FIELD AND BACKGROUND OF THE INVENTION

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.

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).

Poly(glycolic acid) (PGA) 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. Despite its low solubility, 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.

Polycaprolactone (PCL) is a semicrystalline polymer that has been extensively investigated as scaffolds for tissue engineering. It has excellent biocompatibility. PCL has low tensile strength (approximately 23 MPa) but an extremely high elongation at breakage (> 700%). Due to its long degradation rate (approximately 2-3 years), it has been considered as a useful polymer for the development of hernia meshes.

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. In contrast, 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.

The use of 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.

The use of animal collagen is further exacerbated due to species differences. In addition, allergic reactions to animal collagen have been documented. Collagen, extracted from either animal or human cadavers, has typically undergone irreversible crosslinking and harsh processing methods, both of which compromise its biological and mechanical functions. Recent studies have supported the use of recombinant collagen which is added to the composite in order to provide a permissive environment for cell growth and differentiation.

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 β- 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. 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.

Hong et al., [Biomaterials, Volume 26, Issue 32, November 2005, Pages 6305-6313] discloses collagen-coated polylactide (PLA) microspheres.

Kim et al., [Journal of Applied Polymer Science, Volume 92, Issue 4, pages 2082- 2092, 2004] discloses collagen coated porous membranes composed of the biodegradable polyesters poly(D,L-lactide) (PLA) and poly(D,L-lactide-coglycolide) (PLGA).

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.

Xiang et al., Journal of Biobased Materials and Bioenergy, Volume 3, Number 2, June 2009, pp. 147-155(9) teaches electro spinning of Poly(lactic acid)/Cellulose Nanocrystals (PLA/CNs) to fabricate a novel renewable and biocompatible nanocomposite as potential scaffold for bone tissue engineering.

SUMMARY OF THE INVENTION

According to an aspect of some embodiments of the present invention there is provided an isolated fiber comprising:

i. an internal core which comprises a synthetic polymer; ii. an outer layer which comprises collagen; and

iii. an intermediate layer disposed between the internal core and the outer layer which comprises cellulose nanocrystals.

According to an aspect of some embodiments of the present invention there is provided 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.

According to an aspect of some embodiments of the present invention there is provided a method of generating the fiber the present invention, comprising:

(a) contacting a synthetic polymer fiber with cellulose nanocrystals to generate a coated synthetic polymer fiber; and

(b) contacting the coated synthetic polymer fiber with collagen, thereby generating the fiber of the present invention.

According to an aspect of some embodiments of the present invention there is provided a fabric comprising the isolated fibers described herein.

According to an aspect of some embodiments of the present invention there is provided a scaffold comprising the isolated fibers described herein.

According to an aspect of some embodiments of the present invention there is provided an electrospun element comprising the isolated fiber described herein.

According to an aspect of some embodiments of the present invention there is provided an implantable device comprising the fiber described herein.

According to some embodiments of the invention, the collagen comprises recombinant collagen.

According to some embodiments of the invention, the isolated fiber further comprises an adhesive layer situated between the internal core and the intermediate layer.

According to some embodiments of the invention, the adhesive layer comprises gelatin.

According to some embodiments of the invention, a diameter of the internal core is between 50-150 μιη. According to some embodiments of the invention, the thickness of the intermediate layer is between 0.1-10μιη.

According to some embodiments of the invention, the thickness of the outer layer is between 1-11 μιη.

According to some embodiments of the invention, the recombinant collagen is human collagen.

According to some embodiments of the invention, the recombinant collagen is generated in plants.

According to some embodiments of the invention, the synthetic polymer is biodegradable.

According to some embodiments of the invention, 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.

According to some embodiments of the invention, the synthetic polymer is nonbiodegradable.

According to some embodiments of the invention, 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.

According to some embodiments of the invention, the collagen comprises liquid crystal collagen.

According to some embodiments of the invention, the collagen comprises recombinant collagen.

According to some embodiments of the invention, the method further comprises crosslinking the collagen following step (b).

According to some embodiments of the invention, 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).

According to some embodiments of the invention, the method further comprises plasma treating the outer surface of the synthetic polymer fiber prior to step (a).

According to some embodiments of the invention, the plasma treating comprises oxygen plasma treating or ammonia plasma treating.

According to some embodiments of the invention, the scaffold is seeded with cells.

According to some embodiments of the invention, the cells comprise stem cells. According to some embodiments of the invention, the stem cells comprise mesenchymal stem cells.

According to some embodiments of the invention, the implantable device is a surgical mesh.

