparts the desired characteristics of collagen (see U.S. Pat. Nos.
  • proteolytic enzymes such as porcine mucosal pepsin, bromelain, chymopapain, chymotrypsin, collagenase, ficin, papain, peptidase, proteinase A, proteinase K, trypsin, microbial proteases, and, similar enzymes or
  • the present invention also contemplates genetically modified forms of collagen/atelocollagen - for example collagenase-resistant collagens and the like [Wu et al., Proc Natl. Acad Sci, Vol. 87, p.5888-5892, 1990].
  • Recombinant collagen may be expressed in any non-animal cell, including but not limited to plant cells and other eukaryotic cells such as yeast and fungus.
  • Plants in which human collagen may be produced may be of lower (e.g. moss and algae) or higher (vascular) plant species, including tissues or isolated cells and extracts thereof (e.g. cell suspensions).
  • Preferred plants are those which are capable of accumulating large amounts of collagen chains, collagen and/or the processing enzymes described herein below. Such plants may also be selected according to their resistance to stress conditions and the ease at which expressed components or assembled collagen can be extracted.
  • Examples of plants in which human procollagen may be expressed include, but are not limited to tobacco, maize, alfalfa, rice, potato, soybean, tomato, wheat, barley, canola, carrot, lettuce and cotton.
  • Production of recombinant procollagen is typically effected by stable or transient transformation with an exogenous polynucleotide sequence encoding human procollagen.
  • Exemplary polynucleotide sequences encoding human procollagen are set forth by SEQ ID NOs: 7, 8, 9 and 10.
  • Production of human telocollagen is typically effected by stable or transient transformation with an exogenous polynucleotide sequence encoding human procollagen and at least one exogenous polynucleotide sequence encoding the relevant protease.
  • the stability of the triple-helical structure of collagen requires the hydroxylation of prolines by the enzyme prolyl-4-hydroxylase (P4H) to form residues of hydroxyproline within the collagen chain.
  • P4H prolyl-4-hydroxylase
  • plants are capable of synthesizing hydroxyproline-containing proteins
  • the prolyl hydroxylase that is responsible for synthesis of hydroxyproline in plant cells exhibits relatively loose substrate sequence specificity as compared with mammalian P4H.
  • production of collagen containing hydroxyproline only in the Y position of Gly -X-Y triplets requires co-expression of collagen and human or mammalian P4H genes [Olsen et al, Adv Drug Deliv Rev. 2003 Nov 28;55(12): 1547-67].
  • the collagen is directed to a subcellular compartment of a plant that is devoid of endogenous P4H activity so as to avoid incorrect hydroxylation thereof.
  • the phrase "subcellular compartment devoid of endogenous P4H activity" refers to any compartmentalized region of the cell which does not include plant P4H or an enzyme having plant-like P4H activity.
  • the subcellular compartment is a vacuole.
  • Accumulation of the expressed collagen in a subcellular compartment devoid of endogenous P4H activity can be effected via any one of several approaches.
  • the expressed collagen can include a signal sequence for targeting the expressed protein to a subcellular compartment such as the vacuole. Since it is essential that P4H co-accumulates with the expressed collagen chain, the coding sequence thereof is preferably modified accordingly (e.g. by addition or deletion of signal sequences). Thus, P4H is co-expressed with the collagen in the plant, whereby the P4H also includes a signal sequence for targeting to the same subcellular compartment such as the vacuole.
  • both the collagen sequence and the P4H sequence are devoid of an endoplasmic reticulum retention signal, such that it passes through the ER and is retained in the vacuole, where it is hydroxylated.
  • the present invention therefore contemplates genetically modified cells co- expressing both human collagen and a P4H, capable of correctly hydroxylating the collagen alpha chain(s) [i.e. hydroxylating only the proline (Y) position of the Gly -X- Y triplets].