According to some embodiments of the invention, the implantable device is configured for pelvic floor repair.

According to some embodiments of the invention, the implantable device is configured for hernia repair.

According to some embodiments of the invention, the implantable device is configured for plastic surgery.

According to some embodiments of the invention, the implantable device is configured for breast reconstruction.

According to some embodiments of the invention, the implantable device is configured for urinary or fecal incontinence repair.

According to some embodiments of the invention, the implantable device is configured for cardiovascular procedures.

According to some embodiments of the invention, 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. Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying images. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.

In the drawings:

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).

FIGs. 2A-C are confocal micrographs (blue field) of naked PLA fiber (A), blue food coloring-gelatin coated PLA fiber (B) and NCC/ blue food coloring-gelatin coated PLA fiber (C). (n=5)

FIGs. 3A-C are SEM micrographs of pretreated PLA fiber with N ₂ plasma jet (A), NCC coated naked PLA fiber (B), and NCC coated pretreated PLA fiber with N ₂ plasma jet (C). (n=5).

FIGs. 4A-B are SEM micrographs of pretreated PLA fiber with 0 ₂ plasma jet (A), and RhcoUagen coated pretreated PLA fiber with 0 ₂ plasma jet (B). (n=5).

FIGs. 5A-B are SEM micrographs of pretreated PLA fiber with 0 ₂ plasma jet with different collagen concentrations: 18% (w/v) (A), and 4.5% (w/v) (B). (n=5).

FIGs. 6A-D SEM micrographs of pretreated PLA fiber with 0 ₂ plasma jet (A), RhcoUagen coated pretreated PLA fiber with 0 ₂ plasma jet (B), EDC/Rhcollagen coated pretreated PLA fiber with 0 ₂ plasma jet (C), and EDC/NHS/Rhcollagen coated pretreated PLA fiber with 0 ₂ plasma jet (D). (n=5).

FIGs. 7A-D SEM micrographs of pretreated PCL fiber with 0 ₂ plasma jet (A), Rhcollagen coated pretreated PCL fiber with 0 ₂ plasma jet (B), EDC/Rhcollagen coated pretreated PCL fiber with 0 ₂ plasma jet (C), and EDC/NHS/Rhcollagen coated pretreated PCL fiber with 0 ₂ plasma jet (D). (n=5).

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates to collagen coated synthetic polymers for tissue repair and regenerative medicine and fabrics comprising same.

Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.

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. Furthermore, polymers can be designed with chemical functional groups that can induce tissue in-growth.

In order to improve the biocompatibility and tensile strength of such synthetic polymers, the present inventors propose coating synthetic polymers with nano cellulose crystals (NCC) followed by coating with collagen. The three-layered fibers have improved bio-compatibility due to the presence of collagen at the surface and improved mechanical properties and longer degradation time due to the inner NCC coating.

Thus, according to one aspect of the present invention there is provided an isolated fiber comprising:

i. an internal core which comprises a synthetic polymer;

ii. an outer layer which comprises collagen; and iii. an intermediate layer disposed between the internal core and the outer layer which comprises cellulose nanocrystals.

As used herein, the term "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.

In order to generate the three-layered fibers of this aspect of the present invention, 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. Examples of 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. Examples of non-biodegradable polymer 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). In 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). As the fibers exit the spinneret, 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.

Further, 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. In addition, 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. Some high-strength polyethylene and aramid fibers are produced by gel spinning.

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.

Typically, the diameter of the synthetic polymer fiber is between 10-500 μιη, more preferably between 20-200 μιη and more preferably between 50-100 μιη,

The next stage in the generation of the fibers of this aspect of the invention involves coating with nano cellulose crystals.

The term "cellulose" refers to the polysaccharide mainly composed of cellobiose units linked together by β- 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.

Sono-chemical preparation of nano cellulose crystals from lignocellulose derived materials is described in Bioresource Technology, Volume 100, Issue 7, April 2009, Pages 2259-2264.

The nanocellulose crystals may be chemically modified and subsequently dispersed in organic solvents. Alternatively, the nanocellulose crystals may be dispersed in polar aprotic organic solvents, such as DMF and DMSO.

Application of the 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 0.1-10μιη thick.

The synthetic polymeric fibers may optionally be pre-treated in order to enhance the ability of the CN to coat the fibers.

According to one embodiment 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.

According to a preferred embodiment, 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.

According to another embodiment, 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. For surface engineering purposes, 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. When a gas is ionized in this manner, 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.

Typically, 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.

As mentioned, the external layer of the fibers of the present invention comprises collagen.

The term "collagen" as used herein, 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

Table 1 below lists examples of collagen NCBI sequence numbers.

Table 1 According to one embodiment, the collagen of the present invention comprises a sufficient portion of its telopeptides such that under suitable conditions it is capable of forming fibrils.

Thus, for example, the collagen may be atelocollagen, a telocollagen or procollagen. As used herein, the term "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.

The term "procollagen" as used herein, 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.

The term "telocollagen" as used herein, 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.

According to another embodiment, 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.

According to one embodiment, the collagen is recombinant human collagen. Preferably, the recombinant human collagen is generated in plants, as further described herein below.

Irrespective of how it is generated or isolated, 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.

As used herein, the phrase "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.

According to one embodiment 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. Preferably, the PEG is of a molecular weight of 10,000-30,000 g/mol and has a concentration of 25-50 %. According to a particular embodiment, a slide-a-lyzer dialysis cassette (Pierce, MW CO 3500) is used. Typically, 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. According to one embodiment, the solution of monomeric collagen is at a concentration of about 100-200 mg/ml or between 0.7-0.3mM.

In most cases dialysis for 2-16 hours is sufficient, depending on volume and concentration.

According to another embodiment, 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 μιη domains, while remaining in solution (R. Martin et al., J. Mol. Biol. 301: 11-17 (2000)).

In another embodiment, 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 Ιμτη.

Following coating of the nano cellulose crystal-coated fiber with the collagen, the collagen may optionally be fibrillated so as to generate fibrils and/or crosslinked.

The term "fibrillogenesis" as used herein 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). Typically, 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).

Following formation of the fibers of the present invention may be braided, twisted, aligned, or otherwise joined to form a fabric. In embodiments, at least two fibers may form a yarn for use in forming the fabric. In other embodiments, 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.

According to one embodiment, the fibers of the present invention may be used to fabricate a scaffold.

As used herein, 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).

Following generation, the scaffolds of the present invention are typically sterilized, for example by oxygen plasma, following which they may be seeded with cells.

As used herein, the term "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. Alternatively, 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. Furthermore, 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.

As used herein, the phrase "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".

According to one embodiment, the fibers of the present invention may be used to fabricate an implantable device.

Thus, 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. For example, 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.

According to a particular embodiment, the implantable device is a surgical mesh.

The term "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. The term "bioactive agent", as used herein, is used in its broadest sense and includes any substance or mixture of substances that have clinical use. Typically, 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. Moreover, any agent which may enhance tissue repair, limit the risk of sepsis, and modulate the mechanical properties of the fabric (e.g., the swelling rate in water, tensile strength, etc.) may be added during the preparation of the mesh or may be coated on or into the major spaces or pores of the fabric. Examples of classes of bioactive agents which may be utilized in accordance with the present disclosure 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.

Other 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 naltrexone and naloxone; anti-cancer agents; anti-convulsants; anti-emetics; antihistamines; anti-inflammatory agents such as hormonal agents, hydrocortisone, prednisolone, prednisone, non-hormonal agents, allopurinol, indomethacin, phenylbutazone and the like; prostaglandins and cytotoxic drugs; estrogens; antibacterials; antibiotics; anti-fungals; anti-virals; anticoagulants; anticonvulsants; antidepressants; antihistamines; and immunological agents.

Other examples of suitable bioactive agents which may be included in the present disclosure 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., tumoral, bacterial and viral antigens); somatostatin; antigens; blood coagulation factors; growth factors (e.g., nerve growth factor, insulin-like growth factor); protein inhibitors; protein antagonists; protein agonists; nucleic acids such as antisense molecules, DNA, and RNA; oligonucleotides; and ribozymes.

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.

Thus, for example, for hernia repair, a mesh should have sufficient tensile strength to support a fascial wall during repair of a defect in the fascial wall causing a hernia.

In embodiments, 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. In some embodiments, 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. In other embodiments, 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. In some embodiments the strands may be arranged to form a net mesh which has isotropic or near isotropic tensile strength and elasticity.

In embodiments, the fibers/yarns may be nonwoven and formed by mechanically, chemically, or thermally bonding the fibers/yarns into a sheet or web. For example, 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. In other embodiments, 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. In another embodiment, 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. Following suitable placement of the surgical implant, 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. During open surgery, the rigidity of the surgical mesh will allow for ease of handling by the surgeon. Following application and attachment of the mesh, 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.

Alternatively, 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. During laparoscopic procedures, 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. 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.