  • P4H is an enzyme composed of two subunits, alpha and beta as set forth in Genbank Nos. P07237 and P13674. Both subunits are necessary to form an active enzyme, while the beta subunit also possesses a chaperon function.
  • the P4H expressed by the genetically modified cells of the present invention is preferably a human P4H which is encoded by, for example, SEQ ID Nos: 11 and 12.
  • P4H mutants which exhibit enhanced substrate specificity, or P4H homologues can also be used.
  • collagen is also modified by Lysyl hydroxylase, galactosyltransferase and glucosyltransferase. These enzymes sequentially modify lysyl residues in specific positions to hydroxylysyl, galactosylhydroxylysyl and glucosylgalactosyl hydroxylysyl residues at specific positions.
  • Lysyl hydroxylase 3 LH3
  • Genbank No. 060568 can catalyze all three consecutive modifying steps as seen in hydroxylysine-linked carbohydrate formation.
  • the genetically modified cells of the present invention may also express mammalian LH3.
  • An LH3 encoding sequence such as that set forth by SEQ ID No: 13, can be used for such purposes.
  • the procollagen(s) and modifying enzymes described above can be expressed from a stably integrated or a transiently expressed nucleic acid construct which includes polynucleotide sequences encoding the procollagen alpha chains and/or modifying enzymes (e.g. P4H and LH3) positioned under the transcriptional control of functional promoters.
  • a nucleic acid construct (which is also termed herein as an expression construct) can be configured for expression throughout the whole organism (e.g. plant, defined tissues or defined cells), and/or at defined developmental stages of the organism.
  • Such a construct may also include selection markers (e.g. antibiotic resistance), enhancer elements and an origin of replication for bacterial replication.
  • nucleic acid constructs into both monocotyledonous and dicotyledenous plants
  • Potrykus I., Annu. Rev. Plant. Physiol., Plant. Mol. Biol. (1991) 42:205-225; Shimamoto et al., Nature (1989) 338:274-276.
  • Such methods rely on either stable integration of the nucleic acid construct or a portion thereof into the genome of the plant, or on transient expression of the nucleic acid construct, in which case these sequences are not inherited by the plant's progeny.
  • a nucleic acid construct can be directly introduced into the DNA of a DNA-containing organelle such as a chloroplast.
  • the telopeptide-comprising collagen is typically harvested.
  • Plant tissues/cells are preferably harvested at maturity, and the procollagen molecules are isolated using extraction approaches.
  • the harvesting is effected such that the procollagen remains in a state that it can be cleaved by protease enzymes.
  • a crude extract is generated from the transgenic plants of the present invention and subsequently contacted with the protease enzymes.
  • the propeptide or telopeptide- comprising collagen may be purified from the genetically engineered cells prior to incubation with protease, or alternatively may be purified following incubation with the protease. Still alternatively, the propeptide or telopeptide-comprising collagen may be partially purified prior to protease treatment and then fully purified following protease treatment. Yet alternatively, the propeptide or telopeptide-comprising collagen may be treated with protease concomitant with other extraction/purification procedures.
  • Exemplary methods of purifying or semi-purifying the telopeptide-comprising collagen of the present invention include, but are not limited to salting out with ammonium sulfate or the like and/or removal of small molecules by ultrafiltration.
  • the protease used for cleaving the recombinant propeptide or telopeptide comprising collagen is not derived from an animal.
  • Exemplary proteases include, but are not limited to certain plant derived proteases e.g. ficin (EC 3.4.22.3) and certain bacterial derived proteases e.g. subtilisin (EC 3.4.21.62), neutrase.
  • plant derived proteases e.g. ficin (EC 3.4.22.3) and certain bacterial derived proteases e.g. subtilisin (EC 3.4.21.62), neutrase.
  • the present inventors also contemplate the use of recombinant enzymes such as rhTrypsin and rhPepsin. Several such enzymes are commercially available e.g.
  • Ficin from Fig tree latex (Sigma, catalog #F4125 and Europe Biochem), Subtilisin from Bacillus licheniformis (Sigma, catalog #P5459) Neutrase from bacterium Bacillus amyloliquefaciens (Novozymes, catalog # PW201041) and TrypZeanTM, a recombinant human trypsin expressed in corn (Sigma catalog #T3449).
  • compositions, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.
  • range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
  • the term "method” refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.
  • the term “treating” includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.
  • Nano Cellulose Crystals were produced by H 2 SO 4 hydrolysis of 200 ⁇ Micro Crystalline Cellulose (MCC, Avicel). The process involved suspension of the NCC powder in water, hydrolysis in controlled temperatures and acid concentration, washing cycles in water and followed by sonication until a clear was achieved.
  • MCC Micro Crystalline Cellulose
  • Cellulose micro-crystals were suspended in DDW at a very low temperature (iced water). Acid hydrolysis of the cellulose was carried out using sulfuric acid at a final concentration of 45 % at a temperature of 40 °C for 45 minutes followed by 30 minutes at 60 °C. After the hydrolysis process, the suspension was centrifuged at 8000 rpm for 10 minutes. Excess aqueous acid was removed and the resultant precipitate was resuspended in DDW. The washing procedure was repeated 4 to 5 times, until the supernatant emerging from the centrifuge was turbid. The resultant precipitate was resuspended in DDW and dialyzed against DDW until neutral pH is achieved. The cellulose suspension was then sonicated until the solution become optically clear.
  • gelatin solution 20 % w/v rhCollagen (CollPlant, Ltd.) was dissolved in 1 mL 10 mM hydrochloric acid in a water bath at 70 °C. The resulting degradation product was gelatin.
  • Plasma pretreated PLA fibers The oxygen and nitrogen plasma treatment was carried out on PICO low pressure plasma system (DIENER, 13.56 MHz: Power 0 - 300 W) PLA fiber was placed over the electrode in the plasma chamber. The chamber was evacuated to 10 Pa before filling with the desired gas. After the pressure of the chamber had stabilized to a proper value, glow discharge plasma was created by controlling the electrical power at a radio frequency of 13.56 MHz for a predetermined time. The plasma-treated sample was further exposed to the desired gas for another 10 minutes before the sample was taken out from the chamber.
  • PICO low pressure plasma system DIENER, 13.56 MHz: Power 0 - 300 W
  • PLA fibers (number 002, 6.7 Tex) were supplied by Centexbel (Belgium).
  • PLA fibers were coated by the following stages, first PLA fibers were immersed in 20 % (w/v) gelatin solution at RT for 1 hour, followed by air-drying. Then, the gelatin coated PLA fibers were immersed in 2.5 % (w/v) NCC solution for 40 minutes at room temperature. Next, the gelatin/NCC coated PLA fibers were air-dried. Finally, for the shell layer, PLA fibers reinforced by NCC coating as core materials were immersed in 18 % (w/v) collagen solution for 1 hour at room temperature (RT). Finally, the obtained core-shell fibers were air-dried. Every coating treatment was repeated at least 5 times.
  • the microarchitecture of the prepared fibers was evaluated by scanning electron microscopy (SEM).
  • Figure 1 demonstrates that the PLA fiber has a ridge surface morphology (Figure 1A). The same morphology was obtained when the PLA fiber was apparently coated with the NCC particles (Figure IB) in the absence of gelatin, indicating that the fiber was not coated with the NCC composites. NCC-coated PLA fiber was obtained (Figure ID) only when the fiber was first coated with gelatin ( Figure 1C). Moreover, Figure ID clearly indicates that a thin smooth layer of gelatin/NCC with a densely packed structure was obtained. Figure IE also demonstrated smooth surface morphology of the obtained gelatin/NCC/rhcollagen-PLA fiber, but with a thicker layer comparison to the gelatin/NCC coating layer (Figure 1C).