It may be desirable to provide a variety of implants having different sizes so that a surgeon can select an implant of suitable size to treat a particular patient. This allows implants to be completely formed before delivery, ensuring that the smooth edge of the implant is properly formed under the control of the manufacturer. The surgeon would thus have a variety of differently sized (and/or shaped) implants to select the appropriate implant to use after assessment of the patient.

In another embodiment 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. In other words, 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.

More broadly, the present disclosure recognizes that 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.

Typically an anterior uterovaginal prolapse is elliptical in shape or a truncated ellipse, whereas a posterior prolapse is circular or ovoid in shape. Accordingly, 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. In this regard, while 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.

In some embodiments, it may be desirable to secure 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.

In embodiments it may be advantageous to use a biocompatible glue since it is fairly quick to apply glue to the area around the surgical implant. Additionally, the mesh may include at least one capsule containing a biocompatible glue for securing the implant in place. Any biocompatible glue within the purview of one skilled in the art may be used. In embodiments useful glues include fibrin glues and cyanoacrylate glues.

In another embodiment, the mesh of the present disclosure may be secured to tissue using 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.

In embodiments, it may be advantageous to use surgical tacks as a surgical fastener to secure the mesh. Tacks are known to resist larger removal forces compared with other fasteners. In addition, 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, may be used alone or in combination with other fastening methods described herein to secure the mesh to the hernia, prolapse, or other repair site. For example, 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. In embodiments, the ends of the mesh may be directly attached to the fastener(s). In other embodiments, the mesh may be curled around the fastener(s) prior to implantation. In yet another embodiment, 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.

Below is a description of various methods of obtaining collagen used for coating the fibers of the present invention, and generating the optional gelatin coating.

Methods of isolating collagen from animals are known in the art. 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. 3,934,852; 3,121,049; 3,131,130; 3,314,861; 3,530,037; 3,949,073; 4,233,360 and 4,488,911 for general methods for preparing purified soluble collagen). The resulting soluble collagen can be subsequently purified by repeated precipitation at low pH and high ionic strength, followed by washing and re- solublization at low pH.

Plants expressing collagen chains and procollagen are known in the art, see for example, WO06035442A3; Merle et al., FEBS Lett. 2002 Mar 27;515(1-3): 114-8. PMID: 11943205; and Ruggiero et al., 2000, FEBS Lett. 2000 Mar 3;469(1): 132-6. PMID: 10708770; and U.S. Pat. Applications 2002/098578 and 2002/0142391 as well as U.S. Patent Nos. 6,617,431 each of which are incorporated herein by reference.

It will be appreciated that 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 (i.e. expressed) 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. Although 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. Thus, 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].

Thus, according to one embodiment, 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. As is used herein, 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. According to one embodiment, 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.

For example, 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. Preferably, 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. In addition, P4H mutants which exhibit enhanced substrate specificity, or P4H homologues can also be used.

In mammalian cells, 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. A single human enzyme, Lysyl hydroxylase 3 (LH3), as set forth in Genbank No. 060568, can catalyze all three consecutive modifying steps as seen in hydroxylysine-linked carbohydrate formation.

Thus, 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. Such 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.

There are various methods for introducing 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. In addition, several methods exist in which a nucleic acid construct can be directly introduced into the DNA of a DNA-containing organelle such as a chloroplast.

There are two principle methods of effecting stable genomic integration of exogenous sequences, such as those included within the nucleic acid constructs of the present invention, into plant genomes:

(i) Agrobacterium-mediated gene transfer: Klee et al. (1987) Annu. Rev. Plant Physiol. 38:467-486; Klee and Rogers in Cell Culture and Somatic Cell Genetics of Plants, Vol. 6, Molecular Biology of Plant Nuclear Genes, eds. Schell, J., and Vasil, L. K., Academic Publishers, San Diego, Calif. (1989) p. 2-25; Gatenby, in Plant Biotechnology, eds. Kung, S. and Arntzen, C. J., Butterworth Publishers, Boston, Mass. (1989) p. 93-112.