  • Gelatin solution coloring procedure 2.5 ⁇ 1 of 0.1 % blue food dye was added to 350 ⁇ of 20 % (w/v) gelatin solution. Then the PLA fiber was immersed in this solution for 1 hr at RT. Next, the gelatin coated PLA fibers were immersed in 2.5 % (w/v) NCC solution for 40 min at RT. Finally the resulting fibers were air-dried.
  • PLA fibers (number 002, 6.7 Tex, were pretreated with N 2 plasma jet at Centexbel, Belgium) were immersed in 2.5 % (w/v) NCC solution for 40 min at RT. Finally the fibers were air-dried.
  • Rhcollagen coating on pretreated PLA fibers First PLA fibers were pretreated by 0 2 plasma jet (10 min 500W) and subsequently stored under nitrogen gas atmosphere until further use. Next, the pretreated PLA fibers were immersed in 4.5 % (w/v) Rhcollagen solution for 1 hour at RT. Finally, the fibers were air-dried.
  • EDC/NHS crosslinking of collagen coated PLA/PCL fibers PLA/PCL fibers were pretreated by 0 2 plasma jet (10 min 500W, 1 min 300W, respectively) stored under nitrogen gas atmosphere until further use. Next, the pretreated PLA/ PCL fibers were immersed in 4.5 % (w/v) RhcoUagen solution for 1 hr at RT and subsequently air- dried. Afterward, two crosslinking methods were studied. First, RhcoUagen coated pretreated PLA/PCL fibers were immersed in 1.1 M EDC solution (90 % ethanol) for 3 hrs at RT under constant shaking followed by DDW washing and finally air-drying. In the second method, RhcoUagen coated pretreated PLA/PCL fibers were immersed in 1.1 M EDC and 0.55 M NHS solution (90 % ethanol) for 3 hrs at RT under constant shaking followed by DDW washing and finally air-drying.
  • Plasma technique is a convenient method for modifying polymeric materials without altering their bulk properties. This treatment results in incorporation of positively charged groups on the PLA fiber surface. It allows direct coating of the NCC composite due to the interaction between PLA fiber surface and the negatively charged NCC coating (NCC contains sulphate group).
  • Figures 3A-C displays SEM micrographs of a pretreated PLA fiber with N 2 plasma jet (A), NCC coated naked PLA fiber (B) and NCC coated, N 2 plasma jet- pretreated PLA fiber (C).
  • Figure 3C demonstrates that direct NCC coating of approximately 3 ⁇ thickness on pretreated PLA fiber with N 2 plasma jet may be successfully performed
  • Plasma pretreatment 0 2 plasma jet
  • This pretreatment resulted in negatively charged groups that are incorporated on the PLA fiber surface. It allows direct coating of the collagen due to the interaction PLA between fiber surface and the positively charged collagen coating.
  • Figures 4A-B displays SEM micrographs of pretreated PLA fiber with 0 2 plasma jet (A), and RhcoUagen coated pretreated PLA fiber with 0 2 plasma jet (B).
  • Figure 4B demonstrates that direct RhcoUagen coating of approximately 1 ⁇ thickness on pretreated PLA fiber with 0 2 plasma jet may be successfully performed. Different Rhcollagen concentrations were studied in order to achieve uniform coating.
  • Figures 5A-B display HRSEM micrographs of pretreated PLA fibers with 0 2 plasma jet that were coated with different Rhcollagen concentrations: 18 % (w/v) (A), and 4.5% (w/v) (B).
  • Rhcollagen concentrations 18 % (w/v) (A), and 4.5% (w/v) (B).
  • a collagen concentration of 18 % (w/v) was used, a thick homogenous Rhcollagen coating (of 5 ⁇ ) was obtained.
  • Figure 5B a 1 ⁇ thickness Rhcollagen coating was obtained.
  • Figures 6 and 7 demonstrate that crosslinking of RhCollagen (4.5 %) coating on pretreated PLA or PCL fibers with 0 2 plasma jet, respectively may be successfully performed.

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Abstract

L'invention concerne une fibre isolée comprenant : i. une âme interne qui contient un polymère synthétique; ii. une couche externe qui contient du collagène; et iii. une couche intermédiaire qui est placée entre l'âme interne et la couche externe et qui contient des nanocristaux de cellulose.
PCT/IL2012/050543 2011-12-20 2012-12-20 Fibres de polymère synthétique enrobées de collagène WO2013093921A1 (fr)

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CN104629276A (zh) * 2013-11-12 2015-05-20 珠海市红旌发展有限公司 一种纳米晶体纤维素复合材料的制备方法、由其制成的产品及其用途
WO2015077262A1 (fr) * 2013-11-19 2015-05-28 Guill Tool & Engineering Entrées d'impression 3d coextrudées, multicouche et multicomposant
WO2016042211A1 (fr) 2014-09-17 2016-03-24 University Of Helsinki Matériaux implantables et leur utilisation
CN105758911A (zh) * 2016-03-30 2016-07-13 中国科学院兰州化学物理研究所 基于纳米纤维素和半胱氨酸修饰的金电极及其应用
WO2016123207A1 (fr) * 2015-01-29 2016-08-04 University Of Connecticut Fibres composites et matrices formées avec celles-ci
EP3159017A1 (fr) * 2015-10-19 2017-04-26 Bioenergy Capital AG Matrice resorbable destinee au recouvrement de plaie
JP2017516601A (ja) * 2014-03-14 2017-06-22 スクリップス ヘルス 軟骨および半月板のマトリックスポリマーのエレクトロスピニング
CN108210994A (zh) * 2018-03-06 2018-06-29 福建工程学院 一种均匀化聚多巴胺涂层修饰生物支架的制备方法
WO2019237267A1 (fr) * 2018-06-13 2019-12-19 浙江晶通塑胶有限公司 Plancher dégradable utilisant une résine de pla et son procédé de production
WO2020035703A1 (fr) * 2018-08-17 2020-02-20 Raft Enterprises Ltd. Échafaudage tissulaire
US10617787B2 (en) 2017-05-16 2020-04-14 Embody Inc. Biopolymer compositions, scaffolds and devices
US10653817B2 (en) 2017-10-24 2020-05-19 Embody Inc. Method for producing an implantable ligament and tendon repair device
US11020509B2 (en) 2019-02-01 2021-06-01 Embody, Inc. Microfluidic extrusion
CN113183594A (zh) * 2021-04-07 2021-07-30 广东工业大学 用于表皮修复的静电纺丝功能性纳米纤维薄膜的制备方法
WO2021222699A1 (fr) 2020-04-30 2021-11-04 Board Of Regents Of The University Of Nebraska Mailles pour hernie multicouches et leurs procédés de fabrication et d'utilisation
CN113730557A (zh) * 2021-09-03 2021-12-03 山西锦波生物医药股份有限公司 重组i型人源化胶原蛋白在盆底修复中的用途
US11352614B2 (en) * 2017-06-14 2022-06-07 R.J. Reynolds Tobacco Company RuBisCO protein fibers
US11369465B2 (en) 2013-01-14 2022-06-28 Scripps Health Tissue array printing
US11634448B2 (en) 2016-06-15 2023-04-25 The General Hospital Corporation Metabolic labeling and molecular enhancement of biological materials using bioorthogonal reactions
US12115276B2 (en) 2017-06-09 2024-10-15 Collplant Ltd. Additive manufacturing using recombinant collagen-containing formulation

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WO2015077262A1 (fr) * 2013-11-19 2015-05-28 Guill Tool & Engineering Entrées d'impression 3d coextrudées, multicouche et multicomposant
US20160297104A1 (en) * 2013-11-19 2016-10-13 Guill Tool & Engineering Coextruded, multilayer and multicomponent 3d printing inputs field
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US11497830B2 (en) 2014-03-14 2022-11-15 Scripps Health Electrospinning of cartilage and meniscus matrix polymers
WO2016042211A1 (fr) 2014-09-17 2016-03-24 University Of Helsinki Matériaux implantables et leur utilisation
WO2016123207A1 (fr) * 2015-01-29 2016-08-04 University Of Connecticut Fibres composites et matrices formées avec celles-ci
EP3159017A1 (fr) * 2015-10-19 2017-04-26 Bioenergy Capital AG Matrice resorbable destinee au recouvrement de plaie
CN105758911A (zh) * 2016-03-30 2016-07-13 中国科学院兰州化学物理研究所 基于纳米纤维素和半胱氨酸修饰的金电极及其应用
US11634448B2 (en) 2016-06-15 2023-04-25 The General Hospital Corporation Metabolic labeling and molecular enhancement of biological materials using bioorthogonal reactions
US10617787B2 (en) 2017-05-16 2020-04-14 Embody Inc. Biopolymer compositions, scaffolds and devices
US11116870B2 (en) 2017-05-16 2021-09-14 Embody Inc. Biopolymer compositions, scaffolds and devices
US10835639B1 (en) 2017-05-16 2020-11-17 Embody Inc. Biopolymer compositions, scaffolds and devices
US11331410B2 (en) 2017-05-16 2022-05-17 Embody, Inc. Biopolymer compositions, scaffolds and devices
US12115276B2 (en) 2017-06-09 2024-10-15 Collplant Ltd. Additive manufacturing using recombinant collagen-containing formulation
US11352614B2 (en) * 2017-06-14 2022-06-07 R.J. Reynolds Tobacco Company RuBisCO protein fibers
US12065681B2 (en) 2017-06-14 2024-08-20 R.J. Reynolds Tobacco Company Method of producing ribulose-1,5-bisphosphate oxygenase protein fibers
US10653817B2 (en) 2017-10-24 2020-05-19 Embody Inc. Method for producing an implantable ligament and tendon repair device
US11213610B2 (en) 2017-10-24 2022-01-04 Embody Inc. Biopolymer scaffold implants and methods for their production
CN108210994A (zh) * 2018-03-06 2018-06-29 福建工程学院 一种均匀化聚多巴胺涂层修饰生物支架的制备方法
WO2019237267A1 (fr) * 2018-06-13 2019-12-19 浙江晶通塑胶有限公司 Plancher dégradable utilisant une résine de pla et son procédé de production
WO2020035703A1 (fr) * 2018-08-17 2020-02-20 Raft Enterprises Ltd. Échafaudage tissulaire
JP2021533908A (ja) * 2018-08-17 2021-12-09 ラフト エンタープライジズ リミテッド 組織足場
US11338056B2 (en) 2019-02-01 2022-05-24 Embody, Inc. Microfluidic extrusion
US11020509B2 (en) 2019-02-01 2021-06-01 Embody, Inc. Microfluidic extrusion
US11338057B2 (en) 2019-02-01 2022-05-24 Embody, LLC Microfluidic extrusion
WO2021222699A1 (fr) 2020-04-30 2021-11-04 Board Of Regents Of The University Of Nebraska Mailles pour hernie multicouches et leurs procédés de fabrication et d'utilisation
EP4142645A4 (fr) * 2020-04-30 2024-03-27 Board of Regents of the University of Nebraska Mailles pour hernie multicouches et leurs procédés de fabrication et d'utilisation
CN113183594A (zh) * 2021-04-07 2021-07-30 广东工业大学 用于表皮修复的静电纺丝功能性纳米纤维薄膜的制备方法
CN113730557B (zh) * 2021-09-03 2023-12-22 山西锦波生物医药股份有限公司 重组i型人源化胶原蛋白在盆底修复中的用途
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