(ii) Direct DNA uptake: Paszkowski et al., in Cell Culture and Somatic Cell Genetics of Plants, Vol. 6, Molecular Biology of Plant Nuclear Genes eds. Schell, J., and Vasil, L. K., Academic Publishers, San Diego, Calif. (1989) p. 52-68; including methods for direct uptake of DNA into protoplasts, Toriyama, K. et al. (1988) Bio/Technology 6: 1072-1074. DNA uptake induced by brief electric shock of plant cells: Zhang et al. Plant Cell Rep. (1988) 7:379-384. Fromm et al. Nature (1986) 319:791-793. DNA injection into plant cells or tissues by particle bombardment, Klein et al. Bio/Technology (1988) 6:559-563; McCabe et al. Bio/Technology (1988) 6:923- 926; Sanford, Physiol. Plant. (1990) 79:206-209; by the use of micropipette systems: Neuhaus et al., Theor. Appl. Genet. (1987) 75:30-36; Neuhaus and Spangenberg, Physiol. Plant. (1990) 79:213-217; or by the direct incubation of DNA with germinating pollen, DeWet et al. in Experimental Manipulation of Ovule Tissue, eds. Chapman, G. P. and Mantell, S. H. and Daniels, W. Longman, London, (1985) p. 197-209; and Ohta, Proc. Natl. Acad. Sci. USA (1986) 83:715-719.

There are various methods of direct DNA transfer into plant cells. In electroporation, protoplasts are briefly exposed to a strong electric field. In microinjection, the DNA is mechanically injected directly into the cells using very small micropipettes. In microparticle bombardment, the DNA is adsorbed on microprojectiles such as magnesium sulfate crystals, tungsten particles or gold particles, and the microprojectiles are physically accelerated into cells or plant tissues. Regardless of the transformation technique employed, once procollagen- expressing progeny are identified, such plants are further cultivated under conditions which maximize expression thereof. Progeny resulting from transformed plants can be selected, by verifying presence of exogenous mRNA and/or polypeptides by using nucleic acid or protein probes (e.g. antibodies). The latter approach enables localization of the expressed polypeptide components (by for example, probing fractionated plants extracts) and thus also verifies the plant's potential for correct processing and assembly of the foreign protein.

Following cultivation of such plants, the telopeptide-comprising collagen is typically harvested. Plant tissues/cells are preferably harvested at maturity, and the procollagen molecules are isolated using extraction approaches. Preferably, the harvesting is effected such that the procollagen remains in a state that it can be cleaved by protease enzymes. According to one embodiment, a crude extract is generated from the transgenic plants of the present invention and subsequently contacted with the protease enzymes.

For the generation of atelocollagen or collagen, 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.

According to one embodiment, 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. 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 TrypZean™, a recombinant human trypsin expressed in corn (Sigma catalog #T3449).

As used herein the term "about" refers to ± 10 %.

The terms "comprises", "comprising", "includes", "including", "having" and their conjugates mean "including but not limited to".

The term "consisting of means "including and limited to".

The term "consisting essentially of" means that the composition, 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.

Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in 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.

Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases "ranging/ranges between" a first indicate number and a second indicate number and "ranging/ranges from" a first indicate number "to" a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.

As used herein 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. As used herein, 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.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley- Liss, N. Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; "Oligonucleotide Synthesis" Gait, M. J., ed. (1984); "Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., eds. (1985); "Transcription and Translation" Hames, B. D., and Higgins S. J., eds. (1984); "Animal Cell Culture" Freshney, R. I., ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al., "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference.

EXAMPLE 1

Generation of collagen coated fiber

MATERIALS AND METHODS

Preparation of Nano Cellulose Crystals: Nano Cellulose Crystals were produced by H ₂ SO ₄ hydrolysis of 200μιη 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.

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.

Preparation of 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.

Coating of PLA fibers: The 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).

RESULTS

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).

EXAMPLE 2

Imaging of collagen coated fiber

MATERIALS AND METHODS

Gelatin solution coloring procedure: 2.5μ1 of 0.1 % blue food dye was added to 350 μΐ 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.

NCC coating on pretreated PLA fibers: 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.

RESULTS

Confocal microscopy was used in order to identify the gelatin and NCC layers coating the PLA fiber. According to the literature, blue food coloring binds specifically to gelatin therefore, this coloring can be used to identify the different coating layers. As illustrated in Figure 2B, a clear blue radiance is observed when the fiber is coated with gelatin. When, the gelatin-coated fiber is covered with NCC, no blue radiance is observed (Figure 2C). Thus, it can be concluded that the gelatin coating is masked by the NCC layer. EXAMPLE 3

Plasma modified fibers

MATERIALS AND METHODS

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.

RESULTS

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 μιη thickness on pretreated PLA fiber with N ₂ plasma jet may be successfully performed

Plasma pretreatment, 0 ₂ plasma jet, was also performed. 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 μιη 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). When a collagen concentration of 18 % (w/v) was used, a thick homogenous Rhcollagen coating (of 5μιη) was obtained. At the lower Rhcollagen concentration (Figure 5B), a 1 μιη 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.

Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting.