Skin fillers and their uses

Abstract

The present invention provides improved compositions with adjustable rheological and mechanical properties, and methods for preparing the same. [Solution] A composition is provided, wherein the composition is a double crosslinked composition, comprising (a) plant-derived type I recombinant human collagen (plant-derived rh collagen) and (b) chemically crosslinked hyaluronic acid, wherein the plant-derived rh collagen is chemically crosslinked with the crosslinked hyaluronic acid.

Description

Technical Field

[0001] A photoinitiable double-crosslinked dermal filler comprising plant-derived human collagen and a cell growth promoting scaffold, and in some cases, methods of using a dermal filler for soft tissue augmentation are disclosed herein.

Background Art

[0002] Collagen is a major protein involved in the structural integrity of vertebrates and many other multicellular organisms. Collagen constitutes the major component of connective tissue, is the most abundant protein in mammals, and makes up approximately 30% of the proteins found in the body. The disappearance or degradation of collagen can occur as a result of aging or injury (Olsen et al., Adv Drug Deliv Rev. 2003 Nov. 28;55(12):1547-67).

[0003] One common aspect of aging is the development of muscle, fine wrinkles, or wrinkles. Treatments involving the use of collagen extracted from tissues have been used to reduce or eliminate muscle, fine wrinkles, or wrinkles. Similar treatments have been used to reduce scars.

[0004] Collagen is also a component of tendons. Tendon disorders are common injuries, usually associated with sports and physical activity, and are related to the degeneration and disruption of the arrangement of collagen fibers in tendons. Healing of damaged tendons requires organized activity of specific cells and the long-term presence of relevant growth factors (GFs) in the vicinity of the injury. Tendon disorders have recently become a leading cause of musculoskeletal complaints (Kaux et al. (January 2011) J.Sport.Sci.Med.January:238-253). Tendon disorders refer to a variety of painful conditions that develop within and around tendons and ligaments, which may result from an imbalance between pathological changes caused by overuse of tendons and the resulting regenerative response (Andres et al. (2008) Clin.Orthop.Relat.Res.466:1539-1554). Tendon disorders are associated with the degeneration and disruption of collagen arrangement (Maffulli et al. (2003) Clin.Sport.Med.22:675-692), and may also be associated with the presence of micro-tears in the fibers, increased vascular distribution, and mild inflammation (Khan et al. (1999) Sport.Med.27(6):393-408). Clinically, they are characterized by the onset of tendon stiffness, activity-related pain, decreased function, and sometimes localized swelling (Kaux 2011; Andres 2008). The collagen fibers are not the usual dense, parallel bundled appearance, but rather exhibit increased uneven and irregular compression, loosening, and undulation (Mafulli 2003). As people continue to be active even in old age, the incidence of tendon injuries is expected to increase in the coming decades. A wide variety of treatments are available for tendon disorders, including physiotherapy, pharmacological treatment, and combinations thereof, but clinical outcomes are unsatisfactory, and symptom recurrence is common (Kaux 2011).The infusion of autologous platelet-rich plasma (PRP) for the treatment of tendon disorders has received widespread attention over the past few decades (Delong et al. (2016) Curr. Orthpaedic Pract. 22:514-523, Kaux et al. (2012) Wound Repair Regen. 20:748-756, Yuan et al. (2013) Muscles. Ligaments Tendons J. 3(3):139-49, Di Matteo et al. (2015) Musculoskelet. Surg. 99(1):1-9). PRP is a plasma fraction of blood containing a high concentration of platelets. When injected into the site of injury, platelets release various types of growth factors (GFs) that are thought to promote the healing process. Among the growth factors associated with PRP, vascular endothelial growth factor (VEGF), transforming β growth factor (TGF-β), platelet-derived growth factor (PDGF), platelet-derived epidermal growth factor (PDEGF), fibroblast growth factor (bFGF), epidermal growth factor (EGF), and hepatocyte growth factor (HGF) have been reported (Delong 2016, Yuan 2013, Harrison et al. (2011) Am.J.Sports Med.39(4):729-734). Numerous in vitro studies and in vivo models have shown that PRP therapy enhances collagen expression and extracellular matrix production, stimulates angiogenesis, increases cell migration, differentiation, and proliferation, and thus aids in the healing of tendon injuries (Yuan 2013, Kajikawa et al. (2008) J.Cell.Physiol.215(3):837-845, Zhang et al. (2010) Am.J.Sports Med.38(12):2477-2486). However, clear clinical evidence regarding the efficacy of PRP therapy is limited (Delong 2016, Yuan 2013, Moraes et al. (2014)).

[0005] Collagen functions as a major component and key structural-mechanical determinant of the extracellular matrix (ECM) of most tissues [see, e.g., Kadler K. Birth Defects Res C Embryo Today. 2004;72:1-11; Kadler KE, Baldock C, Bella J, Boot-Handford RP. J Cell Sci. 2007;120:1955-1958; Kreger ST. Biopolymers. 2010 93(8):690-707]. Tropocollagen typically consists of three left-handed helices of procollagen (usually two identical helices and a third distinct helix) that combine to form right-handed triple helix tropocollagen, which then forms the profibrils.

[0006] The morphology and most properties of natural collagen are determined by the triple helix domain, which makes up more than 95% of the molecule. This domain consists of three α-chains, each containing approximately 1,000 amino acids, which are wrapped in a rope-like manner to form a dense triple helix structure. Because this triple helix is ​​wound in such a way that peptide bonds linking adjacent amino acids are embedded within the molecule, collagen molecules are resistant to attack by proteases such as pepsin.

[0007] Type I collagen is a typical fibrillary collagen and is the dominant collagen type in most tissues, including bone, tendons, skin, aorta, and lungs. Type I collagen fibers offer excellent tensile strength and limited extensibility. The most abundant molecular form of type I collagen is the heterotrimer, composed of two distinct α chains [α1(I)]2 and α2(I) (Inkinen, Connective Tissue Formation in Wound Healing an Experimental Study, Academic Dissertation, September 2003, University of Helsinki, Faculty of Science, Department of Biosciences, Division of Biochemistry).

[0008] In all fibrous collagen molecules, three polypeptide chains are constructed from repeating Gly-XY triplets, where X and Y may be any amino acids, but are often the imino acids proline and hydroxyproline. Collagen is particularly rich in glycine, proline, and hydroxyproline amino acid residues, and the protein sequence of collagen chains often has repeating amino acid sequences. Procollagen is modified by adding hydroxyl groups to proline and lysine residues. These hydroxylation reactions are catalyzed by prolyl-4-hydroxylase and lysyl-hydroxylase, respectively. The hydroxyl groups on the lysine residues are then glycosylated, followed by the formation of a triple helix.

[0009] A key characteristic of the collagen that forms the profibrils is that they are synthesized as precursor procollagen, containing spherical N-terminal and C-terminal elongated propeptides. Procollagen biosynthesis is a complex process involving several different post-translational modifications, including hydroxylation of proline and lysine, N-linked and O-linked glycosylation, and the formation of both intra- and inter-chain disulfide bonds. The enzymes that perform these modifications act in a coordinated manner to ensure the folding and organization of a correctly aligned and thermally stable triple-helical molecule.

[0010] Three-component polypeptide chains are organized within the rough endoplasmic reticulum (RER) to form procollagen. Because the polypeptide chains move simultaneously across the ER membrane, prolyl-4-hydroxylase (P4H)-dependent hydroxylation of proline and lysine residues occurs within the Gly-XY repeat region. The stability of the final triple helix structure of collagen is highly dependent on P4H-mediated hydroxylation of the collagen chains. Lysyl hydroxylase (LH, EC 1.14.11.4), galactosyltransferase (EC 2.4.1.50), and glucosyltransferase (EC 2.4.1.66) are enzymes involved in post-translational modification of collagen. These sequentially modify lysyl residues at specific positions to hydroxylysyl, galactosylhydroxylysyl, and glucosylgalactosylhydroxylysyl residues. These structures are unique to collagen and essential for their functional activity (Wang et al. (2002) Matrix Biology, 21(7):559-566). A single human enzyme, lysyl hydroxylase 3 (LH3), can catalyze all three consecutive steps in hydroxylysine-linked carbohydrate formation (Wang et al. (2002) Matrix Biology, 21(7):559-566). Once the polypeptide chain has fully moved into the lumen of the endoplasmic reticulum, the three pro-α chains link via C-propeptides to form a trimer molecule, with the Gly-XY repeat region forming a nucleation site at its C-terminus, ensuring correct chain alignment. The Gly-XY region then folds from the C-direction to the N-direction to form a triple helix (Khoshnoodi et al. (2006) J. Biol. Chem. 281:38117-38121).

[0011] The temporal relationship between polypeptide chain modification and triple helix formation is important because hydroxylation of proline residues is necessary to ensure the stability of the triple helix at body temperature. Once formed, the triple helix no longer functions as a substrate for hydroxylases. C-propeptides (and to a lower degree, N-propeptides) maintain the solubility of procollagen during the pathway out of the cell (Bulleid et al. (2000) Biochem.Socy.Transact., 28(4):350-353). After or during the secretion of procollagen molecules into the extracellular matrix, the propeptides are removed by procollagen N-proteinases and C-proteinases, thereby triggering spontaneous self-assembly from collagen molecules into fibrils (Hulmes, 2002, J.Struct.Biol.January-February;137(1-2):2-10). Removal of propeptides from procollagen by N-proteinases and C-proteinases reduces the solubility of procollagen by more than 1 / 10,000, which is necessary to initiate self-assembly from collagen to profibril at 37°C. Crucial to this assembly process are short telopeptides, which are non-triple-helical remnants of the N-terminal and C-terminal propeptides remaining after digestion by N / C proteinases. These peptides, through their crosslinkable aldehydes, ensure the correct covalent positioning of collagen molecules within the profibril structure and act to lower the critical concentration for self-assembly (Bulleid et al. (2000) Biochem. Socy. Transact., 28(4):350-353).

[0012] Natural collagen generally exists in connective tissue as collagen molecules containing telopeptides packed together in the form of profibrils. Each longitudinal pathway has a space that is slightly staggered longitudinally with respect to the next consecutive transversely adjacent longitudinal pathway, and is composed of molecules aligned in an end-to-end configuration. In this way, a gap is created between the opposing terminal regions of consecutive molecules in a given longitudinal pathway, which are bound by the staggered sides of molecules in a transversely adjacent, parallel longitudinal pathway.

[0013] The dispersion and solubilization of natural animal collagen can be achieved using various proteolytic enzymes that break intermolecular bonds and remove immunogenic non-helical telopeptides without affecting the fundamental rigid triple-helical structure that gives collagen its desirable properties (see, for example, U.S. Patents 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 atelocollagen can then be purified by repeated precipitation at low pH and high ionic strength, followed by washing at low pH and resolubilization. Nevertheless, soluble preparations are typically contaminated with cross-linked collagen chains, which reduces the homogeneity of the protein preparation.

[0014] Due to its unique characteristics and diverse profiles in human bodily functions, collagen is selected from a variety of biocompatible materials for use in tissue repair, aiding structural integrity, inducing cell infiltration, and promoting tissue regeneration. Of the five major types of collagen, type I collagen is the most abundant form of collagen in the human body.

[0015] Type I collagen can self-organize into fibrous hydrogels capable of supporting tissue cells via bioactive adhesion sites. Adding methacrylate groups to collagen generates collagen methacrylate (CMA), making it more resistant to degradation (Gaudet et al., Biointerphases (2012) 7:25-33). Thiolation of collagen may improve aggregation and mucosal adhesion, and may affect its swelling capacity (Duggan et al., Eur.J.Pharm.Biopharm. (April 2015) 91:75-81).

[0016] The unique properties of collagen contribute to its use in regenerative medicine products. Collagen provides a biomaterial with the characteristics needed for countless applications, including pharmaceuticals (hemostatic compression, sponges, healing bandages), medical (heart valves, tendons and ligaments, skin substitutes, fillers and other prostheses), dental (gingival grafts / periodontal disease), and cosmetic (additives, anti-wrinkle agents, microcontainers for fragrance substances). Collagen-based products manufactured in all of the aforementioned markets require large quantities of collagen raw materials for their production.

[0017] Human and animal-derived collagen, such as that from cadavers or animals (bovine, porcine, or horse), and collagen-based products have been used for application, injection, implantation, and oral administration. Uses include pre-shaping to desired forms for the repair or partial replacement of damaged bone or cartilage structures, injection into damaged joints, and injection as skin fillers.

[0018] The use of animal-derived collagen (including human-derived collagen) is problematic due to the potential risk of contamination by unconventional infectious agents. While the risks caused by bacterial and viral contamination can be fully controlled, prions are difficult to contain and pose a significant health risk. These infectious agents, which appear to have protein-like properties, are involved in the development of degenerative animal encephalopathy (sheep tremor, bovine spongiform encephalopathy) and human encephalopathy (Creutzfeldt-Jakob disease, Gerstmann-Stroussler syndrome, and Kuru disease). Other diseases (e.g., acquired immunodeficiency syndrome [AIDS], hepatitis, rabies, and some cancers) can also infect recipients. Formal control is difficult because it takes a long time for some encephalopathy and other diseases to develop. (See Castrow et al. (1983) J.Am.Acad.Dermatol.9(6):889-93 and Siegle et al. (1984) Arch.Dermatol.120(2):183-187 in general.)

[0019] Furthermore, in some patients, treatment with human or animal collagen can trigger cellular or humoral immune responses, including allergies. Additionally, the quality of collagen generally declines with the age of the source cadaver or organism, or can be impaired by other factors. Moreover, the extraction process can cause significant structural damage that impairs its biological and mechanical functions (Stein et al. (2009) Biomacromolecules 10(9):2640-2645, Shilo et al. (2013) Tissue Eng. Part A 19(13-14):1519-1526, Shoseyov et al. (2013) Tiss.Eng. Part A 19(13-14):1527).

[0020] Plants expressing collagen chains are known in the art (see, for example, WO2005 / 035442, U.S. Patent No. 6,617,431, U.S. Publication No. 2002 / 0098578, U.S. Publication No. 2002 / 0142391, Merle et al. (2002) FEBS Letters 515:114-118, Ruggiero et al. (March 3, 2000) FEBS Lett. 469(1):132-6). Using such plants, not only collagen but also collagen chains can be produced, but these chains are not properly hydroxylated, and therefore their self-assembly, whether in the plant or not, produces collagen that is inherently unstable. For example, plants can synthesize proteins containing hydroxyproline, but the prolyl hydroxylase involved in hydroxyproline synthesis in plant cells exhibits relatively loose substrate sequence specificity compared to mammalian P4H. Therefore, co-expression of collagen and the P4H gene in plants is necessary to produce collagen containing hydroxyproline only at the Y position of the Gly-XY triplet (Olsen et al. (2003) Adv. Drug Deliv. Rev., 55(12):1547-1567).

[0021] Processing of animal-derived "insoluble collagen" using plant-derived proteases such as ficin and / or papain is also known in the art (U.S. Patents No. 4,597,762, No. 5,670,369, No. 5,316,942, No. 5,997,895 and No. 5,814,328).

[0022] Attempts to generate human collagen by relying on naturally occurring hydroxylation mechanisms in plants have yielded collagen with poor proline hydroxylation (Merle et al. (2002) FEBS Letters 515:114-118). Such collagen melts or loses its triple helix structure at temperatures below 30°C. Co-expression of collagen and prolyl hydroxylase yields stable hydroxylated collagen that is biologically relevant for application at body temperature (Merle et al. (2002) FEBS Letters 515:114-118).

[0023] Hydroxylysine expressed in human collagen from tobacco forms less than 2% of the hydroxylysine found in bovine collagen (0.04% of residues / 1.88% of residues). This suggests that endogenous lysyl hydroxylase in plants is unable to adequately hydroxylate lysine in collagen.

[0024] Recent technological advancements have led to the development of a system for purifying natural human type I collagen (rh collagen) by introducing five human genes encoding heterotrimer type I collagen into tobacco plants (COLLPLANT®, Israel; SIGMA-ALDRICH®, St. Louis, Missouri, USA). [For example, Stein H. (2009) Biomacromolecules 10:2640-5, Yaari et al. (2013) Tiss.Eng.Part A 19(13 / 14):1502-1506, Willard et al. (2013) Tiss.Eng.Part A 19(13 / 14):1507-1518, Shilo et al. (2013) Tiss.Eng.Part A 19(13 / 14):1519-1526, Shoseyov et al. (2013) Tissue Eng.Part A See 19:1527-1533, and Shoseyov et al. (January / February 2014) Bioengineered 5:1, 1-4. This protein is purified to homogeneity through cost-effective industrial processes that take advantage of the properties inherent to collagen. See also WO2006 / 035442, WO2009 / 053985, and patents and patent applications derived therefrom, all of which are incorporated by reference as if fully described herein.

[0025] Compared to collagen extracted from tissues, which can be partially denatured and have detached cell-binding domains, plant-derived human type I collagen has a more consistent structure and more cell-binding domains (Shoseyov et al. (January / February 2014) Bioengineered 5:1, 1-4, Majumdar et al. (2015) J. Biomed. Mater. Res. Part B:Appl. Biomater. 104B:300-307). In additive manufacturing (AM) applications, where 3D objects are manufactured layer by layer using computer models of objects via 3D bioprinting, rh collagen can form functional three-dimensional (3D) matrices and scaffolds. Furthermore, rh collagen generally does not have the immunogenicity and disease migration problems associated with collagen extracted from tissues.

[0026] A method for producing collagen by expressing at least one type of collagen α chain and enabling its accumulation in intracellular compartments lacking endogenous P4H activity (U.S. Patent No. 8,455,717) is available in the same way as a method for producing atelocollagen from collagen containing human telopeptides derived from non-animal cells by protease treatment (U.S. Patent No. 8,759,487).

[0027] Type I collagen and rh collagen are considered candidate substances for use as main components in 3D bioprinting construction materials. Various types of scaffolds have been used for cosmetic and other reconstructive applications.

[0028] In addition, the use of soft tissue enhancements, such as skin fillers for reducing wrinkles, has been increasing. One possible way to use a skin filler involves injecting a polymeric skin filler material into the desired area and then contouring or shaping the filler into the desired form. Polymerization and crosslinking of the material by various methods can convert the monomers in the injected material to form polymers and chains that can form a network structure and retain the desired shaped form. There are several ways to form and crosslink polymers. One method involves a photoinduced reaction with a photoreactive reagent that generates reactive species in a monomer solution. See, for example, U.S. Patent Nos. 9,795,711, 8,945,624, 6,352,710, and U.S. Publication No. 2009 / 0324722, and Elisseeff et al. (March 1999) Proc. Natl. Acad. Sci. USA 96:3104 - 3107.

[0029] However, at least some of these approaches continue to focus on tissue-derived collagen or non-collagen polymers (e.g., poly(vinyl alcohol), hyaluronic acid, or polyethylene glycol). Additionally, the use of collagen extracted from tissue is limited due to its sensitivity to temperature and ionic strength that promotes natural gel formation at temperatures above 20°C under physiological conditions [see, for example, PureCol, Advanced BioMatrix, Inc.]. The typical temperature-dependent gel formation of collagen extracted from tissue significantly impairs its exact fluidity. Keeping the collagen at low temperature until application is a possible solution to this phenomenon but encompasses significant technical limitations. Another solution is the use of gelatin, a denatured form of collagen that does not gel under these conditions. However, gelatin lacks the true tissue and cell interactions of native collagen, resulting in the loss of important biological functions. Additionally, its viscosity makes it more difficult to inject under the dermis using a thin-gauge needle and also more difficult to spread and shape it within smaller cavities.

[0030] Therefore, there is a need for an improved injectable dermal filler having adjustable rheological and mechanical properties, as well as methods and uses thereof, and it would be highly desirable and advantageous to have the same. SUMMARY OF THE INVENTION MEANS FOR SOLVING THE PROBLEM

[0031] In one aspect, a double-crosslinked dermal filler comprising (a) plant-derived human collagen and (b) crosslinked hyaluronic acid, wherein the plant-derived human collagen is crosslinked to the crosslinked hyaluronic acid, is disclosed herein.

[0032] In related aspects, the plant-derived human collagen comprises (a) type 1 recombinant human collagen (rh collagen), or (b) the crosslinked hyaluronic acid comprises crosslinked hyaluronic acid and non-crosslinked hyaluronic acid, or (c) a combination thereof.

[0033] In related aspects, the crosslinking agent that links the crosslinked hyaluronic acid is different from the crosslinking agent that links the plant-derived human collagen to the crosslinked hyaluronic acid, or the ratio of crosslinked hyaluronic acid to plant-derived human collagen comprises a ratio of 4:1 to 1:2, or a combination thereof. Further related aspects, the crosslinking agent for crosslinking hyaluronic acid and the crosslinking agent for crosslinking plant-derived human collagen are independently selected from 1,4-butanediol diglycidyl ether (BBDE), 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide methiodide (EDC), Ν,Ν'-dicyclohexylcarbodiimide (DCC), Ν,Ν'-diisopropylcarbodiimide (DIC).

[0034] In one aspect, a method for preparing a double-crosslinked dermal filler comprising plant-derived human collagen crosslinked to crosslinked hyaluronic acid, (a) A process of crosslinking hyaluronic acid, (b) A step to neutralize cross-linked hyaluronic acid, (c) A process to neutralize plant-derived human collagen, (d) A step of mixing neutralized cross-linked hyaluronic acid with neutralized plant-derived human collagen, (e) The process of adding low molecular weight hyaluronic acid (MW HA), (f) A process of cross-linking a mixture of cross-linked hyaluronic acid and plant-derived human collagen, A method is disclosed herein that includes the step of (g) dialyzing a double-crosslinked crosslinked hyaluronic acid-plant-derived human collagen skin filler.

[0035] In relevant embodiments, the plant-derived human collagen includes type 1 recombinant human collagen (rh collagen), or the crosslinking agent for linking the crosslinked hyaluronic acid in step (a) is different from, or a combination thereof, the crosslinking agent for linking the plant-derived human collagen and the crosslinked hyaluronic acid in step (e). In relevant embodiments, the ratio of crosslinked hyaluronic acid to plant-derived human collagen includes a ratio of 4:1 to 1:2, or the crosslinking agent for crosslinking the hyaluronic acid and the crosslinking agent for crosslinking the plant-derived human collagen are independently selected from, or a combination thereof, 1,4-butanediol diglycidyl ether (BBDE), 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide methoidide (EDC), N,N'-dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide (DIC).

[0036] In addition, in one embodiment, there is a method for filling the tissue space beneath the epidermis, (a) A step of introducing a polymerizable solution into the tissue space, wherein the polymerizable solution is (i) Cross-linkable plant-derived human collagen, (ii) Hyaluronic acid (HA) or its modified derivatives, poly(vinyl alcohol) (PVA) or its modified derivatives, polyethylene glycol (PEG) or its modified derivatives, cellulose oxide (OC) or its modified derivatives, polymethyl methacrylate (PMMA) microspheres or its modified derivatives, tricalcium phosphate (TCP) or its modified derivatives, calcium hydroxyl apatite (CaHA) or its modified derivatives, carboxymethylcellulose or its modified derivatives, crystalline nanocellulose (CNC) or its modified derivatives, or combinations thereof, and (iii) an introduction step including a photoinitiator, (b) A method is disclosed herein that includes the step of inducing polymerization by shining light on the surface of the epidermis on the surface of the space described above.

[0037] In a related embodiment, the components of the polymerizable solution are introduced into the tissue space independently, at substantially the same location, and substantially simultaneously, and the crosslinkable plant-derived human collagen and photoinitiators are introduced together and independently from the above-mentioned hyaluronic acid (HA) or its modified derivatives, poly(vinyl alcohol) (PVA) or its modified derivatives, polyethylene glycol (PEG) or its modified derivatives, oxidized cellulose (OC) or its modified derivatives, polymethyl methacrylate (PMMA) microspheres or its modified derivatives, tricalcium phosphate (TCP) or its modified derivatives, calcium hydroxyl apatite (CaHA) or its modified derivatives, carboxymethyl cellulose or its modified derivatives, crystalline nanocellulose (CNC) or its modified derivatives, or combinations thereof, and are introduced into the tissue space independently and substantially simultaneously. In another related embodiment, the method further comprises the step of molding or shaping the polymerizable solution or components of the polymerizable solution within a desired configuration in the tissue space, which occurs concurrently with or following the step of light application.

[0038] In another related embodiment, the components of the polymerizable solution are introduced together into the tissue space as a mixture, and the crosslinkable plant-derived human collagen and photoinitiator are introduced together with the above-mentioned hyaluronic acid (HA) or its modified derivatives, or the above-mentioned poly(vinyl alcohol) (PVA) or its modified derivatives, or the above-mentioned polyethylene glycol (PEG) or its modified derivatives, or the above-mentioned cellulose oxide (OC) or its modified derivatives, or the above-mentioned polymethyl methacrylate (PMMA) microspheres or its modified derivatives, or the above-mentioned tricalcium phosphate (TCP) or its modified derivatives, or the above-mentioned calcium hydroxyl apatite (CaHA) or its modified derivatives, or the above-mentioned carboxymethylcellulose or its modified derivatives, or the above-mentioned crystalline nanocellulose (CNC) or its modified derivatives, or a combination thereof.

[0039] In another related embodiment, the components of the polymerizable solution are introduced into the tissue space independently of each other, and the crosslinkable plant-derived human collagen and photoinitiators are introduced together and independently of the above-mentioned hyaluronic acid (HA) or its modified derivatives, or the above-mentioned poly(vinyl alcohol) (PVA) or its modified derivatives, or the above-mentioned polyethylene glycol (PEG) or its modified derivatives, or the above-mentioned cellulose oxide (OC) or its modified derivatives, or the above-mentioned polymethyl methacrylate (PMMA) microspheres or its modified derivatives, or the above-mentioned tricalcium phosphate (TCP) or its modified derivatives, or the above-mentioned calcium hydroxyl apatite (CaHA) or its modified derivatives, or the above-mentioned carboxymethylcellulose or its modified derivatives, or the above-mentioned crystalline nanocellulose (CNC) or its modified derivatives, or any combination thereof.

[0040] In another related embodiment, after being introduced into a tissue space, the method further includes a step of molding or shaping the polymerizable solution or components of the polymerizable solution within a desired configuration within the tissue space, which occurs simultaneously with or following the step of illuminating.

[0041] In another related embodiment, the method is non-therapeutic, and the shaping or reshaping steps reduce muscles, folds, fine lines, wrinkles, or scars, or a combination thereof.

[0042] In another related embodiment, (a) Crosslinkable plant-derived human collagen is either methacrylated or thiolated type 1 human recombinant collagen (rh collagen), or (b) Modified derivatives of hyaluronic acid (HA), poly(vinyl alcohol) (PVA), polyethylene glycol (PEG), cellulose oxide (OC), polymethyl methacrylate (PMMA) microspheres, tricalcium phosphate (TCP), calcium hydroxyl apatite (CaHA), carboxymethylcellulose, or crystalline nanocellulose (CNC) include methacrylated or thiolated derivatives, or (c) Hyaluronic acid (HA), poly(vinyl alcohol) (PVA), polyethylene glycol (PEG), cellulose oxide (OC), polymethyl methacrylate (PMMA) microspheres, tricalcium phosphate (TCP), calcium hydroxyl apatite (CaHA), carboxymethylcellulose, or crystalline nanocellulose (CNC) containing cross-linked hyaluronic acid (HA), cross-linked poly(vinyl alcohol) (PVA), cross-linked polyethylene glycol (PEG), cross-linked cellulose oxide (OC), cross-linked polymethyl methacrylate (PMMA) microspheres, cross-linked tricalcium phosphate (TCP), cross-linked calcium hydroxyl apatite (CaHA), cross-linked carboxymethylcellulose, or cross-linked crystalline nanocellulose (CNC), or (d) A combination of (a) and (b), or (a) and (c).

[0043] In more relevant embodiments, when MA-rh collagen is selected, or when hyaluronic acid or its derivatives, or cross-linked hyaluronic acid is selected, the ratio of HA to MA-rh collagen is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 1:2, 1:3, 1:4, 1:5, 1:6, or 0:1.

[0044] In one embodiment, a method for filling a tissue space beneath the epidermis includes the step of introducing a double-crosslinked skin filler into the tissue space, wherein the double-crosslinked skin filler is (a) Plant-derived human collagen, (b) comprising cross-linked hyaluronic acid (HA) or its modified cross-linked derivative, cross-linked poly(vinyl alcohol) (PVA) or its modified cross-linked derivative, cross-linked polyethylene glycol (PEG) or its modified cross-linked derivative, cross-linked cellulose oxide (OC) or its modified cross-linked derivative, cross-linked polymethyl methacrylate (PMMA) microspheres or its modified cross-linked derivative, cross-linked tricalcium phosphate (TCP) or its modified cross-linked derivative, cross-linked calcium hydroxyl apatite (CaHA) or its modified cross-linked derivative, cross-linked carboxymethylcellulose or its modified cross-linked derivative, cross-linked crystalline nanocellulose (CNC) or its modified cross-linked derivative, or a combination thereof, Methods for crosslinking plant-derived human collagen to crosslinked hyaluronic acid (HA) or its modified crosslinked derivatives, crosslinked poly(vinyl alcohol) (PVA) or its modified crosslinked derivatives, crosslinked polyethylene glycol (PEG) or its modified crosslinked derivatives, crosslinked oxidized cellulose (OC) or its modified crosslinked derivatives, crosslinked polymethyl methacrylate (PMMA) microspheres or its modified crosslinked derivatives, crosslinked tricalcium phosphate (TCP) or its modified crosslinked derivatives, crosslinked calcium hydroxyl apatite (CaHA) or its modified crosslinked derivatives, crosslinked carboxymethyl cellulose or its modified crosslinked derivatives, or crosslinked crystalline nanocellulose (CNC) or its modified crosslinked derivatives are disclosed herein.

[0045] In related embodiments, the plant-derived human collagen is type 1 human recombinant collagen (rh collagen), MA or its thiolated derivative, or modified derivatives of hyaluronic acid (HA), poly(vinyl alcohol) (PVA), polyethylene glycol (PEG), cellulose oxide (OC), polymethyl methacrylate (PMMA) microspheres, tricalcium phosphate (TCP), calcium hydroxyl apatite (CaHA), carboxymethylcellulose, or crystalline nanocellulose (CNC), including methacrylated or thiolated derivatives, or a combination thereof.

[0046] In another related embodiment, when cross-linked HA is selected, the ratio of cross-linked HA to plant-derived human collagen is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 1:2, 1:3, 1:4, 1:5, 1:6, or 0:1.

[0047] In related embodiments, the method is non-therapeutic and reduces muscles, folds, fine lines, wrinkles, or scars, or combinations thereof.

[0048] In one embodiment, a polymerizable or non-polymerizable solution for use in tissue enhancement, (a) The polymerizable solution contains crosslinkable plant-derived human collagen and a photoinitiator for inducing polymerization, either before or simultaneously with the application of visible light, or (b) The nonpolymerizable solution comprises a double crosslinked skin filler containing plant-derived human collagen and crosslinked hyaluronic acid or crosslinked PVA, or crosslinked PGE, or crosslinked OC, wherein the plant-derived human collagen is crosslinked with crosslinked hyaluronic acid or crosslinked PVA, or crosslinked PGE, or crosslinked OC. The above-described use includes the steps of injecting the polymerizable or nonpolymerizable solution into the tissue space beneath the epidermis, and subsequently shaping or forming the polymerizable or nonpolymerizable solution within a desired configuration to reduce muscles, folds, fine lines, wrinkles, or scars.

[0049] In relevant embodiments, the crosslinkable plant-derived human collagen is methacrylated or thiolated, or the polymerizable solution further comprises hyaluronic acid (HA) or its modified derivatives or its photopolymerizable derivatives, poly(vinyl alcohol) (PVA) or its modified derivatives or its photopolymerizable derivatives, polyethylene glycol (PEG) or its modified derivatives or its photopolymerizable derivatives, polymethyl methacrylate (PMMA) microspheres or its modified derivatives or its photopolymerizable derivatives, tricalcium phosphate (TCP) or its modified derivatives or its photopolymerizable derivatives, calcium hydroxyl apatite (CaHA) or its modified derivatives or its photopolymerizable derivatives, carboxymethylcellulose or its modified derivatives or its photopolymerizable derivatives, crystalline nanocellulose (CNC) or its modified derivatives or its photopolymerizable derivatives, or combinations thereof, wherein the derivatives may include methacrylated or thiolated derivatives, or combinations thereof.

[0050] In another related aspect, tissue augmentation is required as a result of any medical or dental condition (gingival graft / periodontitis). In yet another related aspect, tissue augmentation reduces muscle, folds, fine lines, wrinkles, or scars, or a combination thereof.

[0051] One embodiment provides a method for inducing a cell proliferation-promoting scaffold in the tissue space beneath the epidermis, comprising the step of introducing a solution into the tissue space, wherein the solution is (a) Plant-derived human collagen, (b) comprising at least one growth factor or a source thereof, A method for promoting the healing or replacement of collagen-containing tissue is disclosed herein.

[0052] In related embodiments, the plant-derived collagen includes type 1 recombinant human collagen (rh collagen), or the source of at least one growth factor includes plasma or platelet-rich plasma, or the collagen-containing tissue includes skin, or any combination thereof.

[0053] In another embodiment, the method is non-therapeutic, and the cell proliferation-promoting scaffold fills the tissue space, reducing muscles, folds, fine lines, wrinkles, or scars, or a combination thereof.

[0054] In one embodiment, a solution for use for inducing a cell proliferation-promoting scaffold is disclosed herein, the solution comprising plant-derived human collagen and at least one growth factor or source thereof, the use comprising the step of injecting the solution into a tissue space beneath the epidermis, the use being for promoting healing or replacement resulting from the breakdown or damage of collagen-containing skin tissue.

[0055] In related embodiments, the plant-derived collagen includes type 1 recombinant human collagen (rh collagen), or the source of at least one growth factor includes plasma or platelet-rich plasma, or the collagen-containing tissue includes skin, or any combination thereof.

[0056] In another related embodiment, rh collagen comprises its methacrylate or thiol derivative.

[0057] In related embodiments, the solution used in this method further comprises hyaluronic acid (HA) or a modified derivative thereof, poly(vinyl alcohol) (PVA) or a modified derivative thereof, polyethylene glycol (PEG) or a modified derivative thereof, cellulose oxide (OC) or a modified derivative thereof, polymethyl methacrylate (PMMA) microspheres or a modified derivative thereof, tricalcium phosphate (TCP) or a modified derivative thereof, calcium hydroxyl apatite (CaHA) or a modified derivative thereof, carboxymethylcellulose or a modified derivative thereof, crystalline nanocellulose (CNC) or a modified derivative thereof, or a combination thereof, and a photoinitiator for inducing polymerization before or simultaneously with the application of visible light, or crosslinked hyaluronic acid or crosslinked PVA, or crosslinked PGE, or crosslinked OC, wherein plant-derived human collagen is crosslinked to crosslinked hyaluronic acid or crosslinked PVA, or crosslinked PGE, or crosslinked OC.

[0058] In related embodiments, the method is non-therapeutic, and the cell proliferation-promoting scaffold fills the tissue space, reducing muscles, folds, fine lines, wrinkles, or scars, or combinations thereof.

[0059] A solution for use in inducing a cell proliferation-promoting scaffold is disclosed herein, the solution comprising plant-derived human collagen and at least one growth factor or its source, the use comprising the step of injecting the solution into a tissue space beneath the epidermis, the use being for promoting healing or replacement resulting from the degradation or damage of collagen-containing tissue.

[0060] In related embodiments, the source of at least one growth factor is plasma or platelet-rich plasma, or plant-derived collagen is type 1 recombinant human collagen (rh collagen), or collagen-containing tissue is skin, or a combination thereof.

[0061] In another related embodiment, the solution for use further comprises hyaluronic acid (HA) or a modified derivative thereof, poly(vinyl alcohol) (PVA) or a modified derivative thereof, polyethylene glycol (PEG) or a modified derivative thereof, cellulose oxide (OC) or a modified derivative thereof, polymethyl methacrylate (PMMA) microspheres or a modified derivative thereof, tricalcium phosphate (TCP) or a modified derivative thereof, calcium hydroxyl apatite (CaHA) or a modified derivative thereof, carboxymethylcellulose or a modified derivative thereof, crystalline nanocellulose (CNC) or a modified derivative thereof, or a combination thereof, and a photoinitiator for inducing polymerization before or simultaneously with the application of visible light, or crosslinked hyaluronic acid or crosslinked PVA, or crosslinked PGE, or crosslinked OC, wherein plant-derived human collagen is crosslinked to crosslinked hyaluronic acid or crosslinked PVA, or crosslinked PGE, or crosslinked OC.

[0062] In one embodiment, a method for filling a tissue space beneath the epidermis is disclosed herein, comprising the steps of (a) introducing a polymerizable solution into the tissue space, wherein the polymerizable solution comprises (i) crosslinkable plant-derived human collagen and (ii) a photoinitiator, and inducing polymerization by shining light on the surface of the epidermis on the surface of the space.

[0063] In related embodiments, the polymerizable solution further comprises a step of molding or shaping the polymerizable solution within a desired configuration in a tissue space, the step of which occurs simultaneously with or following the step of illuminating.

[0064] In another related embodiment, the method is non-therapeutic, and the shaping or reshaping steps reduce muscles, folds, fine lines, wrinkles, or scars, or a combination thereof.

[0065] In another related embodiment, the crosslinkable plant-derived human collagen is methacrylated or thiolated type 1 human recombinant collagen (rh collagen).

[0066] Other objects, features, and advantages of the present invention will become apparent from the following description and drawings. [Brief explanation of the drawing]

[0067] [Figure 1A] This document describes the construction of various expression cassettes and vectors that have been used to transform test plants. All coding sequences synthesized as part of the study were optimized for expression in tobacco. Cloning schemes for type I collagen α1 or type II collagen α2 chains in plant expression vectors according to several embodiments of the present invention are shown. [Figure 1B] This document describes the construction of various expression cassettes and vectors that have been used to transform test plants. All coding sequences synthesized as part of the research were optimized for expression in tobacco. Cloning schemes for the enzyme prolyl-4-hydroxylase (P4H) in plant expression vectors according to several embodiments of the present invention are shown. [Figure 1C] This document describes the construction of various expression cassettes and vectors that have been used to transform test plants. All coding sequences synthesized as part of the research were optimized for expression in tobacco. Cloning schemes for proteinase C or proteinase N in plant expression vectors according to several embodiments of the present invention are shown. [Figure 1D] This document describes the construction of various expression cassettes and vectors that have been used to transform test plants. All coding sequences synthesized as part of the research were optimized for expression in tobacco. Cloning schemes for lysyl hydroxylase 3 (LH3) in plant expression vectors according to several embodiments of the present invention are shown. [Figure 2]This section describes various cotransformation approaches used to date. Each expression cassette is represented by a short name of its coding sequence. The coding sequences are specified in Table 1. Each cotransformation was performed using two pBINPLUS binary vectors. Each rectangle represents a single pBINPLUS vector containing one, two, or three expression cassettes. The promoter and terminator are specified in Example 1. [Figure 3] This is a conventional multiplex PCR screening of transformants showing positive results for collagen α1 (324 bp fragment) or collagen α2 (537 bp fragment) or both. [Figure 4]This is a conventional Western blot analysis of transgenic plants generated by simultaneous transformations 2, 3, and 4. Total soluble proteins were extracted from tobacco cotransformers 2, 3, and 4 and tested with an anti-collagen type I antibody (Chemicon Inc., no. AB745). The size marker was Fermentas Inc., no. SM0671. WT represents wild-type tobacco. A positive collagen band can be seen in plants that are PCR-positive for collagen type I α1 or α2, or both. A positive control band of 500 ng of human placenta-derived collagen type I (Chemicon Inc., no. CC050, extracted from human placenta by pepsin digestion) corresponds to approximately 0.3% (approximately 150 μg) of total soluble proteins in the transgenic plant sample. The larger band of approximately 140 kDa in the human collagen sample is procollagen containing its C-propeptide, as detected by the anti-carboxy-terminal propeptide of the collagen type I antibody (Chemicon Inc., number MAB1913). The smaller band of approximately 120 kDa in the human collagen sample is collagen without the propeptide. Due to their unusual composition, proline-rich proteins (including collagen) consistently migrate on the polyacrylamide gel as bands with higher molecular weights than expected. Therefore, collagen chains without the propeptide, with a molecular weight of approximately 95 kDa, migrate as a band of approximately 120 kDa. [Figure 5] This is a conventional Western blot analysis of transgenic plants produced by cotransformation of cell 8 (possessing an apoplast signal translated and fused to the collagen chain). Total soluble proteins were extracted from transgenic tobacco leaves and tested with an anti-collagen type I antibody (Chemicon Inc., no. AB745). A positive collagen α2 band was observed in plants 8-141. Human placental type I collagen (Chemicon Inc., no. CC050) was used as a control. [Figure 6A]Collagen triple helix organization and previously approved thermal stability are observed through heat treatment and trypsin or pepsin digestion. Total soluble proteins from tobacco 2-9 (expressing only col α1 and not P4H) and 3-5 (expressing col α1+2 and both human P4Hα and β subunits) were heat-treated (15 minutes at 38°C or 43°C), followed by trypsin digestion (20 minutes at room temperature [RT]), and tested with anti-collagen type I antibody in a Western blotting procedure. The positive control was a 500 ng sample of human collagen type I + wt tobacco total soluble protein. [Figure 6B] Collagen triple helix reorganization and previously approved thermal stability are observed after heat treatment and digestion with trypsin or pepsin. Total soluble protein was extracted from transgenic tobacco 13-6 (expressing collagen type I α1 and α2 chains (indicated by arrows), human P4Hα and β subunits, and human LH3), heat-treated (20 minutes at 33°C, 38°C, or 42°C), immediately cooled on ice to prevent triple helix reorganization, incubated with pepsin at room temperature (approximately 22°C) for 30 minutes, and then tested with anti-collagen type I antibody (Chemicon Inc., no. AB745) using a standard Western blotting procedure. A positive control was obtained by adding a 50 ng sample of human collagen type I (Chemicon Inc., no. CC050, extracted from human placenta by pepsin digestion) to total soluble protein extracted from wild-type (wt, wt) tobacco. [Figure 7] Conventional Northern blot analysis performed on wild-type tobacco is shown. The blot was probed with tobacco P4H cDNA. [Figure 8] Conventional Western blot analysis of transgenic plants produced by simultaneous transformations 2, 3, and 13. Total soluble proteins were extracted from tobacco cotransformers and tested with anti-human P4Hα and β and anti-collagen type I antibodies. [Figure 9]Conventional Western blot analysis (lane 1) of hybrid vacuolar target plant A (2-300+20-279) grown under a normal light regimen and vacuolar target plant 13-652 grown in the dark for 8 days. All plants express exogenous col1, col2, P4H-α and P4H-β, as well as LH3 (PCR validated). [Figure 10] This shows purified collagen derived from tobacco leaves after digestion with trypsin. The collagen was purified from tobacco plant transgenic leaves strain 13-6, which were pulverized in 100 mM Tris buffer, centrifuged, proteolytic, and precipitated in a high-salt buffer, as detailed in the Materials and Methods chapter. After resuspension, the collagen-containing pellet was washed, dialyzed, and concentrated to obtain the final product. This gel shows Coomassie staining analysis of the collected collagen samples, with lanes 1 and 2 representing collagen obtained after digestion of procollagen with 300 mg / L trypsin. Pig-derived collagen (0.5 mg / ml) without propeptides was loaded and run as a positive control for collagen type 1 α1 and α2 chains. [Figure 11] The purified collagen derived from tobacco leaves after digestion with various concentrations of trypsin is shown. After digestion with 20 mg / L trypsin (lanes 1-7) or 30 mg / L trypsin (lanes 8-10), collagen was extracted and purified as shown in Figure 10. The products were separated on 10% SDS-PAGE and analyzed with Coomassie stains. Propeptide-free porcine collagen (0.5 mg / ml) was loaded and used as a positive control for collagen type 1 α1 and α2 chains. [Figure 12] The purified collagen derived from tobacco leaves after digestion with trypsin and pepsin is shown. After digestion with 30 mg / L of trypsin and 1 μg / 200 ml of pepsin, the collagen was extracted and purified as shown in Figure 10 (lanes 1-2). The products were separated on 10% SDS PAGE and analyzed with Coomassie stain. Propeptide-free porcine collagen (0.5 mg / ml) was loaded and used as a positive control for collagen type 1 α1 and α2 chains. [Figure 13]The collagen chains obtained by digesting procollagen with subtilisin or bromelain are shown. Collagen was purified from tobacco plant transgenic leaves strain 13-361, pulverized in 100 mM Tris buffer, centrifuged, and proteolytically incubated with either subtilisin (1–25 mg / L) or bromelain (1–25 mg / L) for 3 or 6 hours. Samples were separated on 10% SDS-PAGE and blotted onto nitrocellulose membranes. Collagen chains were immunodetected using anti-collagen type I. The untreated supernatant collected after homogenization and centrifugation was used as a negative control free of collagen (lanes 3–4 sup). Propeptide-free porcine collagen (2.5 μg) was used as a positive control for α1 and α2 chains (lane 1). [Figure 14] The collagen chains obtained by digesting procollagen with papain are shown. Collagen was purified from tobacco plant transgenic leaves strain 13-361, pulverized in 100 mM Tris buffer, centrifuged, and proteolytically bisphagated with papain (1–25 mg / L) for 3 or 6 hours of incubation. Samples were separated on 10% SDS-PAGE and blotted onto nitrocellulose membranes. Collagen chains were immunodetected using anti-collagen type I. The untreated supernatant, collected after homogenization, centrifugation, and incubation at 15°C for 3 hours (lane 3) or 6 hours (lane 2) without enzymes, was used as a negative control free of collagen. Propeptide-free porcine collagen (2.5 μg) was used as a positive control for α1 and α2 chains (lane 1). [Figure 15]Collagen chains obtained by digesting procollagen with ficin or sabinase are shown. Collagen was purified from tobacco plant transgenic leaves strain 13-361, pulverized in 100 mM Tris buffer, centrifuged, and proteolytically bisphosphonated with ficin (1–25 mg / L) or sabinase (1–25 mg / L) for 3 or 6 hours of incubation time. Samples were separated on 10% SDS-PAGE and blotted onto nitrocellulose membranes. Collagen chains were immunodetected using anti-collagen type I. The untreated supernatant collected before proteolysis was used as a collagen-free control sample (lane 3). Propeptide-free porcine collagen (2.5 μg) was used as a positive control for α1 and α2 chains (lane 1). [Figure 16] Collagen chains obtained by digesting procollagen with Protamex or Alcalase are shown. Collagen was purified from tobacco plant transgenic leaves strain 13-361, pulverized in 100 mM Tris buffer, centrifuged, and proteolytically bisphosphonated with Protamex (1–25 mg / L) or Alcalase (1–25 mg / L) for 3 or 6 hours of incubation time. Samples were separated on 10% SDS PAGE and blotted onto nitrocellulose membranes. Collagen chains were immunodetected using anti-collagen type I. The untreated supernatant collected before proteolysis was used as a collagen-free control sample (lane 14). Propeptide-free porcine collagen (2.5 μg) was used as a positive control for α1 and α2 chains (lane 1). [Figure 17]The collagen chains obtained by digesting procollagen with esperase or neutrase are shown. Collagen was purified from tobacco plant transgenic leaves strain 13-361, pulverized in 100 mM Tris buffer, centrifuged, and proteolytically digested with esperase (1–25 mg / L) or neutrase (1–25 mg / L) after 3 or 6 hours of incubation. Samples were separated on 10% SDS-PAGE and blotted onto nitrocellulose membranes. Collagen chains were immunodetected using anti-collagen type I. Propeptide-free porcine collagen (2.5 μg) was used as a positive control for α1 and α2 chains (lane 1). [Figure 18] The collagen chains obtained by digesting procollagen with 8.0 L of esperase or alcalase are shown. Collagen was purified from tobacco plant transgenic leaves strain 13-361, pulverized in 100 mM Tris buffer, centrifuged, and proteolytically fermented with esperase (1–25 mg / L) or neutrase (1–25 mg / L) after 3 or 6 hours of incubation. Samples were separated on 10% SDS-PAGE and blotted onto nitrocellulose membranes. Collagen chains were immunodetected using anti-collagen type I. The untreated supernatant, collected after homogenization, centrifugation, and incubation at 15°C for 3 hours (lane 3) or 6 hours (lane 2) without proteolytic enzymes, was used as a negative control free of collagen. Propeptide-free porcine collagen (2.5 μg) was used as a positive control for α1 and α2 chains (lane 1). [Figure 19]The collagen chains obtained at various purification stages after digestion of procollagen with ficin are shown. Collagen was purified from tobacco plant transgenic leaves strain 13-361, pulverized in 100 mM Tris buffer, centrifuged, and proteolytically bisphosphonated with ficin (5 mg / L) after a 3-hour incubation time at 15°C. Samples were separated on 10% SDS PAGE and blotted onto nitrocellulose membranes. Collagen chains were immunodetected using anti-collagen type I. Samples collected after pulverization, centrifugation, and incubation of the supernatant with ficin were loaded into lane 5. Lanes 6-14 show samples of ficin-treated collagen at various stages of the purification process. Lane 6: Sample incubated and centrifuged after ficin. Lane 7: After salt precipitation and resuspending in 0.5 M acetic acid. Lane 8: Same sample as lane 7 with the addition of a centrifugation step. Lane 9: Same sample as lane 8 after resuspending in 0.5 M acetic acid and centrifugation. Lane 10: Mature collagen after resuspending in 10 mM HCl and dialyzing. Lane 11: Same sample as Lane 10 with an added filtration step. Lane 12: Same sample as Lane 11 with an added 5x concentration step. Lane 13: Same sample as Lane 11 with an added 20x concentration step. Lane 14: Same sample as Lane 13 with an added 5x concentration step. Untreated procollagen samples (lanes 3-4) were used as negative controls. Propeptide-free porcine collagen (2.5 μg) was used as a positive control for α1 and α2 chains (lane 1). [Figure 20]The collagen chains obtained at various purification stages after digestion of procollagen with ficin are shown. Collagen was purified from tobacco plant transgenic leaves strain 13-361, pulverized in 100 mM Tris buffer, centrifuged, and proteolytically bisphosphonated with ficin (5 mg / L) after a 3-hour incubation time at 15°C. Samples were separated on 10% SDS PAGE and blotted onto nitrocellulose membranes. Collagen chains were immunodetected using anti-collagen type I. Samples collected after pulverization, centrifugation, and incubation of the supernatant with ficin were loaded into lane 5. Lanes 6-14 show samples of ficin-treated collagen at various stages of the purification process. Lane 6: Sample incubated and centrifuged after ficin. Lane 7: After salt precipitation and resuspending in 0.5 M acetic acid. Lane 8: Same sample as lane 7 with the addition of a centrifugation step. Lane 9: Same sample as lane 8 after resuspending in 0.5 M acetic acid and centrifugation. Lane 10: Mature collagen after resuspending in 10 mM HCl and dialyzing. Lane 11: Same sample as Lane 10 with an added filtration step. Lane 12: Same sample as Lane 11 with an added 5x concentration step. Lane 13: Same sample as Lane 11 with an added 20x concentration step. Lane 14: Same sample as Lane 13 with an added 5x concentration step. Untreated procollagen samples (lanes 3-4) were used as negative controls. Propeptide-free porcine collagen (2.5 μg) was used as a positive control for α1 and α2 chains (lane 1). [Figure 21] This section shows the collagen content of samples after ficin treatment at various stages of purification. Collagen-containing samples were collected at each extraction and purification stage of the reactor-sized AMS purification procedure described in the Materials and Methods section. Samples were treated with ficin (5 mg / L, 15°C, 3 hours) for propeptide removal, separated on 10% SDS PAGE, and analyzed with Coomassie staining solutions. [Figure 22]This report describes the optimization of procollagen cleavage using food-grade ficin (optimization of ficin concentration and reaction time). AMS-pelletized tobacco leaf extract expressing procollagen was resuspended in extraction buffer and then incubated with increasing concentrations of food-grade ficin (5–15 mg / L). The reaction mixture was then incubated at 15°C for 1–3 hours. Cleavage was completed by centrifugation, and the protein samples were separated on 8% SDS-PAGE, transferred to a nitrocellulose membrane, and immunoblotting of α-1 and α-2 collagen chains was performed using an anti-collagen type I antibody. Procollagen bands are indicated by white arrows, while red arrows indicate bands of cleaved collagen. [Figure 23A] This report describes the optimization of procollagen cleavage using pharmaceutical-grade ficin (optimization of ficin concentration and reaction time). AMS-pelletized tobacco leaf extract expressing procollagen was resuspended in extraction buffer and then incubated with increasing concentrations of pharmaceutical-grade ficin (2.5–10 mg / L). The reaction mixture was then incubated at 15°C for 0.5–3 hours. Cleavage was completed by centrifugation, and the protein samples were separated on 8% SDS-PAGE, transferred to a nitrocellulose membrane, and immunoblotting of α-1 and α-2 collagen chains was performed using an anti-collagen type I antibody. Arrows indicate the procollagen and collagen bands. [Figure 23B] This report describes the optimization of procollagen cleavage using pharmaceutical-grade ficin (optimization of ficin concentration and reaction time). AMS-pelletized tobacco leaf extract expressing procollagen was resuspended in extraction buffer and then incubated with increasing concentrations of pharmaceutical-grade ficin (2.5–10 mg / L). The reaction mixture was then incubated at 15°C for 0.5–3 hours. Cleavage was completed by centrifugation, and the protein samples were separated on 8% SDS-PAGE, transferred to a nitrocellulose membrane, and immunoblotting of α-1 and α-2 collagen chains was performed using an anti-collagen type I antibody. Arrows indicate the procollagen and collagen bands. [Figure 23C]This report describes the optimization of procollagen cleavage using pharmaceutical-grade ficin (optimization of ficin concentration and reaction time). AMS-pelletized tobacco leaf extract expressing procollagen was resuspended in extraction buffer and then incubated with increasing concentrations of pharmaceutical-grade ficin (2.5–10 mg / L). The reaction mixture was then incubated at 15°C for 0.5–3 hours. Cleavage was completed by centrifugation, and the protein samples were separated on 8% SDS-PAGE, transferred to a nitrocellulose membrane, and immunoblotting of α-1 and α-2 collagen chains was performed using an anti-collagen type I antibody. Arrows indicate the procollagen and collagen bands. [Figure 24A] This document describes the optimization of procollagen cleavage using pharmaceutical-grade ficin (optimization of reaction buffer pH and salt concentration). AMS-pelletized tobacco leaf extract expressing procollagen was resuspended in an extraction buffer containing 10 mg / L pharmaceutical-grade ficin at various pH values ​​(5.5–9.5) while increasing the NaCl concentration (0.5–3 M). The reaction mixture was then incubated at 15°C for 1 hour. Cleavage was completed by centrifugation, and both the resulting pellet and supernatant protein samples were separated on an 8% SDS-PAGE and transferred to a nitrocellulose membrane. α-1 and α-2 collagen chains were immunoblotted using an anti-collagen type I antibody. Arrows indicate collagen bands. [Figure 24B] This document describes the optimization of procollagen cleavage using pharmaceutical-grade ficin (optimization of reaction buffer pH and salt concentration). AMS-pelletized tobacco leaf extract expressing procollagen was resuspended in an extraction buffer containing 10 mg / L pharmaceutical-grade ficin at various pH values ​​(5.5–9.5) while increasing the NaCl concentration (0.5–3 M). The reaction mixture was then incubated at 15°C for 1 hour. Cleavage was completed by centrifugation, and both the resulting pellet and supernatant protein samples were separated on an 8% SDS-PAGE and transferred to a nitrocellulose membrane. α-1 and α-2 collagen chains were immunoblotted using an anti-collagen type I antibody. Arrows indicate collagen bands. [Figure 25] This report describes the optimization of procollagen cleavage using pharmaceutical-grade ficin (optimization of EDTA and L-cysteine ​​concentrations in the reaction buffer). AMS-pelletized tobacco leaf extract expressing procollagen was resuspended in extraction buffer (pH 7.5) containing various concentrations of L-cysteine ​​(10–100 mM – upper panel of concentrations) and EDTA (8–80 mM – lower panel of concentrations). The samples were then incubated with 1 mg / L of pharmaceutical-grade ficin at 15°C for 1 hour. Cleavage was terminated by centrifugation, and the protein samples were separated on an 8% SDS-PAGE, transferred to a nitrocellulose membrane, and immunoblotting of α-1 and α-2 collagen chains using anti-collagen type I was performed. [Figure 26] This study demonstrates effective procollagen digestion by recombinant trypsin at pH 7.5. AMS-pelletized tobacco leaf extract expressing procollagen was resuspended in an extraction buffer (pH 7.5) containing L-cysteine ​​and EDTA. The sample was then incubated with 30–100 mg / L recombinant trypsin at 15°C for 1–3 hours. Clenching was completed, and the protein samples were separated on 8% SDS-PAGE, transferred to a nitrocellulose membrane, and immunoblotting of α-1 and α-2 collagen chains was performed using anti-collagen type I. [Figure 27] Viscosity (eta[η], cP) as a function of shear rate is shown. The solid line represents 2.7 mg / mL bovine collagen in phosphate-buffered saline (PBS), and the dashed line represents 2.79 mg / mL rh collagen in PBS. ▼: measured at 4°C, ▲: measured at 37°C. [Figure 28] This graph shows the viscosity of 3.4 mg / mL bovine collagen in FB as a function of shear rate. ▼: Measured at 4°C, ▲: Measured at 37°C. [Figure 29] This graph shows the viscosity of 10 mg / mL of rh-collagen-MA in PBS as a function of shear rate. ▲: measured at 4°C, ▼: measured at 37°C. [Figure 30]This shows the viscosity measurements of rh-collagen-MA in DMEM with and without the addition of HA / HAMA. [Figure 31] The storage modulus and loss modulus, as well as the tan phase shift angle, of rh-collagen-MA formulations at different concentrations before (upper graph) and after (lower graph) photocrosslinking are shown. [Figure 32] The G' and G'' values ​​at 37°C, recorded in a frequency sweep test and plotted at 1 Hz, are shown. [Figure 33A] A flowchart is provided for the processing of rh collagen and rh collagen methacrylate. It shows the upstream isolation and processing of procollagen and collagen (steps A-H). [Figure 33B] A flowchart is provided for the processing of rh collagen and rh collagen methacrylate. It shows two stages of downstream processing (steps IM and N-P and Z, respectively). [Figure 33C] A flowchart is provided for the processing of rh collagen and rh collagen methacrylate. It shows two stages of downstream processing (steps IM and N-P and Z, respectively). [Figure 34] The viscosity (eta[η], cP) of 5 mg / ml rh collagen methacrylate (CollMA) (solid black curve) and 5 mg / ml collagen MA + polyvinyl alcohol methacrylate (PVMA) (light gray curve) is shown, with CollMA:PVMA ratios of 5:1 (solid curve), 2:1 (dashed curve), and 1:2 (dotted curve). For comparison, the viscosity of 5 mg / ml rh collagen methacrylate is reported (black curve). [Figure 35]The viscosity of 5 mg / ml CollMA (solid black curve) and 5 mg / ml Collagen MA + Hyaluronic Acid Methacrylate (HAMA) (gray curve) is shown, with CollMA:HAMA ratios of 5:1 (solid curve) and 2:1 (dashed curve). For comparison, the viscosity of 5 mg / ml rh-collagen methacrylate is reported (solid black curve). These materials are not yet cross-linked but will be cross-linked after injection. Viscosity represents the injectability of the material. (HAMA-HA methacrylate; Collagen MA (ColMA)-rh-collagen methacrylate.) [Figure 36] The viscosities of 5 mg / ml CollMA (solid black curve) and 5 mg / ml Collagen MA + Oxidized Cellulose (OC) (gray curve) are shown for CollMA:OC ratios of 5:1 (solid curve), 2:1 (dashed curve), and 1:2 (dotted curve). For comparison, the viscosity of 5 mg / ml rh Collagen Methacrylate is reported (solid black curve). [Figure 37] Figures 33-35 provide a comparison of the data. The viscosities of 5 mg / ml Collagen MA (solid black curve) and 5 mg / ml Collagen MA + various additives (light gray to dark gray curves as shown in the figure) are shown in ratios of 5:1 (solid curve), 2:1 (dashed curve), and 1:2 (dotted curve). For comparison, the viscosity of 5 mg / ml rh Collagen Methacrylate is reported (solid black curve). [Figure 38] ColMA: This shows the polymerizable scaffold of rh collagen methacrylate (ColMA) + additive at an additive ratio of 2:1. ColMA alone was compared with ColMA combined with polyvinyl alcohol methacrylate (PVMA), hyaluronic acid methacrylate (HAMA), or oxidized cellulose (OC). The solution was mixed with the photoinitiator 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (0.1%) and irradiated with ultraviolet (UV) light (365 nm) for 20 seconds. [Figure 39]The survival rate test is shown. Upper graph: Comparison of the survival rate (and proliferation) of normal human fibroblasts (nHDF) when cultured in the presence of GF released from an rh-collagen-PRP matrix (black), in the presence of GF released from activated PRP (gray), and under starvation conditions (white). This data is the mean of two different fibroblast proliferation assays performed on PRP extracted from two different donors. *Significant difference (p<0.0002). Lower inset: Microscopic images of nHDF cells grown in the presence of GF released from an rh-collagen matrix combined with PRP (A), grown in the presence of GF released from activated PRP (B), and cultured under starvation conditions (C). Images were taken 7 days after cell seeding. [Figure 40] The weight of the scaffolds is shown as a function of time (each point represents the average of 6 scaffolds with 2 rats per time point and 3 injections per rat). [Figure 41A] Studies using a subcutaneous rat model (PDGF content as a function of time) are shown. *Significant differences between rh collagen matrix combined with PRP and PRP alone (p<0.038), and between rh collagen matrix alone and PRP alone (p<0.004). **Significant differences between rh collagen matrix alone and PRP alone, and between rh collagen matrix alone and rh collagen matrix combined with PRP (p<0.021). [Figure 41B] This study uses a subcutaneous rat model and shows the VEGF content as a function of time in the subcutaneous rat model. **Significant differences (p<0.007) were observed between rh collagen matrix combined with PRP and PRP alone, and between rh collagen matrix combined with PRP and rh collagen matrix alone.** [Figure 42] This shows the integral of the nominal PDGF and VEGF content in the injected matrix over 45 (or 30) days in a rat model. [Figure 43A]This shows the histopathological scoring of the Achilles tendon in a rat model of tendon disorder (mature fibrosis) treated with PRP or rh-collagen / PRP matrix. [Figure 43B] This shows the histopathological scoring of the Achilles tendon in a rat model of tendon injury (presence of mononuclear inflammatory cells) treated with PRP or rh-collagen / PRP matrix. [Figure 43C] This shows the histopathological scoring of the Achilles tendon in a rat model of tendon injury (presence of immature granulation tissue) treated with PRP or rh-collagen / PRP matrix. [Figure 44] This shows a comparison of the expression force (Newton, N) required to inject cross-linked hyaluronic acid (HA) (black curve - □), cross-linked hyaluronic acid (HA) + monomer collagen (▽), or cross-linked hyaluronic acid (HA) + profibrillated collagen (△) from a 32-gauge needle and a 1 ml syringe (Becton Dickinson [BD], reference no. 309628). Both cross-linked HA + monomer collagen and cross-linked HA + profibrillated collagen have a semi-interpenetrating network structure, and the collagen is not cross-linked in either case. [Figure 45] This shows a comparison of the expression power (Newton, N) required for injecting cross-linked hyaluronic acid (HA) (black) or a double cross-linked network structure of cross-linked hyaluronic acid (HA) and collagen (gray) from a 32-gauge needle and a 1 ml syringe (Becton Dickinson [BD], reference no. 309628). [Figure 46] This shows a comparison of the viscosity (eta[η], cP) of cross-linked hyaluronic acid (HA) (black), cross-linked hyaluronic acid (HA) + monomeric collagen (▽), or cross-linked hyaluronic acid (HA) + profibrillated collagen (△). [Figure 47] This shows a comparison of the viscosity (eta[η], cP) of cross-linked hyaluronic acid (HA) (black) or the double cross-linked network structure of cross-linked hyaluronic acid (HA) and collagen (gray). [Figure 48A] The image shows a mouse patch placed on top of a methacrylated collagen (collMA / rhcollagenMA) solution. [Figure 48B]This image shows methacrylated collagen (collMA / rh-collagenMA) polymerized and incorporated within skin tissue after being irradiated through the skin with a white light-emitting diode (LED) torch. [Figure 49] Two examples of dermal filler components are shown. The left diagram is a schematic of a semi-interpenetrating dermal filler containing cross-linked hyaluronic acid (HA) and rh collagen. The right diagram is a schematic of a double-crosslinked dermal filler containing cross-linked hyaluronic acid and rh collagen, where the cross-linked HA is further cross-linked with rh collagen. The light gray rods represent the HA cross-linker, the black strings represent HA, the rh collagen is represented as a light gray string, and the second cross-linker, which cross-links the cross-linked HA and rh collagen, is represented as a black circle. [Figure 50] The graph shows rheological measurements of storage modulus and loss modulus for various bicrosslinked formulations, measured using a HAAKE-RHEO STRESS 600™ instrument (THERMO SCIENTIFIC™) with a cone (1°) versus plate configuration (C35 / 1). Frequency sweep measurements were performed at a constant deformation of 0.8% and frequencies ranging from 0.02 to 100 Hz. Storage modulus (solid line) and loss modulus (dashed line) of representative double-crosslinked formulations (see Table 7) compared with commercially available skin fillers (solid and dashed lines: solid black line - commercial product; solid ▽ ​​- formulation 2; solid square - formulation 2A; solid upward pentagon - formulation 3; solid downward pentagon - formulation 1A; solid circle - formulation 1; dashed triangle - commercial product G"; dashed ▽ - formulation 2G"; dashed square - formulation 2A-G"; dashed circle - formulation 1G"; dashed downward pentagon - formulation 1A G"; dashed upward pentagon - formulation 3)). [Figure 51] Figure 50 shows a graph comparing the storage modulus and loss modulus of the reported formulations at f=1Hz. (White bar graph G'[Pa]; gray bar graph G[Pa].) [Figure 52]The graphs show the injectability of selected bicrosslinked formulations, measured using a MULTITEST 1-i MECMESIN® compression tester with a 1 ml LUER-LOK® syringe (BECTON-DICKINSON®) and a 30 G needle, as used for formulations 2, 2A, and 3 (Table 8). A commercially available dermal filler is included for comparison with the bicrosslinked formulations. The injectability of representative bicrosslinked formulations as a function of plunger displacement (12 mm / min) was compared with that of the commercially available dermal filler. (Black triangles - commercially available dermal filler; gray squares - formulation 3; gray upward-pointing pentagons - formulation 2; gray triangles - formulation 2A.) [Figure 53] Graphs show rheological measurements of storage modulus and loss modulus for various combinations of highly crosslinked hyaluronic acid (HA), rh collagen methacrylate (MA), and / or rh collagen (see Table 10) before (dashed lines) and after (solid lines) photocuring with visible light, compared to highly crosslinked HA (horizontal triangles of black discontinuous lines). Before photocuring, storage modulus and loss modulus were measured using a HAAKE-RHEO STRESS 600™ instrument (THERMO SCIENTIFIC™) with a cone (1°) pair plate configuration (C35 / 1). Frequency sweep measurements were performed at a constant deformation of 0.8% and frequencies ranging from 0.02 to 100 Hz. After photocuring (irradiation with visible light for 6 minutes using a white LED flashlight), the storage modulus and loss modulus were measured using a HAAKE-RHEO STRESS 600™ instrument (THERMO SCIENTIFIC™) with a serrated plate and plate configuration (PP20). Frequency sweep measurements were performed with a constant shear stress of 3 Pa, a frequency in the range of 0.02 to 100 Hz, and a constant vertical load of 0.3 N. (Solid triangles - Formulation 4 - after; solid triangles - Formulation 5 - after; solid squares - Formulation 6 - after; dashed upward pentagons - Formulation 4 - before; dashed downward pentagons - Formulation 5 - before; dashed circle - Formulation 6 - before; dashed black dotted horizontal triangles - highly bridged HA.) [Figure 54]The graphs shown at a frequency of F=1Hz compare the storage modulus and loss modulus of formulations 4, 5, and 6 in Table 10 before and after photocuring, as well as the storage modulus and loss modulus of the uncured, highly crosslinked HA. (White bar graphs G'[Pa]; gray bar graphs G[Pa].) [Figure 55] The graph shows the injectability of selected bicrosslinked formulations, measured using a MULTITEST 1-i MECMESIN® compression tester with a 1 ml LUER-LOK® syringe (BECTON-DICKINSON®) and a 30 G needle, which were used for all samples. The expression rate of representative bicrosslinked formulations as a function of plunger displacement (12 mm / min) was compared with that of highly crosslinked HA. (Black triangles - highly crosslinked HA; gray triangles - formulation 4; gray squares - formulation 5; gray circles - formulation 6.) [Figure 56] Representative histological images are shown 7 days after subcutaneous injection of formulations 2, 2A, and a control (commercial dermal filler) into the backs of Sprague dawley rats. In all cases, the arrows indicate elevated (but not severe) inflammatory responses to formulations 2 and 2A, suggesting the initiation of tissue regeneration. [Figure 57] Figure 56 shows the histological scores of formulations 2, 2A, and the control at day 7 from the analyzed tissues. (Black - control; light gray - formulation 2; dark gray - formulation 2A.) [Figure 58] The photocurable histological scoring results for photocurable dermal fillers on days 7, 14, and 20 are shown. (Black - highly crosslinked HA control; Gray - Formulation 4, highly crosslinked HA and rhColMA.) [Figure 59] The results of fibrosis scores 7 and 14 days after injection of formulation 4 (gray - highly crosslinked HA + rhColMA) and the control (black - highly crosslinked HA) are shown. [Modes for carrying out the invention]

[0068] Photoinitiated dermal fillers and double-crosslinked dermal fillers, as well as cell proliferation-promoting scaffolds, and methods of using them, for example, for soft tissue enhancement, are disclosed herein.

[0069] While collagen-producing plants can be used to produce not only collagen but also collagen chains, these chains are not properly hydroxylated, and therefore, their self-assembly, whether within the plant or not, results in inherently unstable collagen, in contrast to the plant-derived human collagen of this application.

[0070] To implement the polymerizable solution and double-crosslinked solution of the present invention, and the method of use, while reducing costs, the implementer devised a plant expression approach that ensures the correct hydroxylation of collagen, thereby enabling the in-plant production of collagen that closely mimics the characteristics of human type I collagen (e.g., molecular structure, temperature stability, cell interactions).

[0071] In one embodiment, a method for filling the tissue space beneath the epidermis, (a) A step of introducing a polymerizable solution into the tissue space, wherein the polymerizable solution is (i) Crosslinkable plant-derived human collagen, and (ii) A step of introducing a photoinitiator, (b) A method is disclosed herein that includes the step of inducing polymerization by shining light on the surface of the epidermis on the surface of the space described above.

[0072] In certain embodiments, the method further includes a step of molding or shaping the polymerizable solution within a desired configuration in a tissue space, either before or simultaneously with the light-exposure step. In another specific embodiment, the molding or shaping step reduces muscles, folds, fine lines, wrinkles, or scars.

[0073] In yet another specific embodiment, the polymer solution further comprises a filler containing hyaluronic acid (HA) or a modified derivative thereof, poly(vinyl alcohol) (PVA) or a modified derivative thereof, polyethylene glycol (PEG) or a modified derivative thereof, oxidized cellulose (OC) or a modified derivative thereof, or a combination thereof. In one specific embodiment, isolated plant-derived human collagen is optionally formulated using, for example, hyaluronic acid (HA), poly(vinyl alcohol) (PVA), polyethylene glycol (PEG), oxidized cellulose (OC), polymethyl methacrylate (PMMA) microspheres, tricalcium phosphate (TCP), calcium hydroxyl apatite (CaHA), carboxymethylcellulose, crystalline nanocellulose (CNC), or a combination thereof.

[0074] Modified derivatives include, but are not limited to, photopolymerizable versions of HA, PVA, PEG, or OC. Modifications include, but are not limited to, methacrylate or thiolation. In yet another specific embodiment, the light source is selected from light-emitting diodes (LEDs), lasers, xenon lamps, and the like.

[0075] In some embodiments, in the formulations disclosed herein, methacrylate rh collagen crosslinks only to itself under irradiation conditions. In some embodiments, in the formulations disclosed herein, methacrylate rh collagen crosslinks to thiolated rh collagen under irradiation conditions. In some embodiments, in the formulations disclosed herein, methacrylate rh collagen crosslinks to any MA / thiolated additive under irradiation conditions. In some embodiments, in the formulations disclosed herein, methacrylate rh collagen crosslinks to methacrylated HA under irradiation conditions. In some embodiments, in the formulations disclosed herein, methacrylate rh collagen crosslinks to thiolated HA under irradiation conditions. In some embodiments, in the formulations disclosed herein, methacrylate rh collagen crosslinks to methacrylated PVA under irradiation conditions. In some embodiments, in the formulations disclosed herein, methacrylate rh collagen crosslinks to thiolated PVA under irradiation conditions. In some embodiments, in the formulations disclosed herein, methacrylate rh collagen crosslinks to methacrylated PEG under irradiation conditions. In some embodiments, in the formulations disclosed herein, methacrylate rh collagen is crosslinked to thiolated PEG under irradiation conditions. In some embodiments, in the formulations disclosed herein, methacrylate rh collagen is crosslinked to methacrylated OC under irradiation conditions. In some embodiments, in the formulations disclosed herein, methacrylate rh collagen is crosslinked to thiolated OC under irradiation conditions.

[0076] Those skilled in the art will understand that a photocurable formulation is actually semi-IPN before curing and becomes an IPN (interpenetrated network) after curing. An IPN may contain two intertwined network structures, each one cross-linking with itself but not with the other.

[0077] In some embodiments, the crosslinking formulation includes a ratio of unmodified rh-collagen to adjust the stiffness after (photo-crosslinking) without reducing the final total amount of rh-collagen, since unmodified rh-collagen cannot crosslink under irradiation and therefore does not increase the final stiffness. Methacrylated HA may also be added to this final formulation.

[0078] In some embodiments, HA or MA-HA may be crosslinked with a crosslinking agent, for example, BDDE, as described in Example 23, for example, but not limited to BDDE. In some embodiments, the crosslinking agent for HA or MA-HA includes divinyl sulfone (DVS) or glutaraldehyde. In certain embodiments, HA or MA-HA crosslinked with BDDE does not further crosslink with rh collagen or MA-rh collagen, but instead forms a so-called interpenetrating network structure (left side of Figure 49).

[0079] In yet another specific embodiment, the plant-derived collagen includes rh collagen. In yet another specific embodiment, the plant-derived collagen is obtained from a genetically modified plant. In yet another specific embodiment, the genetically modified plant is selected from the group consisting of tobacco, corn, alfalfa, rice, potato, soybean, tomato, wheat, barley, canola, carrot, and cotton. In particular, the genetically modified plant is the tobacco plant.

[0080] In yet another specific embodiment, the genetically modified plant comprises an expressible sequence of at least one gene sequence of human deoxyribonucleic acid (DNA) selected from the group consisting of COL1, COL2, P4H-α, P4H-β, and LH3. In yet another specific embodiment, the plant-derived human collagen comprises at least one modified human collagen α-1 chain shown in SEQ ID NO: 3 and expressed in the genetically modified plant, and at least one modified human collagen α-2 chain shown in SEQ ID NO: 6 and expressed in the genetically modified plant, wherein the genetically modified plant further expresses exogenous prolyl-4-hydroxylase (P4H). In yet another specific embodiment, the method further comprises the step of expressing an exogenous polypeptide selected from the group consisting of lysyl hydroxylase (LH), protease N, and protease C. In yet another specific embodiment, the human collagen α-1 chain is encoded by the sequence described in SEQ ID NO: 1. In yet another specific embodiment, the human collagen α-2 chain is encoded by the sequence described in SEQ ID NO: 4.

[0081] In yet another embodiment, the exogenous P4H is mammalian P4H. In particular, the exogenous P4H is human P4H. In yet another embodiment, the method further comprises the steps of targeting human collagen α-1 to a vacuole of a plant or genetically modified plant and digesting it with ficin. In yet another embodiment, the method further comprises the steps of targeting human collagen α-2 to a vacuole of a plant or genetically modified plant and digesting it with ficin.

[0082] In yet another embodiment, the plant-derived human collagen is atelocollagen. In yet another embodiment, the plant-derived human collagen is atelocollagen having an amino acid (AA) sequence derived from SEQ ID NOs: 1 and SEQ ID NOs: 4. The atelocollagen is derived from the enzymatic digestion (e.g., by ficin) of procollagen, which is the product of SEQ ID NOs: 1 and SEQ ID NOs: 4.

[0083] In yet another embodiment, the photoinitiator induces polymerization of the polymerizable solution in response to visible light. In particular, the visible light has a wavelength of 390 to 800 nm. Specifically, the photoinitiator is selected from the group consisting of eosin Y + triethanolamine, riboflavin, and the like.

[0084] In another embodiment, a photoinitiator induces polymerization of a polymerizable solution in response to ultraviolet (UV) light. In particular, the photoinitiator is selected from the group consisting of lithium phenyl-2,4,6-trimethylbenzoyl phosphinate (LAP) or 1-[4 2-hydroxy-1-[4-(2-hydroxyethoxy)phenyl]-2-methylpropan-1-one (IRGACURE® 2959).

[0085] In another embodiment, the photoinitiator induces polymerization of a polymerizable solution in response to infrared light.

[0086] In yet another embodiment, the polymerizable solution is introduced into the tissue space via a hollow needle or cannula ranging from 27 gauge to 33 gauge.

[0087] In yet another embodiment, the polymerizable solution within the tissue space is molded or shaped into a desired configuration by manual massage. In yet another embodiment, the polymerizable solution within the tissue space is molded or shaped into a desired configuration using a molding or shaping instrument.

[0088] In yet another embodiment, the polymerizable solution in the tissue space is essentially non-gelatable at room temperature. In yet another embodiment, the polymerizable solution in the tissue space is essentially non-gelatable at 37°C. In yet another embodiment, the polymerizable solution containing plant-derived human collagen has a lower viscosity at room temperature compared to a similar polymerizable solution containing human or animal-derived collagen extracted from tissue, for example, bovine, porcine, or horse collagen, at the same concentration and formulation. In yet another embodiment, the polymerizable solution containing plant-derived human collagen has a lower viscosity at 37°C compared to a similar polymerizable solution containing human or animal-derived collagen extracted from tissue, at the same concentration and formulation.

[0089] As used throughout, the term “animal-derived collagen” may include bovine, porcine, or horse collagen or rat tail collagen, which is in contrast to human-derived collagen.

[0090] In yet another embodiment, a polymerizable solution containing plant-derived human collagen is introduced into the tissue space with less force at room temperature compared to a similar polymerizable solution containing human or animal-derived collagen extracted from tissue, at the same concentration and formulation. In yet another embodiment, a polymerizable solution containing plant-derived human collagen is introduced into the tissue space with less force at 37°C compared to a similar polymerizable solution containing human or animal-derived collagen extracted from tissue, at the same concentration and formulation.

[0091] In another embodiment, the use of a polymerizable solution injected into the tissue space beneath the epidermis to reduce muscles, folds, fine lines, wrinkles, or scars is disclosed herein, wherein the polymerizable solution comprises methacrylated or thiolated crosslinkable plant-derived human collagen and a photoinitiator for inducing polymerization before or simultaneously with the application of visible light, and further comprises the step of shaping or forming the polymerizable solution into a desired configuration for reducing muscles, folds, fine lines, wrinkles, or scars. In certain embodiments, the polymerizable solution further comprises a filler comprising hyaluronic acid (HA) or a modified derivative thereof, poly(vinyl alcohol) (PVA) or a modified derivative thereof, polyethylene glycol (PEG) or a modified derivative thereof, cellulose oxide (OC) or a modified derivative thereof, polymethyl methacrylate (PMMA) microspheres or a modified derivative thereof, tricalcium phosphate (TCP) or a modified derivative thereof, calcium hydroxyl apatite (CaHA) or a modified derivative thereof, carboxymethylcellulose or a modified derivative thereof, crystalline nanocellulose (CNC) or a modified derivative thereof, or any combination thereof.

[0092] Modified derivatives include, but are not limited to, photopolymerizable versions of HA, PVA, PEG, OC, PMMA, TCP, CaHA, carboxymethylcellulose, or CNC. Modifications include, but are not limited to, methacrylate or thiolation.

[0093] In another embodiment, a method for filling the tissue space beneath the epidermis, (a) A method is disclosed herein that includes the step of introducing a polymerizable solution into a tissue space, wherein the polymerizable solution comprises crosslinkable plant-derived human collagen.

[0094] This technology relates to a collagen-based polymerizable filler for cosmetic and medical applications that forms a moldable composition that can be polymerized by photoactivation using a light source, such as a visible light source. The polymerizable filler contains crosslinkable plant-derived human collagen together with a photoinitiator.

[0095] The intended technology has the advantage of enabling the in situ formation of skin fillers or implants with custom contours, typically without invasive surgical intervention or general anesthesia. Generally, collagen-based polymerizable solutions are introduced into the tissue space beneath the epidermis (i.e., below the epidermis), and polymerization is induced by exposure to visible light on the skin surface, i.e., from outside the body or skin, or applied to the epidermis.

[0096] The in situ polymerization method provides cosmetic and medical correction and / or enhancement procedures using a polymerizable solution containing polymer components capable of forming a water-insoluble crosslinked network structure upon photoactivation with a visible light source.

[0097] In some embodiments, the skin fillers or cell proliferation-promoting scaffolds disclosed herein are for cosmetic applications. In some embodiments, the skin fillers or cell proliferation-promoting scaffolds disclosed herein are for medical orthodontic applications. In some embodiments, the skin fillers or cell proliferation-promoting scaffolds disclosed herein are for use in augmentation procedures, e.g., tissue augmentation, but not limited to these. In some embodiments, the double-crosslinked skin fillers disclosed herein are for cosmetic applications. In some embodiments, the double-crosslinked skin fillers disclosed herein are for medical orthodontic applications. In some embodiments, the double-crosslinked skin fillers disclosed herein are required as a result of a medical or dental condition (gingival graft / periodontitis). In some embodiments, the double-crosslinked skin fillers disclosed herein are required as a result of a medical condition requiring skin augmentation. In some embodiments, the double-crosslinked skin fillers disclosed herein are for use in augmentation procedures, e.g., tissue augmentation, but not limited to these. In some embodiments, the photocurable skin fillers disclosed herein are for cosmetic applications. In some embodiments, the photocurable skin fillers disclosed herein are for medical orthodontic applications. In some embodiments, the photocurable skin fillers disclosed herein are required as a result of a medical or dental condition (gingival graft / periodontitis). In some embodiments, the photocurable skin fillers disclosed herein are required as a result of a medical condition requiring skin augmentation. In some embodiments, the photocurable skin fillers disclosed herein are for use in augmentation procedures, e.g., tissue augmentation, but not limited to these. In some embodiments, the cell proliferation-promoting scaffolds disclosed herein are for cosmetic applications. In some embodiments, the cell proliferation-promoting scaffolds disclosed herein are for medical orthodontic applications. In some embodiments, the cell proliferation-promoting scaffold skin fillers disclosed herein are required as a result of a medical or dental condition (gingival graft / periodontitis).In some embodiments, the cell proliferation-promoting scaffold skin fillers disclosed herein are required as a result of medical conditions necessitating skin augmentation. In some embodiments, corrective medical applications include treating tendinitis. In some embodiments, the cell proliferation-promoting scaffolds disclosed herein are intended for use in augmentation procedures, for example, tissue augmentation, but are not limited to these.

[0098] In some embodiments, tissue enhancement is the enhancement of skin tissue.

[0099] In some embodiments, the use of skin fillers containing the cell proliferation-promoting scaffolds disclosed herein is in humans. In some embodiments, the use of skin fillers containing the cell proliferation-promoting scaffolds disclosed herein in humans reduces muscles, folds, fine lines, wrinkles, or scars, or any combination thereof. In some embodiments, the reduction of muscles, folds, fine lines, wrinkles, or scars, or any combination thereof, is for cosmetic purposes. In some embodiments, the reduction of muscles, folds, fine lines, wrinkles, or scars, or any combination thereof, is for cosmetic purposes. In some embodiments, the use of skin fillers containing the cell proliferation-promoting scaffolds disclosed herein in humans enhances tissue, for example, epidermis or skin tissue, but not limited to these. In some embodiments, tissue enhancement is for cosmetic purposes. In some embodiments, tissue enhancement is for medical treatment. In some embodiments, tissue enhancement is part of an enhancement procedure. In some embodiments, tissue enhancement is part of a skin enhancement procedure.

[0100] In some embodiments, tissue augmentation is required as a result of any medical or dental condition (gingival graft / periodontitis).

[0101] In certain embodiments, the skin fillers for use described herein include interpenetrating (IPN) network structures or semi-interpenetrating (semi-IPN) network structures, where different components can be crosslinked to themselves but not to each other. In some embodiments, the IPN or semi-IPN skin filler comprises rh collagen and a filler, e.g., hyaluronic acid (HA), poly(vinyl alcohol) (PVA), polyethylene glycol (PEG), oxidized cellulose (OC), or derivatives thereof, or combinations thereof. In some embodiments, the IPN or semi-IPN comprises rh collagen and crosslinked HA. In some embodiments, the IPN or semi-IPN comprises rh collagen derivatives, e.g., methacrylated rh collagen or thiol rh collagen and / or derivatives of the filler, e.g., methacrylated HA, PVA, PEG or OC, or thiol HA, PVA, PEG or OC, or combinations thereof.

[0102] In some embodiments, the IPN or semi-IPN network structure or double-crosslinked network structure containing a skin filler includes a ratio of filler, for example, but not limited to HA, PVA, PEG, or OC, to rh collagen of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 1:2, 1:3, 1:4, 1:5, 1:6, or 0:1. In some embodiments, the IPN or semi-IPN network structure containing a skin filler includes a ratio of MA filler, for example, but not limited to HA, PVA, PEG, or OC, to rh collagen of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 1:2, 1:3, 1:4, 1:5, 1:6, or 0:1. In some embodiments, the IPN or semi-IPN network structure containing a skin filler includes a ratio of the filler, e.g., HA, PVA, PEG, or OC, to MA-rh collagen of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 1:2, 1:3, 1:4, 1:5, 1:6, or 0:1.

[0103] In some embodiments, the IPN or semi-IPN or double-crosslinked network structure containing a skin filler includes a ratio of HA to rh collagen of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 1:2, 1:3, 1:4, 1:5, 1:6, or 0:1. In some embodiments, the IPN or semi-IPN or double-crosslinked network structure containing a skin filler includes a ratio of MA-HA to rh collagen of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 1:2, 1:3, 1:4, 1:5, 1:6, or 0:1. In some embodiments, the IPN or semi-IPN network structure containing a skin filler includes a ratio of HA to MA-rh collagen of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 1:2, 1:3, 1:4, 1:5, 1:6, or 0:1. In some embodiments, the IPN or semi-IPN network structure containing the skin filler includes MA-HA to MA-rh collagen ratios of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 1:2, 1:3, 1:4, 1:5, 1:6, or 0:1.

[0104] In some embodiments, the IPN or semi-IPN or double-crosslinked network structure containing a skin filler includes a ratio of filler to rh collagen of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 1:2, 1:3, 1:4, 1:5, 1:6, or 0:1. In some embodiments, the IPN or semi-IPN or double-crosslinked network structure containing a skin filler includes a ratio of MA-HA, or MA-PVA, or MA-PEG, or MA-OC, to rh collagen of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 1:2, 1:3, 1:4, 1:5, 1:6, or 0:1. In some embodiments, the IPN or semi-IPN network structure containing the skin filler includes a ratio of MA-HA, MA-PVA, MA-PEG, or MA-OC to MA-rh collagen of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 1:2, 1:3, 1:4, 1:5, 1:6, or 0:1.

[0105] In some embodiments, an IPN or semi-IPN network structure comprising a skin filler or a double-crosslinked skin filler includes a cell proliferation-promoting scaffold.

[0106] In certain embodiments, the skin fillers for use described herein include photocurable skin fillers comprising at least one component, for example, rh collagen, a methacrylate-rh collagen derivative, or a thiol-rh collagen derivative. In some embodiments, the curable skin filler comprises rh collagen and a filler, for example, hyaluronic acid (HA), poly(vinyl alcohol) (PVA), polyethylene glycol (PEG), oxidized cellulose (OC), or derivatives thereof, or combinations thereof. In some embodiments, the photocurable skin filler comprises MA-rh collagen and HA or a derivative thereof. In some embodiments, the photocurable skin filler comprises rh collagen derivatives, for example, methacrylated rh collagen or thiol rh collagen and / or derivatives of the filler, for example, methacrylated HA, PVA, PEG, or OC, or thiolized HA, PVA, PEG, or OC, or combinations thereof.

[0107] In some embodiments, the photocurable dermal filler comprises a filler, for example, not limited to HA, PVA, PEG, or OC, or derivatives thereof, in a ratio of rh-collagen to 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 1:2, 1:3, 1:4, 1:5, 1:6, or 0:1. In some embodiments, the photocurable dermal filler comprises a filler, for example, not limited to HA, PVA, PEG, or OC, or derivatives thereof, in a ratio of rh-collagen to MA-rh-collagen to 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 1:2, 1:3, 1:4, 1:5, 1:6, or 0:1. In some embodiments, the photocurable dermal filler comprises a filler, for example, but not limited to HA, PVA, PEG, or OC, and thiol-rh collagen in a ratio of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 1:2, 1:3, 1:4, 1:5, 1:6, or 0:1. In some embodiments, the photocurable dermal filler comprises an MA-filler and MA-rh collagen in a ratio of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 1:2, 1:3, 1:4, 1:5, 1:6, or 0:1.

[0108] In some embodiments, the photocurable dermal filler includes a ratio of HA to MA-rh collagen of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 1:2, 1:3, 1:4, 1:5, 1:6, or 0:1, 1:3, 1:4, 1:5, or 0:1. In some embodiments, the photocurable dermal filler includes a ratio of MA-HA to MA-rh collagen of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 1:2, 1:3, 1:4, 1:5, 1:6, or 0:1. In some embodiments, the photocurable dermal filler includes a ratio of PVA, PEG, or OC to MA-rh collagen of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 1:2, 1:3, 1:4, 1:5, 1:6, or 0:1. In some embodiments, the photocurable dermal filler includes MA-PVA, MA-HA- or OC and MA-rh collagen in a ratio of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 1:2, 1:3, 1:4, 1:5, 1:6, or 0:1. In some embodiments, the HA component of the photocurable dermal filler includes cross-linked HA or cross-linked MA-HA.

[0109] Throughout this application, various embodiments of the dermal filler and their uses may be described in range form. It should be understood that the range form is merely for convenience and conciseness and should not be interpreted as an inflexible limitation on the scope of the invention. Therefore, range descriptions should be considered to specifically disclose all possible sub-ranges and the individual numbers within those ranges. For example, a range description such as 1:1 to 6:1 should be considered to specifically disclose sub-ranges such as 1.1:1, 1.2:1, 1.3:1 to 5.9:1, 1:1.1 to 1:1.9, and the individual numbers included in those ranges and parts thereof, such as 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0110] Whenever a numerical range is given herein, it is understood that any cited digit (fraction or integer) is included within that range. The expressions “range between” the first digit and the second digit, and “range from” the first digit to the second digit, are used interchangeably herein and mean all the first and second digits, as well as all the fractions and integers between them.

[0111] For example, the present disclosure provides a skin filler comprising crosslinkable plant-derived human collagen, either alone or in combination with fillers such as hyaluronic acid (HA), poly(vinyl alcohol) (PVA), polyethylene glycol (PEG), oxidized cellulose (OC), or a combination thereof, for tissue spaces beneath the epidermis, wherein these are crosslinked to form water-insoluble crosslinked polymer preparations in situ upon visible light activation in the presence of a photoinitiator. In some embodiments, the collagen is methacrylated or thiolated.

[0112] In some embodiments, the skin filler provides the uses described herein and forms an IPN or semi-IPN network structure. In some embodiments, the skin filler provides the uses described herein and forms a double-crosslinked network structure.

[0113] In certain embodiments, the double-crosslinked dermal fillers provided herein for use include rh collagen crosslinked to a crosslinked filler, such as crosslinked hyaluronic acid (HA), crosslinked poly(vinyl alcohol) (PVA), crosslinked polyethylene glycol (PEG), crosslinked oxidized cellulose (OC), or their crosslinked derivatives, or combinations thereof. In certain embodiments, the double-crosslinked dermal fillers provided herein for use include rh collagen further crosslinked to a methacrylated or thiolated crosslinked filler, such as HA, PVA, PEG, or OC.

[0114] In certain embodiments, in a double-crosslinked dermal filler, the ratio of the crosslinked filler to rh collagen is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 1:2, 1:3, 1:4, 1:5, 1:6, or 0:1. In certain embodiments, in a double-crosslinked dermal filler, the ratio of the MA filler to rh collagen is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 1:2, 1:3, 1:4, 1:5, 1:6, or 0:1. In certain embodiments, in a double-crosslinked dermal filler, the ratio of the crosslinked filler to MA-rh collagen is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 1:2, 1:3, 1:4, 1:5, 1:6, or 0:1. In certain embodiments, the ratio of MA-filler to MA-rh collagen in a double-crosslinked dermal filler is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 1:2, 1:3, 1:4, 1:5, 1:6, or 0:1. In certain embodiments, the ratio of thiolated filler to rh collagen in a double-crosslinked dermal filler is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 1:2, 1:3, 1:4, 1:5, 1:6, or 0:1. In certain embodiments, the ratio of crosslinked filler to thiolated rh collagen in a double-crosslinked dermal filler is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 1:2, 1:3, 1:4, 1:5, 1:6, or 0:1. In certain embodiments, the ratio of thiolated filler to thiolated rh collagen in a double-crosslinked skin filler is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 1:2, 1:3, 1:4, 1:5, 1:6, or 0:1.

[0115] In certain embodiments, in a double-crosslinked dermal filler, crosslinked HA or crosslinked MA-HA is further crosslinked with rh collagen or methacrylated rh collagen or thiol rh collagen to produce a double-crosslinked dermal filler. In certain embodiments, in a double-crosslinked dermal filler, the ratio of crosslinked HA to rh collagen or methacrylated rh collagen or thiol rh collagen is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 1:2, 1:3, 1:4, 1:5, 1:6, or 0:1. In certain embodiments, in a double-crosslinked dermal filler, the ratio of crosslinked MA-HA to rh collagen or methacrylated rh collagen or thiol rh collagen is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 1:2, 1:3, 1:4, 1:5, 1:6, or 0:1. In certain embodiments, the ratio of cross-linked MA-HA to rh collagen or methacrylated rh collagen or thiol rh collagen in the double cross-linked skin filler is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 1:2, 1:3, 1:4, 1:5, 1:6, or 0:1.

[0116] In certain embodiments, in a double-crosslinked dermal filler, crosslinked PVA, PEG, or OC, or crosslinked MA-PVA, MA-PEG, or MA-OC, is further crosslinked with rh collagen or methacrylated rh collagen to produce a double-crosslinked dermal filler. In certain embodiments, in a double-crosslinked dermal filler, the ratio of crosslinked PVA, PEG, or OC to rh collagen is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 1:2, 1:3, 1:4, 1:5, 1:6, or 0:1. In certain embodiments, in a double-crosslinked dermal filler, the ratio of crosslinked MA-PVA, MA-PEG, or MA-OC to rh collagen is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 1:2, 1:3, 1:4, 1:5, 1:6, or 0:1. In certain embodiments, the ratio of cross-linked PVA, PEG, or OC to MA-rh collagen in a double-crosslinked dermal filler is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 1:2, 1:3, 1:4, 1:5, 1:6, or 0:1. In certain embodiments, the ratio of cross-linked MA-PVA, MA-PEG, or MA-OC to MA-rh collagen or thiol-rh collagen in a double-crosslinked dermal filler is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 1:2, 1:3, 1:4, 1:5, 1:6, or 0:1.

[0117] In certain embodiments, in a double-crosslinked dermal filler, crosslinked thiol-PVA, thiol-PEG, or thiol-OC is further crosslinked with rh collagen or methacrylated rh collagen to produce a double-crosslinked dermal filler. In certain embodiments, in a double-crosslinked dermal filler, the ratio of crosslinked thiol-PVA, thiol-PEG, or thiol-OC to rh collagen is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 1:2, 1:3, 1:4, 1:5, 1:6, or 0:1. In certain embodiments, in a double-crosslinked dermal filler, the ratio of crosslinked thiol-PVA, thiol-PEG, or thiol-OC to MArh collagen or thiol-rh collagen is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 1:2, 1:3, 1:4, 1:5, 1:6, or 0:1.

[0118] In some embodiments, any water-soluble coupling agent capable of crosslinking hyaluronic acid to collagen can be used. Some non-limiting examples of coupling agents include carbodiimides such as N,N'-dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide (DIC), or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). Carbodiimide coupling agents may facilitate the formation of ester or amide bonds without becoming part of the bond. In other words, the ester or amide bond may contain atoms from a carboxylate group from one of the hyaluronic acid or collagen and a hydroxyl or amine group from the other. However, other coupling agents that become part of the crosslinking group may be used. The concentration of the coupling agent may vary. In some embodiments, the coupling agent may be present at about 2 mM to about 150 mM, about 2 mM to about 50 mM, about 20 mM to about 100 mM, or about 50 mM. In some embodiments, the coupling agent is EDC present at concentrations of approximately 20 mM to approximately 100 mM, approximately 2 mM to approximately 50 mM, or approximately 50 mM. In some embodiments, the coupling agent is EDC present in amounts equal to 10 to 100 times the number of free amines in rh collagen. In some embodiments, the coupling agent is EDC present in amounts equal to 50 times the number of free amines in rh collagen. Increasing the carbodiimide concentration up to approximately 50 mM may result in a crosslinked polymer matrix with high hydrogel rigidity and / or less swelling.

[0119] Those skilled in the art will understand that a dermal filler containing double crosslinking, which is crosslinked to itself and then to rh collagen, is different from a dermal filler that involves directly crosslinking collagen and HA using a single type of crosslinking agent in a single reaction. The properties of such dermal fillers are different.

[0120] For example, this polymerizable solution can be used to block or fill various lumens and voids just below the skin surface. Thus, the present technology provides a method for tissue enhancement in a host such as a human patient, wherein the polymerizable solution of the purpose described above is introduced into the target site using a method known in the art, for example, by injecting the polymerizable solution into or on a tissue site requiring enhancement, and once applied, shining visible light on the body surface above it to polymerize the deposited polymerizable solution.

[0121] "Augmentation" means the repair, prevention, or mitigation of defects, particularly defects resulting from the loss or absence of tissue, by providing, enhancing, or replacing such tissue with polymers, networks, or objects. Augmentation also means the reinforcement of natural structures or features, i.e., constructs added to existing body parts (e.g., lips, nose, breasts, ears, organ parts, jaw, cheeks, etc.) to increase their size. Therefore, examples of tissue augmentation include filling or reducing muscles, folds, wrinkles, scars, small facial depressions, cracked lips, and epidermal wrinkles in the mid or superficial parts of the face, neck, hands, feet, fingers, and toes; correcting mild deformities caused by aging or disease, including those in the hands and feet, fingers, and toes; augmenting the vocal cords or glottis to restore speech; filling the skin of muscles and facial lines due to sleep; replacing skin and subcutaneous tissue lost due to aging; lip augmentation; filling wrinkles around the eyes and orbital sulcus; breast augmentation; chin augmentation; cheek and / or nose augmentation; filling depressions in the skin or subcutaneous soft tissue caused by excessive liposuction or other external factors, such as acne or traumatic scars and wrinkles; and filling nasolabial folds, nasolabial folds, glabellar ridges, and sphincter ridges below the mouth.

[0122] In some embodiments, the polymerizable solution of interest encompasses a polymerizable solution having a viscosity suitable for easy extrusion by a delivery means such as a fine surgical needle (e.g., a needle having a gauge of at least 27 gauge, at least 33 gauge, or a finer needle) at the temperature of use. Thus, a "injectable" solution is one having a texture and viscosity that allows it to flow through a suitable delivery device, e.g., a surgical needle, other surgical instruments, or other delivery means such as an endoscope or instruments used in percutaneous discectomy. Accordingly, the polymerizable solution of interest is injectable by a suitable applicator, e.g., a catheter, cannula, needle, syringe, tubular device, etc., as is known in the art.

[0123] Once injected into a tissue space, the polymerizable solution may be manipulated, massaged, molded, or shaped within the desired contours of the tissue space, typically after photoinitiation of polymerization is facilitated. In one embodiment, manipulation, massaging, molding, or shaping is performed during the gelation process. The polymerizable solution, the solution in polymerization, or the partially polymerized solution can be molded by external manipulation, for example, using molding means such as a surgical compressor, or other tools or instruments using a flat or curved surface, fingers, palms, knuckles, etc.

[0124] Remarkably, the genetically modified, crosslinkable plant-derived human collagen of this method provides improved collagen-containing dermal fillers and improved methods of dermal filling by enabling the use of smaller gauge needles and reduced injection force, and by its ability to fill smaller tissue spaces.

[0125] The "force" (Newton, N) of injection includes the force required for injection from the needle or cannula.

[0126] Absolute viscosity (or dynamic viscosity) is the resistance of a fluid to flow when a force is applied. Absolute viscosity is proportional to the ratio of force to velocity. The Greek letter η (eta) represents absolute viscosity in calculations. Since the viscosity of many common fluids is between 0.5 cP and 1000 cP, it is usually measured in cP.

[0127] A "gel" is a semi-rigid slab or cylindrical form of an organic polymer used as a medium for separating polymers. A gel is essentially a thin crosslinked system and does not exhibit fluidity in a steady state. A gel is primarily liquid by weight, but behaves partially solid due to a three-dimensional crosslinked network structure within the liquid, while retaining some liquid properties, such as deformability. It is the crosslinking within the fluid that gives the gel its structure (hardness) and contributes to its adhesiveness (tackiness). As a result, a gel can be considered as a dispersion of liquid molecules in a solid, i.e., liquid particles dispersed in a solid medium. "Gelation time" is the time it takes for a polymerizable solution to form a gel.

[0128] A "hydrogel" is a polymer network structure that is hydrophilic and sometimes found as a colloidal gel with water as the dispersion medium. Hydrogels are highly absorbent polymer network structures (for example, they can contain more than 90% water) and, due to their remarkable water content, possess flexibility very similar to that of natural tissues.

[0129] A polymer is a macromolecule composed of a series of repeating subunits. The basic repeating subunits are known as monomers. Collectively, polymers are known for their tensile strength and elasticity.

[0130] A "photoinitiator" is a molecule that generates reactive species (free radicals, cations, or anions) when exposed to radiation (UV or visible light). The photoinitiators of the present invention induce polymerization of polymerizable solutions. Examples of photoinitiators useful in this method include, but are not limited to, lithium phenyl-2,4,6-trimethylbenzoyl phosphinate (LAP) or 1-[4 2-hydroxy-1-[4-(2-hydroxyethoxy)phenyl]-2-methylpropan-1-one (IRGACURE® 2959), eosin Y + triethanolamine, or riboflavin.

[0131] Methacrylic acid is an ester or salt derived from methacrylic acid. Methacrylates are common monomers in polymer plastics and form acrylate polymers. The addition of methacrylate groups to collagen yields photocurable collagen methacrylate (rh-collagen-MA or MA-rh-collagen). The addition of methacrylate groups to hyaluronic acid (HA) yields photocurable hyaluronic acid-methacrylate (HAMA or MA-HA).

[0132] In some embodiments, the rh collagen used in the skin fillers described herein includes a combination of unmodified rh collagen and MA-rh collagen. In some embodiments, the ratio of unmodified rh collagen to MA-rh collagen is about 1:0, 1:1, 1:2, 1:3, 1:4, 0:1, 2:1, 3:1, or 4:1. In some embodiments, the final concentration range of MA-rh collagen includes about 0 to 12 mg / ml. In some embodiments, the final concentration range of unmodified rh collagen includes about 0 to 12 mg / ml. In some embodiments, the final concentration range of MA-rh collagen includes about 0 to 12 mg / ml, and the final concentration range of unmodified rh collagen includes about 0 to 12 mg / ml. In some embodiments, the final concentration range of MA-rh collagen includes about 0 mg / ml to 6 mg / ml. In some embodiments, the final concentration range of unmodified rh collagen includes about 0 mg / ml to 6 mg / ml. In some embodiments, the final concentration of MA-rh collagen is approximately 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 mg / ml. In some embodiments, the final concentration of unmodified rh collagen is approximately 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 mg / ml.

[0133] Thiols are organosulfur compounds containing a carbon-bonded sulfhydryl (R-SH) group (wherein R represents an alkyl or other organic substituent). Thiolation of collagen may improve its aggregation and mucosal adhesion properties and affect its swelling capacity.

[0134] Light is a form of electromagnetic radiation. "Visible light" is light with wavelengths in the range of 380-800 nm or at least 390-700 nm. "Ultraviolet light" has shorter wavelengths, and "infrared light" has longer wavelengths.

[0135] The irradiation means may be a light source suitable for activating the photoinitiator used, and the photoinitiator can be activated from outside the body. A thermal initiator can be used, and therefore an infrared source can be used, and an ultraviolet activating initiator can be used, and therefore a suitable ultraviolet source can be used, and a preferred light source is a white light source. Thus, a suitable photoinitiator is used, and as a result, the maximum absorbance of the initiator and the light source is adjusted. As described above herein, one such visible light source is a light-emitting diode (LED). Other suitable light sources can be used, for example, by applying electromagnetic radiation to the body, to a site as needed, or from above the skin surface, insofar as gelation occurs inside the body, at the aforementioned sites, below the skin surface, etc. The electromagnetic radiation is applied with an intensity, time and duration that allows gelation to occur. The light source can be placed on or directly on the skin surface, typically above the location of the polymerizable solution to be molded or shaped.

[0136] The monomer solutions of some embodiments may contain various other materials, as known in the pharmaceutical field, such as inert materials (e.g., preservatives, fillers, excipients, or diluents), pharmacologically active molecules, or drugs (e.g., small molecules or biological cells). Accordingly, suitable inert or biologically active drugs may be added to the monomer solution. In the latter case, the active drug may exert a local pharmacological effect at or near the site of the polymerized or network structure of interest, or it may be released from the formed scaffold, matrix, or network structure and travel through adjacent tissue spaces, or it may enter the circulatory system to reduce the local effect.

[0137] As discussed above, the method for producing the desired polymerizable solution can also be used in combination with other dermatological, orthopedic, cosmetic, and other medical treatments.

[0138] In some embodiments, a polymerizable solution is mixed with known fillers to provide a composition that is moldable, contourable, and has a long residence time. Examples of fillers include, but are not limited to, hyaluronic acid (HA), poly(vinyl alcohol) (PVA), polyethylene glycol (PEG), oxidized cellulose (OC), or modified derivatives thereof, or combinations thereof. In some embodiments, a semi-liquid phase polymerizable solution is injected independently into the dermis, similar to a semi-liquid phase known filler, and together they provide a composition that is moldable, contourable, and has a long residence time. Examples of independently injectable fillers include, but are not limited to, hyaluronic acid (HA), poly(vinyl alcohol) (PVA), polyethylene glycol (PEG), oxidized cellulose (OC), or modified derivatives thereof, or combinations thereof. In some embodiments, a semi-liquid phase polymerizable solution is injected into the dermis as a mixture, and together they provide a composition that is moldable, contourable, and has a long residence time. Examples of fillers that can be injected in combination with rh collagen include, but are not limited to, hyaluronic acid (HA), poly(vinyl alcohol) (PVA), polyethylene glycol (PEG), oxidized cellulose (OC), or modified derivatives thereof, or combinations thereof.

[0139] In yet another embodiment, a method for inducing a cell proliferation-promoting scaffold in a tissue space beneath the epidermis is disclosed herein, the method comprising the step of introducing a solution into the tissue space, the solution comprising (a) human collagen of plant origin and (b) at least one growth factor or a source thereof.

[0140] In one embodiment, the source of at least one growth factor includes plasma or platelet-rich plasma.

[0141] In one embodiment, a cell proliferation-promoting scaffold promotes healing or replacement resulting from the breakdown or damage of collagen-containing tissue. In one embodiment, the collagen-containing tissue is selected from the group consisting of tendons, ligaments, skin, cornea, cartilage, blood vessels, intestines, intervertebral discs, muscles, bones, or teeth. In a specific embodiment, the cell proliferation-promoting scaffold promotes the healing of tendinitis.

[0142] In one embodiment, the plant-derived collagen includes rh collagen. In one embodiment, the plant-derived collagen is obtained from a genetically modified plant. In various embodiments, the genetically modified plant is selected from the group consisting of tobacco, corn, alfalfa, rice, potato, soybean, tomato, wheat, barley, canola, carrot, and cotton. In one embodiment, the genetically modified plant is a tobacco plant.

[0143] In one embodiment, the genetically modified plant contains an expressible sequence of at least one gene sequence of star deoxyribonucleic acid (DNA) selected from the group consisting of COL1, COL2, P4H-α, P4H-β, and LH3.

[0144] In certain embodiments, the plant-derived human collagen comprises at least one modified human collagen α-1 chain, shown in SEQ ID NO: 3 and expressed in the genetically modified plant, and at least one modified human collagen α-2 chain, shown in SEQ ID NO: 6 and expressed in the genetically modified plant, wherein the genetically modified plant further expresses exogenous prolyl-4-hydroxylase (P4H).

[0145] In another specific embodiment, the method further comprises the step of expressing an exogenous polypeptide selected from the group consisting of lysyl hydroxylase (LH), protease N, and protease C.

[0146] In one particular embodiment, the human collagen α-1 chain is encoded by the sequence described in Sequence ID No. 1. In another particular embodiment, the human collagen α-2 chain is encoded by the sequence described in Sequence ID No. 2.

[0147] In one embodiment, the exogenous P4H is mammalian P4H. In one specific embodiment, the exogenous P4H is human P4H.

[0148] In one embodiment, the method further includes the steps of targeting human collagen α-1 to the vacuole of a plant or genetically modified plant and digesting it with ficin. In another embodiment, the method further includes the steps of targeting human collagen α-2 to the vacuole of a plant or genetically modified plant and digesting it with ficin.

[0149] In one particular embodiment, the plant-derived human collagen is atelocollagen.

[0150] Those skilled in the art will understand that the term “dermal filler” in some embodiments encompasses solutions containing plant-derived human collagen, e.g., recombinant human collagen type 1 (rh collagen) or its derivatives. The term “dermal filler” also in some embodiments encompasses solutions containing plant-derived human collagen, e.g., recombinant human collagen type 1 (rh collagen) or its derivatives, fillers or their derivatives, or cross-linked fillers or their derivatives, all having the same meaning and quality, and the dermal filler may be used to enhance tissue structure or to reduce muscle, folds, fine lines, wrinkles, or scars, or a combination thereof.

[0151] Those skilled in the art will understand that the dermal fillers described herein include, for example, different formulations, but are not limited to the following: ●rh collagen or its MA or thiol derivatives, ● An IPN, semi-IPN, or double-crosslinked network structure comprising rh collagen or rh collagen-MA or rh collagen-thiol, and a filler or its derivative, ● An IPN, semi-IPN, or double-crosslinked network structure containing rh collagen or rh collagen-MA or rh collagen-thiol, and HA or MA-HA or thiol-HA. ● An IPN, semi-IPN, or double-crosslinked network structure comprising rh collagen or rh collagen-MA or rh collagen-thiol, and PVA or MA-PVA or thiol-PVA, ● An IPN, semi-IPN, or double-crosslinked network structure comprising rh collagen or rh collagen-MA or rh collagen-thiol, and PEG or MA-PEG or thiol-PEG. ● An IPN, semi-IPN, or double-crosslinked network structure comprising rh-collagen or rh-collagen-MA or rh-collagen-thiol, and OC or MA-OC or thiol-OC. ● An IPN, semi-IPN, or double-crosslinked network structure, or cell proliferation-promoting scaffold, comprising rh collagen and autologous platelet-rich plasma (PRP) fraction containing a high concentration of platelets. ● An IPN, semi-IPN, or double-crosslinked network structure, or cell proliferation-promoting scaffold, each comprising rh collagen and autologous platelet-rich plasma (PRP) fraction of blood containing a high concentration of platelets, wherein the platelets release various types of growth factors (GF), including vascular endothelial growth factor (VEGF), transforming growth factor-β (TGF-β), platelet-derived growth factor (PDGF), platelet-derived epidermal growth factor (PDEGF), fibroblast growth factor (bFGF), epidermal growth factor (EGF), or hepatocyte growth factor (HGF), or a combination thereof. ●A double-crosslinked skin filler containing a crosslinked filler or its derivative, rh collagen, rh collagen-MA, or rh collagen-thiol. ●A double-crosslinked skin filler containing rh-collagen, rh-collagen-MA, or rh-collagen-thiol cross-linked HA, or cross-linked MA-HA, or cross-linked thiol-HA. ●A double-crosslinked skin filler containing crosslinked PVA or crosslinked MA-PVA or crosslinked thiol-PVA, crosslinked rh-collagen or rh-collagen-MA or rh-collagen-thiol, ●A double-crosslinked skin filler containing crosslinked PEG or crosslinked MA-PEG or crosslinked thiol-PEG, or rh collagen-MA or rh collagen-thiol, or ●A double-crosslinked dermal filler containing rh-collagen, rh-collagen-MA, or rh-collagen-thiol crosslinked to cross-linked OC, cross-linked MA-OC, or cross-linked thiol-OC.

[0152] Those skilled in the art will understand that, in some embodiments, the term “proliferative scaffold” encompasses a skin filler comprising collagen and a fraction of the blood’s own platelet-rich plasma (PRP) or a component thereof. In some embodiments, the PRP does not contain “cells” but contains (cell-derived) membrane vesicles containing growth factors and plasma components such as fibrinogen and prothrombin. In some embodiments, the “proliferative scaffold” encompasses a skin filler comprising collagen and a fraction of the blood’s own platelet-rich plasma (PRP) or a component thereof and at least one further filler component.

[0153] In some embodiments, the cell proliferation-promoting scaffold comprises a dermal filler which may be an IPN network structure, a semi-IPN network structure, or a bicrosslinked dermal filler which further comprises an autologous platelet-rich plasma (PRP) fraction of blood containing a high concentration of platelets, wherein the autologous PRP fraction of blood contains a high concentration of platelets, and the platelets release various types of growth factors (GFs), including vascular endothelial growth factor (VEGF), transforming growth factor-β (TGF-β), platelet-derived growth factor (PDGF), platelet-derived epidermal growth factor (PDEGF), fibroblast growth factor (bFGF), epidermal growth factor (EGF), or hepatocyte growth factor (HGF), or a combination thereof. In some embodiments, the cell proliferation-promoting scaffold comprises a skin filler containing an IPN network structure, a semi-IPN network structure, or a double-crosslinked skin filler further comprising at least one growth factor, including vascular endothelial growth factor (VEGF), transforming growth factor-β (TGF-β), platelet-derived growth factor (PDGF), platelet-derived epidermal growth factor (PDEGF), fibroblast growth factor (bFGF), epidermal growth factor (EGF), or hepatocyte growth factor (HGF), or a combination thereof. In some embodiments, the cell proliferation-promoting scaffold comprises a skin filler containing an IPN network structure, a semi-IPN network structure, or a double-crosslinked skin filler further comprising a portion or fraction of a PRP component.

[0154] In some embodiments, the dermal fillers described herein include polymerizable solutions. In some embodiments, the dermal fillers described herein include non-polymerizable solutions. In some embodiments, polymerization of the dermal filler solution occurs in vivo. In some embodiments, the components of a polymerizable dermal filler solution are injected together and then polymerized to form a cured dermal filler. In some embodiments, the components of a polymerizable dermal filler solution are injected independently and then polymerized to form a cured dermal filler. An example of a unique approach to independently injecting dermal filler components may, in some embodiments, include the steps of injecting a filler, e.g., HA or a derivative thereof, into the dermis of the skin, and separately injecting methacrylated or thiol-rh collagen into this dermis, immediately adjacent to the first injection, wherein the component is in a semiliquid phase and then crosslinked in situ. This approach, in some embodiments, allows for easier injection and in situ shaping before curing the dermal filler components together by photopolymerization.

[0155] In some embodiments, the skin fillers provided herein are used in soft tissue augmentation methods. In some embodiments, the skin fillers provided herein improve cell proliferation. In some embodiments, the skin fillers provided and used in soft tissue augmentation methods degrade over time. In some embodiments, the skin fillers provided herein are used in soft tissue augmentation methods in which the skin filler fills the tissue space beneath the epidermis. In some embodiments, the skin fillers provided herein are used in soft tissue augmentation methods, and their use reduces muscles, folds, fine lines, wrinkles, or scars.

[0156] In one embodiment, a solution containing plant-derived human collagen has a lower viscosity at room temperature compared to a similar solution containing human or animal-derived collagen extracted from tissue, at the same concentration and formulation. In another embodiment, a solution containing plant-derived human collagen has a lower viscosity at 37°C compared to a similar solution containing human or animal-derived collagen extracted from tissue, at the same concentration and formulation. In yet another embodiment, a solution containing plant-derived human collagen is introduced into tissue space with less force at room temperature compared to a similar solution containing human or animal-derived collagen extracted from tissue, at the same concentration and formulation. In yet another embodiment, a solution containing plant-derived human collagen is introduced into tissue space with less force at 37°C compared to a similar solution containing human or animal-derived collagen extracted from tissue, at the same concentration and formulation. In one particular embodiment, a solution containing plant-derived human collagen increases scaffold formation or promotes increased cell proliferation compared to a similar solution containing human or animal-derived collagen extracted from tissue, at the same concentration and formulation.

[0157] In yet another embodiment, the use of a solution injected into a subepidermal tissue space to induce a cell proliferation-promoting scaffold is disclosed herein, the solution comprising plant-derived human collagen and at least one growth factor or its source, which promotes healing or replacement resulting from the degradation or damage of collagen-containing tissue. In certain embodiments, the source of at least one growth factor comprises plasma or platelet-rich plasma.

[0158] In one embodiment, the collagen-containing tissue is selected from the group consisting of tendons, ligaments, skin, cornea, cartilage, blood vessels, intestines, intervertebral discs, muscles, bones, or teeth. In another embodiment, the cell proliferation-promoting scaffold promotes the healing of tendinitis. In one embodiment, the collagen-containing tissue is skin.

[0159] In some embodiments, genetically modified plants are provided that express at least one type of collagen α chain and are capable of accumulating it in an intracellular compartment lacking endogenous P4H activity.

[0160] As used herein, the phrase “genetically modified plant” means any lower (e.g., moss) or higher (e.g., vascular) plant, or its tissue or isolated cells (e.g., cell suspensions), that are stably or transiently transformed with an exogenous polynucleotide sequence. Examples of plants include lower plants such as tobacco, maize, alfalfa, rice, potato, soybean, tomato, wheat, barley, canola, cotton, carrot, and moss.

[0161] As used herein, the phrase “collagen chain” refers to a collagen subunit such as an α1 or α2 chain of a collagen fiber, preferably a type I fiber. As used herein, the phrase “collagen” refers to an assembled collagen trimer, which in the case of type I collagen includes two α1 chains and one α2 chain. A collagen fiber is collagen lacking terminal propeptides C and N.

[0162] As used herein, the phrase “intracellular compartment lacking endogenous P4H activity” refers to any compartmentalized region of a cell that does not contain enzymes having plant P4H or plant-like P4H activity. Examples of such intracellular compartments include vacuoles, apoplasts, and cytoplasm, as well as organelles such as chloroplasts and mitochondria.

[0163] Any type of collagen chain can be expressed in the genetically modified plants of the present invention. Examples include collagen that forms profibrils (types I, II, III, V, and XI), collagen that forms network structures (types IV, VIII, and X), collagen that associates with the surface of profibrils (types IX, XII, and XIV), collagen that arises as transmembrane proteins (types XIII and XVII), or collagen that forms 11 nm periodic spherical filaments (type VI).

[0164] In one embodiment, the expressed collagen chains are α1 and / or α2 chains of type I collagen. The expressed collagen α chains can be encoded by any polynucleotide sequence derived from any mammal. In a particular embodiment, the sequence encoding the collagen α chains is human and is indicated by SEQ ID NOs: 1 and 4.

[0165] Typically, α-collagen chains expressed in plants may or may not contain their terminal propeptides (i.e., propeptide C and propeptide N).

[0166] The processing of procollagen by plant proteolytic activity differs from normal processing in humans; the cleavage site is unknown, but the propeptide C is removed by plant proteolytic activity. Cleavage of the C propeptide may occur in the procollagen peptide before trimer organization (the association of three C-propeptides is essential to initiate trimer organization).

[0167] Cleavage of N-propeptides by plant proteolytic activity occurs in mature plants but not in immature plants. This cleavage removes two amino acids from the N-telopeptide (2 out of 17).

[0168] C-propeptides (and to a lesser extent, N-propeptides) maintain the solubility of procollagen while passing through animal cells (Bulleid et al., 2000), and a similar effect is expected in plant cells. After or during the secretion of procollagen molecules into the extracellular matrix, propeptides are removed by procollagen N-proteinases and C-proteinases, thereby triggering spontaneous self-assembly from collagen molecules into fibrils. The removal of propeptides by procollagen N-proteinases and C-proteinases reduces the solubility of procollagen by more than 1 / 10,000, which is necessary and sufficient to initiate self-assembly from collagen into fibrils. Crucial to this organizing process are short, non-triple-helical peptides called telopeptides, located at the ends of the triple-helical domains, which ensure the correct positioning of collagen molecules within the fibril structure and lower the critical concentration for self-assembly. Pepsin can cleave propeptides during collagen production. However, pepsin damages telopeptides, and as a result, collagen extracted by pepsin cannot form a regular fibrous structure.

[0169] The protein disulfide isomerase (PDI), which forms the β-subunit of human P4H, has been shown to bind to the C-propeptide before trimer organization, thereby functioning as a molecular chaperone during chain organization.

[0170] Using human procollagen type I N-proteinases and procollagen C-proteinases expressed in different plants may produce collagen more similar to natural human collagen, which can form a regular fibrous structure.

[0171] If the expressed collagen chain contains either the N or C propeptide, or both, the genetically modified plant of the present invention may also express the respective proteases (i.e., C or N or both). Polynucleotide sequences encoding such proteases are exemplified by SEQ ID NOs: 18 (protease C) and 20 (protease N). Such proteases may be expressed so that they accumulate in the same intracellular compartment as the collagen chain.

[0172] The accumulation of collagen chains expressed in intracellular compartments lacking endogenous P4H activity can be induced through one of several approaches.

[0173] For example, expressed collagen chains may contain signal sequences for targeting the expressed protein into intracellular compartments such as apoplasts or organelles (e.g., chloroplasts). Examples of suitable signal sequences include chloroplast transit peptides (Swiss-Prot entry P07689, contained in amino acids 1-57) and mitochondrial transit peptides (Swiss-Prot entry P46643, contained in amino acids 1-28). The following Examples section provides further examples of suitable signal sequences, as well as guidelines for using such signal sequences in collagen chain expression in plant cells.

[0174] Alternatively, the sequence of collagen chains can be modified in a way that alters the cellular localization of collagen when expressed in plants.

[0175] As described herein, the plant ER contains P4H, which is unable to properly hydroxylate collagen chains. The collagen α chain naturally contains an ER target sequence that induces expressed collagen to be post-translationally modified (including incorrect hydroxylation) ER. Therefore, removal of the ER target sequence would result in cytoplasmic accumulation of collagen chains lacking any post-translational modification, including hydroxylation.

[0176] Example 1 in the following section of Examples describes the generation of a collagen sequence lacking an ER sequence.

[0177] Alternatively, collagen chains may be expressed and accumulated within DNA, including in organelles such as chloroplasts or mitochondria. A further explanation of chloroplast expression is provided below in this specification.

[0178] As described herein, hydroxylation of the α-chain is necessary for the organization of stable type I collagen. Since the α-chain expressed by the genetically modified plants of the present invention accumulates in a compartment lacking endogenous P4H activity, such chains may be isolated from plants, plant tissues, or cells and hydroxylated in vitro. Such hydroxylation can be achieved by the method described by Turpeenniemi-Hujanen and Myllyla (Concomitant hydroxylation of proline and lysine residues in collagen using purified enzymes in vitro. Biochim Biophys Acta. July 16, 1984; 800(1):59-65).

[0179] While such in vitro hydroxylation can produce correctly hydroxylated collagen chains, it is difficult to achieve and can be costly.

[0180] To overcome the limitations of in vitro hydroxylation, the genetically modified plants of the present invention preferably also co-express P4H, which is capable of correctly hydroxylating collagen α chains [i.e., hydroxylating only at the proline (Y) position of the Gly-XY triplet]. P4H is an enzyme composed of two subunits, α and β. The β subunit also possesses chaperone function, but both are necessary to form an active enzyme.

[0181] The P4H expressed by the genetically modified plants of the present invention is preferably human P4H, for example, as encoded by SEQ ID NOs. 12 and 14. Furthermore, P4H mutants or P4H homologs exhibiting improved substrate specificity can also be used.

[0182] A suitable P4H homolog is exemplified by Arabidopsis oxidoreductase, identified by NCBI deposit NP_179363. Pairwise alignment of this protein sequence with the human P4Hα subunit, performed by the inventors of the present invention, revealed the greatest homology between the functional domains of any known plant P4H homolog.

[0183] Since P4H needs to accumulate along with the expressed collagen chain, its coding sequence is preferably modified accordingly (e.g., addition of a signal sequence, deletion that may interfere with ER targeting).

[0184] In mammalian cells, collagen is also modified by lysyl hydroxylase, galactosyltransferase, and glucosyltransferase. These enzymes sequentially modify lysyl residues at specific positions to hydroxylysyl, galactosylhydroxylysyl, and glucosylgalactosylhydroxylysyl residues. A single human enzyme, lysyl hydroxylase 3 (LH3), can catalyze all three consecutive steps in the formation of hydroxylysine-linked carbohydrates.

[0185] Therefore, the genetically modified plants of the present invention preferably also express mammalian LH3. An LH3 encoding sequence, for example, shown by SEQ ID NO: 22, can be used for such purposes.

[0186] The collagen chains and modifying enzymes described above may be expressed from stably incorporated or transiently expressed nucleic acid constructs comprising polynucleotide sequences encoding α chains and / or modifying enzymes (e.g., P4H and LH3) positioned under the transcriptional control of a plant functional promoter. Such nucleic acid constructs (also referred herein as expression constructs) may be configured for expression in a whole plant, defined plant tissue or defined plant cells, or defined developmental stages of a plant. Such constructs may also include a selection marker (e.g., antibiotic resistance), an enhancer element, and a replication origin for bacterial replication.

[0187] A construct containing two expressible inserts (e.g., two different α-procollagen chains, or an α-chain and P4H) may preferably contain individual promoters for each insert, or it will be understood that such a construct may express a single transcriptional chimera containing both insert sequences from a single promoter. In such a case, the chimeric transcript contains an IRES sequence between the two insert sequences so that a downstream insert can be translated from it.

[0188] Numerous plant functional expression promoters and enhancers, which may be tissue-specific, development-specific, constitutive, or inducible, can be utilized by the constructs of the present invention, several examples of which are provided below herein.

[0189] In the chapters of this specification and the claims that follow the phrase “plant promoter” or “promoter,” the term includes, as used herein, a promoter capable of directing gene expression in plant cells (including organelles containing DNA). Such promoters may be of plant, bacterial, viral, fungal, or animal origin. Such promoters may be constitutive (i.e., capable of directing high levels of gene expression in multiple plant tissues), tissue-specific (i.e., capable of directing gene expression in specific plant tissues or multiple tissues), inducible (i.e., capable of directing gene expression by stimulation), or chimeric (i.e., formed from at least two different promoter parts).

[0190] Therefore, the plant promoter used may be a constitutive promoter, a tissue-specific promoter, an inducible promoter, or a chimeric promoter.

[0191] Examples of constitutive plant promoters include, but are not limited to, the CaMV35S and CaMV19S promoters, the FMV34S promoter, the sugarcane rod-shaped Badnavirus promoter, the CsVMV promoter, the Arabidopsis ACT2 / ACT8 actin promoter, the Arabidopsis ubiquitin UBQI promoter, the barley leaf thionine BTH6 promoter, and the rice actin promoter.

[0192] Examples of tissue-specific promoters include, but are not limited to, the soybean phaseolin storage protein promoter, the DLEC promoter, the PHS promoter, the zein storage protein promoter, the soybean-derived conglutinin gamma promoter, the AT2S1 gene promoter, the Arabidopsis-derived ACT11 actin promoter, the Brassica napus-derived napA promoter, and the potato patatin gene promoter.

[0193] Inducible promoters are promoters that are induced by specific stimuli, such as stress conditions including light, temperature, chemicals, drought, high salinity, osmotic shock, and oxidizing agent conditions, or in the case of pathogenicity, and include, but are not limited to, the photo-inducible promoter derived from the soybean rbcS gene, the promoter derived from the alfalfa rbcS gene, the drought-active promoters DRE, MYC, and MYB, the high-salinity and osmotic shock-active promoters INT, INPS, prxEa, Ha hsp17.7G4, and RD21, and the promoters hsr203J and str246C in pathogenic stress.

[0194] Preferably, the promoter used in the present invention is a potent constitutive promoter such that the overexpression of the construct insert occurs after plant transformation.

[0195] It will be understood that any type of construct used in the present invention can be co-transformed within the same plant using the same or different selection markers in each type of construct. Alternatively, a first type of construct can be introduced into a first plant, while a second type of construct can be introduced into a second syngeneic plant, and the resulting transgenic plants can then be crossed to select offspring for double transgenic plants. Further self-hybridization of such offspring can be used to generate homozygous lines for both constructs.

[0196] There are various methods for introducing nucleic acid constructs into both monocots and dicots (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 the stable integration of the nucleic acid construct or a portion thereof into the plant genome, or the transient expression of the nucleic acid construct, in which case these sequences are not inherited by the plant's offspring.

[0197] In addition, there are several methods that allow nucleic acid constructs to be directly introduced into the DNA of organelles containing DNA, such as chloroplasts.

[0198] There are two main methods for achieving stable genomic integration of exogenous sequences into the plant genome, such as those contained within the nucleic acid constructs of the present invention in the plant genome. (i) Gene transfer via Agrobacterium: 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, LK, Academic Publishers, San Diego, Calif. (1989) p. 2-25, Gatenby in Plant Biotechnology (eds.), Kung, S. and Arntzen, CJ, 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, edited by Molecular Biology of Plant Nuclear Genes, Schell, J. and Vasil, LK, Academic Publishers, San Diego, Calif. (1989) pp. 52-68, including a method for direct DNA uptake into protoplasts, Toriyama, K. et al. (1988) Bio / Technology 6:1072-1074. DNA uptake induced by simple electric shock in 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 collision, 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 a micropipette system, Neuhaus et al., Theor.Appl.Genet.(1987)75:30-36; Neuhaus and Spangenberg, Physiol.Plant.(1990)79:213-217; or by direct incubation of DNA using germinated pollen, DeWet et al., in Experimental Manipulation of Ovule Tissue (eds.), Chapman, GP, Mantell, SH, and Daniels, W. Longman, London, (1985)p.197-209; and Ohta, Proc.Natl.Acad.Sci.USA(1986)83:715-719.

[0199] Agrobacterium systems involve the use of plasmid vectors containing predefined DNA segments that are integrated into the plant's genomic DNA. The method of inoculation into plant tissue varies depending on the plant species and the Agrobacterium delivery system. A widely used approach is the leaf disc procedure, which can be performed with any tissue explant that provides an excellent source for initiating whole-plant differentiation. (Horsch et al., in Plant Molecular Biology Manual A5, Kluwer Academic Publishers, Dordrecht (1988) pp. 1-9). A supplementary approach employs the Agrobacterium delivery system in combination with vacuum infiltration. Agrobacterium systems are particularly feasible in the creation of transgenic dicotyledonous plants.

[0200] There are various methods for directly transferring DNA to plant cells. In electroporation, protoplasts are exposed to a strong electric field for a short period of time. In microinjection, a very small micropipette is used to mechanically inject DNA directly into the cells. In microparticle impaction, DNA is adsorbed onto microprojectiles such as magnesium sulfate crystals, tungsten particles, or gold particles, and the microprojectiles are physically accelerated towards the cells or plant tissue.

[0201] Following transformation, plant propagation takes place. The most common method of plant propagation is by seed. However, seed propagation has the disadvantage of lacking uniformity in the crop due to heterozygosity, as seeds are produced by the plant according to genetic differences governed by Mendel's laws. Essentially, each offspring is genetically different, and each grows with its own specific traits. Therefore, it is preferable to produce transformed plants that have the same traits and characteristics as the parent transgenic plant. Thus, it is preferable to regenerate transformed plants by micropropagation, which provides rapid and consistent regeneration of transformed plants.

[0202] Temporary expression methods available for the temporary expression of isolated nucleic acids contained in the nucleic acid constructs of the present invention include, but are not limited to, microinjection and collision under conditions desirable for temporary expression, as described above, and virus-mediated expression, which involves infecting plant tissue or cells with an encapsulated or unencapsulated recombinant viral vector containing the nucleic acid construct so that the established, growing recombinant virus expresses a non-viral nucleic acid sequence.

[0203] Viruses that have been shown to be useful for transforming plant hosts include CaMV, TMV, and BV. Plant transformation using plant viruses is described in U.S. Patent No. 4,855,237 (BGV), EP-A67,553 (TMV), Japanese Publication No. 63-14693 (TMV), EPA 194,809 (BV), EPA 278,667 (BV), and Gluzman, Y. et al., Communications in Molecular Biology: Viral Vectors, Cold Spring Harbor Laboratory, New York, pp. 172-189 (1988). Pseudoviral particles for use in expressing foreign DNA in many hosts, including plants, are described in WO87 / 06261.

[0204] The construction of plant RNA viruses for the introduction and expression of nonviral exogenous nucleic acid sequences in plants is described in the above references, as well as by Dawson, WO et al., Virology (1989) 172:285-292, Takamatsu et al., EMBO J. (1987) 6:307-311, French et al., Science (1986) 231:1294-1297, and Takamatsu et al., FEBS Letters (1990) 269:73-76.

[0205] If the virus is a DNA virus, construction can be carried out on the virus itself. Alternatively, the virus can be first cloned into a bacterial plasmid to facilitate the construction of a desired viral vector using foreign DNA. The virus can then be excised from the plasmid. If the virus is a DNA virus, a bacterial origin of replication may attach to the viral DNA, and then it is replicated by the bacteria. Transcription and translation of this DNA produce a coat protein that encloses the viral DNA in a capsid. If the virus is an RNA virus, the virus is generally cloned as cDNA and inserted into a plasmid. Then, all constructs are made using the plasmid. Next, the RNA virus is produced by transcribing the viral sequence of the plasmid and translating the viral gene to produce a coat protein that encloses the viral RNA in a capsid.

[0206] The construction of plant RNA viruses for the introduction and expression of nonviral exogenous nucleic acid sequences into plants, including those included in the constructs of the present invention, is described in the above-mentioned references and U.S. Patent No. 5,316,931.

[0207] In one embodiment, a plant virus nucleic acid is provided in which a non-natural plant virus coat protein coding sequence and a non-natural promoter, preferably a subgenome promoter of the non-natural coat protein coding sequence, are inserted, thereby enabling expression in a plant host, inclusion of recombinant plant virus nucleic acid, and ensuring systemic infection of the host by recombinant plant virus nucleic acid. Alternatively, the coat protein gene may be inactivated by inserting a non-natural nucleic acid sequence therein so that the protein is produced. The recombinant plant virus nucleic acid may contain one or more further non-natural subgenome promoters. Each non-natural subgenome promoter can transcribe or express adjacent genes or nucleic acid sequences in the plant host and cannot be recombined with each other or with the natural subgenome promoter. The non-natural (foreign) nucleic acid sequence may be inserted adjacent to the natural plant virus subgenome promoter, or, if multiple nucleic acid sequences are included, to the natural and non-natural plant virus subgenome promoters. The non-natural nucleic acid sequence is transcribed or expressed in the host plant under the control of the subgenome promoter to produce the desired product.

[0208] In a second embodiment, recombinant plant virus nucleic acids are provided similarly to the first embodiment, except that the natural coat protein coding sequence is located adjacent to one of the non-natural coat protein subgenome promoters, instead of the non-natural coat protein coding sequence.

[0209] In a third embodiment, recombinant plant virus nucleic acid is provided, wherein a natural coat protein gene is adjacent to its subgenome promoter, and one or more non-natural subgenome promoters are inserted into the viral nucleic acid. The inserted non-natural subgenome promoters can transcribe or express adjacent genes in the plant host and cannot recombinate with each other or with the natural subgenome promoter. The non-natural nucleic acid sequence may be inserted adjacent to the non-natural subgenome plant virus promoter, and as a result, the sequence is transcribed or expressed in the host plant under the control of the subgenome promoter to produce a desired product.

[0210] In the fourth embodiment, recombinant plant virus nucleic acid is provided in the same manner as in the third embodiment, except that the natural coat protein coding sequence is replaced by a non-natural coat protein coding sequence.

[0211] A viral vector is encapsulated in a capsid by a coat protein encoded by recombinant plant virus nucleic acid, producing a recombinant plant virus. Recombinant plant virus nucleic acid or recombinant plant virus is used to infect a suitable host plant. Recombinant plant virus nucleic acid can replicate in the host, spread throughout the host, and transcribe or express the foreign gene (isolated nucleic acid) in the host, producing the desired protein.

[0212] A technique for introducing exogenous nucleic acid sequences into the genome of chloroplasts is known. This technique involves the following steps: First, plant cells are chemically treated to reduce the number of chloroplasts per cell to approximately one. Next, the exogenous nucleic acid is introduced into the cells via particle collision with the aim of introducing at least one exogenous nucleic acid molecule into the chloroplast. The exogenous nucleic acid is selected so that it can be incorporated into the chloroplast genome via homologous recombination, which is readily carried out by enzymes specific to the chloroplast. For this purpose, the exogenous nucleic acid contains at least one nucleic acid elongation portion derived from the chloroplast genome, in addition to the gene of interest. In addition, the exogenous nucleic acid contains a selectable marker, which is aided by a series of selection procedures to ensure that all or substantially all replicas of the chloroplast genome following such selection contain the exogenous nucleic acid. Further details relating to this technique can be found in U.S. Patents 4,945,050 and 5,693,507, which are incorporated herein by reference. Therefore, polypeptides can be produced by chloroplast protein expression systems and incorporated into the inner membrane of chloroplasts.

[0213] Using the transformation approach described above, collagen chains and / or modifying enzymes, as well as organized collagen (with or without propeptides), can be produced in any plant species, or in plant tissues or isolated plant cells derived therefrom.

[0214] Preferred plants are those capable of accumulating large quantities of the collagen chains, collagen, and / or processing enzymes described herein. Such plants may also be selected according to their tolerance to stress conditions and the ease with which the expressed components or organized collagen can be extracted. Examples of preferred plants include tobacco, corn, alfalfa, rice, potato, soybean, tomato, wheat, barley, canola, and cotton.

[0215] Collagen fibers are widely used in the food and beauty industries. Therefore, while collagen fiber components (α-chains) and modifying enzymes expressed by plants find usefulness in the industrial synthesis of collagen, complete collagen production in plants is preferred due to its simplicity and cost-effectiveness.

[0216] Several approaches can be used to generate type I collagen in plants. For example, collagen α1 chains may be isolated from plants expressing collagen α1 and P4H (and optionally LH3) and mixed with collagen α2 chains isolated from plants expressing collagen α2 and P4H (and optionally LH3 and protease C and / or N). Since collagen α1 chains self-assemble into triple helices, it may be necessary to denature such homotrimers before mixing them with collagen α2 chains and regenerating them.

[0217] Preferably, a first plant expressing collagen α1 and P4H (and optionally LH3 and protease C and / or N) can be crossed with a second (and preferably syngeneic) plant expressing collagen α2, or a first plant expressing both α chains can be crossed with a second plant expressing P4H and optionally LH3 and protease C and / or N.

[0218] While the plant breeding approach described above utilizes two individually transformed plants, it should be noted that approaches using three or more individually transformed plants, each expressing one or two components, are also available.

[0219] Those skilled in the art are well aware of various plant breeding techniques, and therefore, no further explanation of such techniques is provided herein.

[0220] While a plant breeding approach is preferred, it should be noted that a single plant expressing collagen α1 and α2, P4H and LH3 (and optionally protease C and / or N) can be generated through several transformation events, each designed to introduce one or more expressible components into the cell. In such cases, the stability of each transformation event can be verified using specific selection markers.

[0221] In any case, transformation and plant breeding approaches can be used to generate any plant expressing any number of components. Currently preferred are plants expressing collagen α1 and α2 chains, P4H, LH3, and at least one protease (e.g., protease C and / or N). As further described in the Examples section below, such plants accumulate collagen that is stable at temperatures up to 42°C.

[0222] Progeny obtained from bred or multiple-transformed plants can be selected by confirming the presence of exogenous mRNA and / or polypeptides using nucleic acid or protein probes (e.g., antibodies). The latter approach is preferred because it allows for the localization of expressed polypeptide components (e.g., by scrutinizing fractionated plant extracts) and therefore also verifies the potential for correct processing and organization. Examples of suitable probes are described in the following Examples section.

[0223] Once collagen-expressing offspring are identified, such plants are further cultured under conditions that maximize the expression of collagen chains and modifying enzymes.

[0224] Since free proline accumulation can promote the overproduction of different proline-rich proteins, including collagen chains, expressed by the genetically modified plants of the present invention, preferred cultivation conditions are those that increase free proline accumulation in the cultivated plants.

[0225] Free proline accumulates in various plants in response to a wide range of environmental stresses, including water deficit, salinity, low temperature, high temperature, pathogen infection, heavy metal toxicity, anaerobic bacteria, nutrient deficiency, air pollution and UV irradiation (Hare and Cress, 1997).

[0226] Free proline may also accumulate in response to treatment of plants or soil with compounds such as ABA, or stress-inducing compounds such as copper salts, paraquat, salicylic acid, etc.

[0227] Therefore, progeny expressing collagen can be grown under different stress conditions (e.g., different concentrations of NaCl ranging from 50 mM to 250 mM). To further improve collagen production, the effects of various stress conditions on collagen expression are investigated and optimized with respect to plant survival rate, biomass and collagen accumulation.

[0228] Plant tissues / cells are preferably harvested at maturity, and collagen fibers are isolated using well-known prior art extraction approaches, one such approach being detailed below.

[0229] Leaves of transgenic plants are ground to powder under liquid nitrogen and the homogenate is extracted in 0.5 M acetic acid containing 0.2 M NaCl at 4 °C for 60 hours. Insoluble materials are removed by centrifugation. The supernatant containing recombinant collagen is salt fractionated with 0.4 M and 0.7 M NaCl. The 0.7 M NaCl precipitate containing recombinant heterotrimeric collagen is dissolved in 0.1 M acetic acid, dialyzed against this and stored at -20 °C (according to Ruggiero et al., 2000).

[0230] In one embodiment, a method for processing procollagen to produce atelocollagen that forms homogeneous and soluble fibrils is disclosed herein.

[0231] In some embodiments, as shown herein by analysis of proteolytic degradation results by SDS-PAGE, certain plant-derived proteases (e.g., papain) are unable to cleave the propeptide portion from soluble procollagen without cleaving the helical region (even if it is possible to remove the telopeptide from telocollagen of animal origin), while other proteases (e.g., esperase, sabinase) do not effectively cleave the propeptide region from soluble procollagen, thereby hindering effective fibril formation. Through meticulous experimentation, the inventors have shown that using only certain plant-derived proteases such as ficin, or bacterial-derived proteases such as neutrase and subtilisin, may correctly cleave the propeptide portion (including the telopeptide) from soluble procollagen, thereby producing a homogeneous preparation of soluble atelocollagen without digesting the helical region of non-animal procollagen (Figures 13, 15, 17, 19, and 20). In addition, the inventors of the present invention demonstrated that recombinant trypsin is also capable of proper cleavage (Figure 26). The inventors further demonstrated that cleavage by ficin allows the resulting atelocollagen to retain its fibrillary formation ability (Table 5 in the Examples section below).

[0232] Accordingly, according to one embodiment, a method for producing atelocollagen is provided. This method comprises contacting collagen containing human recombinant telopeptides with a protease selected from the group consisting of neutrase, subtilisin, recombinant trypsin, recombinant pepsin, and ficin, wherein the collagen containing human recombinant telopeptides is expressed in non-animal cells, thereby producing atelocollagen.

[0233] As used herein, the phrase "collagen containing telopeptides" refers to soluble collagen molecules containing telopeptides that are longer than the telopeptide remnants contained in atelocollagen. Thus, collagen containing telopeptides may be procollagen containing full-length propeptides. Alternatively, collagen containing telopeptides may be procollagen molecules containing partially digested propeptides. Alternatively, collagen containing telopeptides may be atelocollagen.

[0234] As used herein, the term "procollagen" refers to collagen molecules (e.g., human) containing either the N-terminal propeptide, the C-terminal propeptide, or both. Exemplary human procollagen amino acid sequences are set forth by SEQ ID NOs: 30, 31, 36, and 37.

[0235] As used herein, the term "atelocollagen" refers to collagen molecules that lack both the N-terminal and C-terminal propeptides normally contained in procollagen, but still contain telopeptides. As described in the Background section above, the telopeptides of fibrillar collagen are remnants of the N-terminal and C-terminal propeptides after digestion with native N / C proteinase.

[0236] Recombinant human atelocollagen can be produced in cells transformed to express both exogenous human procollagen and the respective protease (i.e., C or N, or both). Polynucleotide sequences encoding such proteases are exemplified by SEQ ID NOs: 39 (protease C) and 40 (protease N). Such proteases can be expressed so that they accumulate in the same intracellular compartment as the collagen chains, as further described herein below.

[0237] As used herein, the term "atelocollagen" refers to a collagen molecule that typically lacks both the N-terminal and C-terminal propeptides found in at least a portion of procollagen and its telopeptides, but contains a sufficient portion of its telopeptides to be capable of forming profibrils under possible preferred conditions.

[0238] Any type of atelocollagen can be produced according to the methods disclosed herein. Examples include collagen that forms profibrils (types I, II, III, V, and XI), collagen that forms a network structure (types IV, VIII, and X), collagen that associates with the profibril surface (types IX, XII, and XIV), collagen that arises as a transmembrane protein (types XIII and XVII), or collagen that forms 11 nm periodic spherical filaments (type VI). According to one embodiment, the atelocollagen comprises the α-1 and / or α-2 chains of type I collagen.

[0239] In some embodiments, it will be understood that genetically modified forms of collagen / atelocollagen, such as collagenase-resistant collagen, are disclosed herein.

[0240] Recombinant human procollagen or telocollagen may be expressed in any non-animal cells, including, but not limited to, plant cells and other eukaryotic cells such as yeast and fungi.

[0241] Plants that can produce (i.e., express) human procollagen or telocollagen may include their tissues or isolated cells and their extracts (e.g., cell suspensions), and may be lower (e.g., mosses and algae) or higher (e.g., vascular tissue) plant species. Preferred plants are those capable of accumulating large amounts of collagen chains, collagen and / or processing enzymes as described below herein. Such plants may also be selected according to their tolerance to stress conditions and the ease with which the expressed components or organized collagen can be extracted. Examples of plants that can express human procollagen include, but are not limited to, tobacco, corn, alfalfa, rice, potato, soybean, tomato, wheat, barley, canola, carrot, lettuce and cotton.

[0242] The production of recombinant human procollagen is typically brought about by stable or transient transformation with exogenous polynucleotide sequences encoding human procollagen.

[0243] Exemplary polynucleotide sequences encoding human procollagen are shown by SEQ ID NOs: 32, 33, 41, and 42.

[0244] As described above, human terocollagen production is typically brought about by stable or transient transformation with an exogenous polynucleotide sequence encoding human procollagen and at least one exogenous polynucleotide sequence encoding an associated protease.

[0245] The stability of the collagen triple helix structure requires hydroxylation of proline by the enzyme prolyl-4-hydroxylase (P4H) to form hydroxyproline residues within the collagen chain. While plants can synthesize proteins containing hydroxyproline, the prolyl hydroxylase involved in hydroxyproline synthesis in plant cells exhibits relatively loose substrate sequence specificity compared to mammalian P4H. Therefore, co-expression of collagen and the human or mammalian P4H gene is necessary to produce collagen containing hydroxyproline only at the Y position of the Gly-XY triplet.

[0246] Accordingly, according to one embodiment, procollagen or telocollagen is expressed in an intracellular compartment of a plant lacking endogenous P4H activity to avoid its incorrect hydroxylation. As used herein, the phrase “intracellular compartment lacking endogenous P4H activity” refers to any compartmentalized region of a cell that does not contain an enzyme having plant P4H or plant-like P4H activity. According to one embodiment, the intracellular compartment is a vacuole.

[0247] The accumulation of procollagen expressed in intracellular compartments lacking endogenous P4H activity can be induced through one of several approaches.

[0248] For example, expressed collagen / telocollagen may contain signal sequences for targeting the expressed protein into intracellular compartments such as apoplasts or organelles (e.g., chloroplasts). Examples of suitable signal sequences include chloroplast transit peptides (Swiss-Prot entry P07689, contained in amino acids 1-57) and mitochondrial transit peptides (Swiss-Prot entry P46643, contained in amino acids 1-28).

[0249] Alternatively, the procollagen sequence can be modified in a way that alters the cellular localization of procollagen when expressed in plants.

[0250] In some embodiments, genetically modified cells are disclosed herein that co-express both human procollagen and P4H and are capable of correctly hydroxylating the procollagen α chain [i.e., hydroxylating only at the proline (Y) position of the Gly-XY triplet]. P4H is an enzyme composed of two subunits, α and β, as described in Genbank numbers P07237 and P13674. Both subunits are required to form the active enzyme, but the β subunit also possesses chaperone function.

[0251] The P4H expressed by the genetically modified cells of the present invention is preferably human P4H, for example, as encoded by SEQ ID NOs. 34 and 35. Furthermore, P4H variants or P4H homologs exhibiting improved substrate specificity can also be used. A suitable P4H homolog is exemplified by Arabidopsis oxidoreductase, identified by NCBI deposit NP_179363.

[0252] Since it is essential that P4H accumulates together with the expressed procollagen chain, its coding sequence is preferably modified accordingly (e.g., by adding or deleting a signal sequence).

[0253] In mammalian cells, collagen is also modified by lysyl hydroxylase, galactosyltransferase, and glucosyltransferase. These enzymes sequentially modify lysyl residues at specific positions to hydroxylysyl, galactosylhydroxylysyl, and glucosylgalactosylhydroxylysyl residues at specific positions. Lysyl hydroxylase 3 (LH3), a single human enzyme described in Genbank number O60568, can catalyze all three sequential modification steps, as seen in the formation of hydroxylysine-linked carbohydrates.

[0254] Thus, the genetically modified cells disclosed herein may also express mammalian LH3. The LH3 coding sequence, such as that shown by SEQ ID NO: 38, can be used for such purposes.

[0255] The above-mentioned procollagen and modifying enzymes may be expressed from a stably integrated or transiently expressed nucleic acid construct comprising a polynucleotide sequence encoding a procollagen α-chain and / or a modifying enzyme (e.g., P4H and LH3) that is placed under the transcriptional control of a functional promoter. Such nucleic acid constructs (also referred to herein as expression constructs) may be configured for expression in the whole organism (e.g., a plant, a defined tissue or a defined cell), and / or at a defined developmental stage of the organism. Such constructs may also include a selectable marker (e.g., antibiotic resistance), an enhancer element, and a replication origin for bacterial replication.

[0256] It will be understood that constructs containing two expressible inserts (e.g., two types of α-procollagen chains, or a procollagen α-chain and P4H) preferably include individual promoters for each insert, or alternatively, such constructs may express a single transcriptional chimera containing both insert sequences using a single promoter. In such cases, the chimeric transcript may include an internal ribosome entry site (IRES) sequence between the two insert sequences such that the downstream insert can be translated therefrom.

[0257] A number of functional expression promoters and enhancers, which can be tissue-specific, development-specific, constitutive or inducible, can be utilized by the constructs of the present invention, and some examples are provided hereinbelow. [[ID=]]

[0258] Regardless of the transformation technique employed, once progeny expressing procollagen are identified, such plants are further cultured under conditions that maximize their expression. Progeny obtained from transformed plants can be selected by confirming the presence of exogenous mRNA and / or polypeptides using nucleic acid or protein probes (e.g., antibodies). The latter approach allows for the localization of the expressed polypeptide components (e.g., by scrutinizing fractionated plant extracts) and thus also validates the plant's potential for the correct processing and organization of foreign proteins.

[0259] Following the cultivation of such plants, collagen containing telopeptides is typically harvested. Plant tissues / cells are preferably harvested at maturity, and procollagen molecules are isolated using an extraction approach. Preferably, harvesting is carried out so that the procollagen remains in a state where it can be cleaved by protease enzymes. According to one embodiment, a crude extract is produced from the transgenic plant of the present invention and subsequently contacted with a protease enzyme. Exemplary methods for producing a crude plant extract are described in the following Examples chapter of this specification.

[0260] It will be understood that collagen containing propeptides or telopeptides may be purified from the genetically modified cells of the present invention before incubation with protease, or after incubation with protease. Alternatively, collagen containing propeptides or telopeptides may be partially purified before protease treatment and then completely purified after protease treatment. Alternatively, collagen containing propeptides or telopeptides may be treated with protease simultaneously with other extraction / purification procedures.

[0261] Exemplary methods for purifying or semi-purifying collagen containing the telopeptides of the present invention include, but are not limited to, the removal of low-molecular-weight materials by salting out with ammonium sulfate and / or ultrafiltration.

[0262] As described in the background above, the use of animal-derived materials for medical purposes carries risks. These risks are also relevant when selecting proteolytic enzymes used in processing procollagens expressed in plants into atelocollagens. The application of enzymes from animal sources, such as trypsin or pepsin, can itself contaminate the final preparation with disease carriers. Therefore, it is desirable to devise production systems in which not all components are of animal origin.

[0263] It is disclosed herein that only specific proteases are capable of correctly cleaving collagen containing recombinant propeptides or telopeptides. Examples of such specific proteases include 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), and neutrases. In some embodiments, the use of recombinant enzymes such as rh-trypsin and rh-pepsin is disclosed herein. Such enzymes are commercially available and include, for example, ficin derived from fig cratex (Sigma, catalog number F4125 and Europe Biochem), subtilisin derived from Bacillus licheniformis (Sigma, catalog number P5459), neutrase derived from bacterium Bacillus amyloliquefaciens (Novozymes, catalog number PW201041) and TrypZean™, and recombinant human trypsin expressed in maize (Sigma catalog number T3449).

[0264] Procollagen or telocollagen is preferably brought into contact with a protease under conditions that allow the protease to cleave the propeptide or telopeptide from the contact point. Typically, these conditions are determined according to the specific protease chosen. For example, procollagen may be incubated with the protease at a concentration of 1–25 mg / ml and a temperature of about 10–20°C for up to 15 hours.

[0265] The atelocollagen produced may be further purified, for example by salt precipitation, as described in the Examples section below, so that the final product includes a purified composition of atelocollagen processed from procollagen produced from plants or plant cells by a protease selected from the group consisting of neutrase, subtilisin, ficin, and recombinant human trypsin, and analyzed using methods known in the art (e.g., size analysis by Coomassie staining, Western dysmetology, etc.).

[0266] After purification, atelocollagen may be resolubilized by adding an acidic solution (e.g., 10 mM HCl). Such an acidic solution is useful for preserving the purified atelocollagen.

[0267] The inventors of the present invention have shown that after digestion by ficin, atelocollagen retains its ability to form profibrils upon neutralization of the aforementioned acidic solution. According to one embodiment, at least 70% of the atelocollagen produced, purified, and resolubilized according to the method of the present invention is capable of forming profibrils. According to another embodiment, at least 88% of the atelocollagen produced, purified, and resolubilized according to the method of the present invention is capable of forming profibrils.

[0268] The ability to form fibrils indicates that the produced atelocollagen is useful for medical purposes including, but not limited to, cosmetic surgery, assisting in the healing of burn patients, bone reconstruction, and a wide variety of dental, orthopedic, and surgical purposes.

[0269] As discussed in the background section, type I collagen is considered a perfect candidate for use as a primary component in 3D bioprinting construction materials. Despite the significant advantages offered by this natural polymer, several factors hinder its use in 3D bioprinting. The use of tissue-extracted collagen for this purpose is limited due to its sensitivity to temperature and ionic strength, which promotes natural gel formation at temperatures above 20°C under physiological conditions [see, e.g., PureCol, Advanced BioMatrix, Inc.]. The typical temperature-dependent gel formation of tissue-extracted collagen significantly impairs precise fluidity during printing. Keeping the printing medium cold until application is a possible solution to this phenomenon, but it involves serious technical limitations. Another solution is the use of gelatin, a denatured form of collagen that does not gel under these conditions. However, gelatin lacks the true tissue-cell interaction of natural collagen, resulting in a loss of important biological functions.

[0270] Recent technological advancements have led to the development of a system for purifying natural human type I collagen (rh collagen) by introducing five human genes encoding heterotrimer type I collagen (COLLPLANT®, Israel, now also available as SIGMA-ALDRICH®, St. Louis, Missouri, USA) into tobacco plants. This protein is purified to a homogeneous state through a cost-effective industrial process that utilizes the inherent properties of collagen. See also WO2006 / 035442, WO2009 / 053985, and the patents and patent applications derived therefrom, which are all incorporated by reference as fully described herein.

[0271] Accordingly, according to one embodiment, a genetically modified plant is disclosed herein that expresses at least one type of collagen α chain and is capable of accumulating it in an intracellular compartment lacking endogenous P4H activity.

[0272] Type I collagen and rh collagen are considered candidate substances for use as main components in 3D bioprinting construction materials. Various types of scaffolds have been used for cosmetic and other reconstructive applications.

[0273] In addition, the use of skin fillers for soft tissue enhancement, such as wrinkle reduction, is increasing. One possible method for using skin fillers involves injecting polymerizable skin filler material into a desired area, followed by the steps of contouring or molding the filler into the desired shape. By polymerization and crosslinking of the material by one of various methods, monomers in the injected material can be converted to form polymers and chains, which can form a network structure and retain the desired molded shape. Several methods exist for forming polymers and crosslinking polymers. One method involves a photo-induced reaction with a photoreactive reagent that generates reactive species in a monomer solution.

[0274] However, at least some of these approaches continue to focus on tissue-derived collagen or non-collagen polymers (e.g., poly(vinyl alcohol) or hyaluronic acid). Furthermore, the use of collagen extracted from tissue is limited due to its sensitivity to temperature and ionic strength, which promotes natural gel formation at temperatures above 20°C under physiological conditions [see, e.g., PureCol, Advanced BioMatrix, Inc.]. The typical temperature-dependent gel formation of collagen extracted from tissue significantly impairs its precise fluidity. Keeping the collagen at a low temperature until application is a possible solution to this phenomenon, but it involves serious technical limitations. Another solution is the use of gelatin, a denatured form of collagen that does not gel under these conditions. However, gelatin lacks the true tissue-cell interaction of natural collagen, thus losing important biological functions. Furthermore, its viscosity makes it more difficult to inject beneath the dermis using fine-gauge needles, and also more difficult to spread and mold within smaller cavities.

[0275] Embodiments and uses of the skin fillers disclosed herein include, but are not limited to, the following: 1. A method for filling the tissue space beneath the epidermis, a. A step of introducing a polymerizable solution into the tissue space, wherein the polymerizable solution is i. Cross-linkable plant-derived human collagen, and ii. A step of introducing the photoinitiator, b. A method comprising the step of irradiating the surface of the epidermis on the surface of the space described above with light to induce polymerization. 2. A method for filling the tissue space beneath the epidermis, (a) A method comprising the step of molding or shaping a polymerizable solution within a desired configuration in a tissue space, wherein the step occurs simultaneously with or following the step of illuminating. 3. A method for filling a tissue space beneath the epidermis, wherein the shaping or reshaping step reduces muscles, folds, fine wrinkles, wrinkles, or scars. 4. A method for filling a tissue space beneath the epidermis, wherein cross-linkable plant-derived human collagen is methacrylated or thiolated. 5. A method for filling tissue space beneath the epidermis, wherein the polymer solution further comprises hyaluronic acid (HA) or a modified derivative thereof, poly(vinyl alcohol) (PVA) or a modified derivative thereof, polyethylene glycol (PEG) or a modified derivative thereof, cellulose oxide (OC) or a modified derivative thereof, polymethyl methacrylate (PMMA) microspheres or a modified derivative thereof, tricalcium phosphate (TCP) or a modified derivative thereof, calcium hydroxyl apatite (CaHA) or a modified derivative thereof, carboxymethylcellulose or a modified derivative thereof, crystalline nanocellulose (CNC) or a modified derivative thereof, or a combination thereof. 6. A method for filling the tissue space beneath the epidermis, wherein the modified derivatives of hyaluronic acid (HA), poly(vinyl alcohol) (PVA), polyethylene glycol (PEG), cellulose oxide (OC), polymethyl methacrylate (PMMA) microspheres, tricalcium phosphate (TCP), calcium hydroxyl apatite (CaHA), carboxymethylcellulose, or crystalline nanocellulose (CNC) include photopolymerizable modified derivatives. 7. The method according to claim 5, wherein the modified derivatives of hyaluronic acid (HA), poly(vinyl alcohol) (PVA), polyethylene glycol (PEG), cellulose oxide (OC), polymethyl methacrylate (PMMA) microspheres, tricalcium phosphate (TCP), calcium hydroxyl apatite (CaHA), carboxymethylcellulose, or crystalline nanocellulose (CNC) include methacrylated or thiolated derivatives. 8. A method for filling the tissue space beneath the epidermis, wherein the plant-derived collagen includes rh-collagen. 9. A method for filling the tissue space beneath the epidermis, wherein the plant-derived collagen is obtained from genetically modified plants. 10. A method for filling the tissue space beneath the epidermis, wherein the genetically modified plant is a genetically modified plant selected from the group consisting of tobacco, maize, alfalfa, rice, potato, soybean, tomato, wheat, barley, canola, carrot, and cotton. 11. A method for filling the tissue space beneath the epidermis, wherein the genetically modified plant is a tobacco plant. 12. A method for filling a tissue space beneath the epidermis, wherein the genetically modified plant comprises an expressible sequence of at least one gene sequence of starfish oxyribonucleic acid (DNA) selected from the group consisting of COL1, COL2, P4H-α, P4H-β, and LH3. 13. A method for filling a tissue space beneath the epidermis, wherein the plant-derived human collagen comprises at least one modified human collagen α-1 chain expressed in a genetically modified plant as shown in SEQ ID NO: 3, and at least one modified human collagen α-2 chain expressed in a genetically modified plant as shown in SEQ ID NO: 6, wherein the genetically modified plant further expresses exogenous prolyl-4-hydroxylase (P4H). 14. A method for filling a tissue space beneath the epidermis, further comprising the step of expressing an exogenous polypeptide selected from the group consisting of lysyl hydroxylase (LH), protease N, and protease C. 15. A method for filling a tissue space beneath the epidermis, wherein human collagen α-1 chain is encoded by the sequence described in Sequence ID No. 1. 16. A method for filling a tissue space beneath the epidermis, wherein human collagen α-2 chain is encoded by the sequence described in Sequence ID No. 2. 17. A method for filling the tissue space beneath the epidermis, wherein the exogenous P4H is mammalian P4H. 18. A method for filling the tissue space beneath the epidermis, wherein the exogenous P4H is human P4H. 19. A method for filling a tissue space beneath the epidermis, further comprising the steps of targeting human collagen α-1 to a vacuole of a plant or genetically modified plant, and digesting the same with ficin. 20. A method for filling a tissue space beneath the epidermis, further comprising the steps of targeting human collagen α-2 to a vacuole of a plant or genetically modified plant, and digesting the same with ficin. 21. A method for filling the tissue space beneath the epidermis, wherein the plant-derived human collagen is atelocollagen. 22. A method for filling a tissue space beneath the epidermis, wherein the light source includes a light-emitting diode (LED), a laser, or a xenon lamp. 23. A method for filling a tissue space beneath the epidermis, wherein a photoinitiator induces polymerization of a polymerizable solution in response to visible light. 24. A method for filling the tissue space beneath the epidermis, wherein the visible light has a wavelength of 390 to 700 nm. 25. A method for filling the tissue space beneath the epidermis, wherein the photoinitiator is selected from the group consisting of eosin Y + triethanolamine or riboflavin. 26. A method for filling a tissue space beneath the epidermis, wherein a photoinitiator induces polymerization of a polymerizable solution in response to ultraviolet (UV) light. 27. A method for filling a subcutaneous tissue space, wherein the photoinitiator is selected from the group consisting of lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) or 1-[4 2-hydroxy-1-[4-(2-hydroxyethoxy)phenyl]-2-methylpropan-1-one (IRGACURE® 2959). 28. A method for filling a tissue space beneath the epidermis, wherein a photoinitiator induces polymerization of a polymerizable solution in response to infrared light. 29. A method for filling a tissue space beneath the epidermis, wherein a polymerizable solution is introduced into the tissue space via a hollow needle or cannula ranging from 27 gauge to 33 gauge. 30. A method for filling a tissue space beneath the epidermis, wherein a polymerizable solution within the tissue space is molded or shaped into a desired configuration by manual massage. 31. A method for filling a tissue space beneath the epidermis, wherein a polymerizable solution within the tissue space can be molded or shaped into a desired configuration using a molding or shaping instrument. 32. A method for filling a tissue space beneath the epidermis, wherein the polymerizable solution within the tissue space is essentially non-gelatable at room temperature. 33. A method for filling a tissue space beneath the epidermis, wherein the polymerizable solution within the tissue space is essentially non-gelatinous at 37°C. 34. A method for filling the tissue space beneath the epidermis, wherein a polymerizable solution containing plant-derived human collagen has a lower viscosity at room temperature compared to a similar polymerizable solution containing human or bovine collagen extracted from tissue, at the same concentration and formulation. 35. A method for filling the tissue space beneath the epidermis, wherein a polymerizable solution containing plant-derived human collagen has a lower viscosity at 37°C compared to a similar polymerizable solution containing human or bovine collagen extracted from tissue, at the same concentration and formulation. 36. A method for filling tissue spaces beneath the epidermis, wherein a polymerizable solution containing plant-derived human collagen is introduced into the tissue space with less force at room temperature compared to a similar polymerizable solution containing human or bovine collagen extracted from tissue, at the same concentration and formulation. 37. A method for filling tissue spaces beneath the epidermis, wherein a polymerizable solution containing plant-derived human collagen is introduced into the tissue space at a lower force at 37°C compared to a similar polymerizable solution containing human or bovine collagen extracted from tissue at the same concentration and formulation. 38. A method for filling the tissue space beneath the epidermis, wherein a polymerizable solution containing plant-derived human collagen provides increased tissue enhancement compared to a similar polymerizable solution containing human or bovine collagen extracted from tissue, at the same concentration and formulation. 39. Use of a polymerizable solution injected into the tissue space beneath the epidermis to reduce muscles, folds, fine lines, wrinkles, or scars, wherein the polymerizable solution comprises crosslinkable plant-derived human collagen and a photoinitiator for inducing polymerization before or simultaneously with the application of visible light, and the use comprises the step of shaping or forming the polymerizable solution within a desired configuration for reducing muscles, folds, fine lines, wrinkles, or scars. 40. Use of a polymerizable solution injected into the tissue space beneath the epidermis to reduce muscle, folds, fine lines, wrinkles, or scars, wherein crosslinkable plant-derived human collagen is methacrylated or thiolated. 41. Use of a polymerizable solution injected into the tissue space beneath the epidermis to reduce muscle, folds, fine lines, wrinkles, or scars, wherein the polymer solution further comprises hyaluronic acid (HA) or a modified derivative thereof, poly(vinyl alcohol) (PVA) or a modified derivative thereof, polyethylene glycol (PEG) or a modified derivative thereof, polymethyl methacrylate (PMMA) microspheres or a modified derivative thereof, tricalcium phosphate (TCP) or a modified derivative thereof, calcium hydroxyl apatite (CaHA) or a modified derivative thereof, carboxymethylcellulose or a modified derivative thereof, crystalline nanocellulose (CNC) or a modified derivative thereof, or a combination thereof. 42. Use of polymerizable solutions injected into the tissue space beneath the epidermis to reduce muscle, folds, fine lines, wrinkles, or scars, wherein modified derivatives of hyaluronic acid (HA), poly(vinyl alcohol) (PVA), polyethylene glycol (PEG), cellulose oxide (OC), polymethyl methacrylate (PMMA) microspheres, tricalcium phosphate (TCP), calcium hydroxyl apatite (CaHA), carboxymethylcellulose, or crystalline nanocellulose (CNC) include photopolymerizable modified derivatives. 43. Use of polymerizable solutions injected into the tissue space beneath the epidermis to reduce muscle, folds, fine lines, wrinkles, or scars, wherein modified derivatives of hyaluronic acid (HA), poly(vinyl alcohol) (PVA), polyethylene glycol (PEG), cellulose oxide (OC), polymethyl methacrylate (PMMA) microspheres, tricalcium phosphate (TCP), calcium hydroxyl apatite (CaHA), carboxymethylcellulose, or crystalline nanocellulose (CNC) include methacrylated or thiolated derivatives. 44. A method for filling a tissue space beneath the epidermis, comprising the step of introducing a polymerizable solution into the tissue space, wherein the polymerizable solution comprises crosslinkable plant-derived human collagen. 45. A method for filling the tissue space beneath the epidermis, (a) A method further comprising the step of molding or shaping a polymerizable solution within a desired configuration in a tissue space. 46. ​​A method for filling a tissue space beneath the epidermis, wherein the shaping or reshaping step reduces muscles, folds, fine wrinkles, wrinkles, or scars. 47. A method for filling the tissue space beneath the epidermis, wherein the polymerizable solution further comprises hyaluronic acid (HA) or a modified derivative thereof, poly(vinyl alcohol) (PVA) or a modified derivative thereof, polyethylene glycol (PEG) or a modified derivative thereof, cellulose oxide (OC) or a modified derivative thereof, polymethyl methacrylate (PMMA) microspheres or a modified derivative thereof, tricalcium phosphate (TCP) or a modified derivative thereof, calcium hydroxyl apatite (CaHA) or a modified derivative thereof, carboxymethylcellulose or a modified derivative thereof, crystalline nanocellulose (CNC) or a modified derivative thereof, or a combination thereof. 48. A method for filling the tissue space beneath the epidermis, wherein the plant-derived collagen includes rh-collagen. 49. A method for filling the tissue space beneath the epidermis, wherein the plant-derived collagen is obtained from genetically modified plants. 50. A method for filling the tissue space beneath the epidermis, wherein the genetically modified plant is selected from the group consisting of tobacco, maize, alfalfa, rice, potato, soybean, tomato, wheat, barley, canola, carrot, and cotton. 51. A method for filling the tissue space beneath the epidermis, wherein the genetically modified plant is a tobacco plant. 52. A method for filling the tissue space beneath the epidermis, wherein the genetically modified plant comprises an expressible sequence of at least one gene sequence of starfish oxyribonucleic acid (DNA) selected from the group consisting of COL1, COL2, P4H-α, P4H-β, and LH3. 53. A method for filling a tissue space beneath the epidermis, wherein the plant-derived human collagen comprises at least one modified human collagen α-1 chain expressed in a genetically modified plant as shown in SEQ ID NO: 3, and at least one modified human collagen α-2 chain expressed in a genetically modified plant as shown in SEQ ID NO: 6, the genetically modified plant further expressing exogenous prolyl-4-hydroxylase (P4H). 54. A method for filling a tissue space beneath the epidermis, further comprising the step of expressing an exogenous polypeptide selected from the group consisting of lysyl hydroxylase (LH), protease N, and protease C. 55. A method for filling a tissue space beneath the epidermis, wherein human collagen α-1 chain is encoded by the sequence described in Sequence ID No. 1. 56. A method for filling a tissue space beneath the epidermis, wherein human collagen α-2 chain is encoded by the sequence described in Sequence ID No. 2. 57. A method for filling the tissue space beneath the epidermis, wherein the exogenous P4H is mammalian P4H. 58. A method for filling the tissue space beneath the epidermis, wherein the exogenous P4H is human P4H. 59. A method for filling a tissue space beneath the epidermis, further comprising the steps of targeting human collagen α-1 to a vacuole of a plant or genetically modified plant, and digesting the same with ficin. 60. A method for filling a tissue space beneath the epidermis, further comprising the steps of targeting human collagen α-2 to a vacuole of a plant or genetically modified plant, and digesting the same with ficin. 61. A method for filling the tissue space beneath the epidermis, wherein the plant-derived human collagen is atelocollagen. 62. A method for filling a tissue space beneath the epidermis, wherein a polymerizable solution is introduced into the tissue space via a hollow needle or cannula ranging from 27 gauge to 33 gauge. 63. A method for filling a tissue space beneath the epidermis, wherein a polymerizable solution within the tissue space is molded or shaped into a desired configuration by manual massage. 64. A method for filling a tissue space beneath the epidermis, wherein a polymerizable solution within the tissue space can be molded or shaped into a desired configuration using a molding or shaping instrument. 65. A method for filling a tissue space beneath the epidermis, wherein the polymerizable solution within the tissue space is essentially non-gelatable at room temperature. 66. A method for filling a tissue space beneath the epidermis, wherein the polymerizable solution within the tissue space is essentially non-gelatable at 37°C. 67. A method for filling the tissue space beneath the epidermis, wherein a polymerizable solution containing plant-derived human collagen has a lower viscosity at room temperature compared to a similar polymerizable solution containing human or bovine collagen extracted from tissue, at the same concentration and formulation. 68. A method for filling the tissue space beneath the epidermis, wherein a polymerizable solution containing plant-derived human collagen has a lower viscosity at 37°C compared to a similar polymerizable solution containing human or bovine collagen extracted from tissue, at the same concentration and formulation. 69. A method for filling tissue spaces beneath the epidermis, wherein a polymerizable solution containing plant-derived human collagen is introduced into the tissue space with less force at room temperature compared to a similar polymerizable solution containing human or bovine collagen extracted from tissue, at the same concentration and formulation. 70. A method for filling tissue spaces beneath the epidermis, wherein a polymerizable solution containing plant-derived human collagen is introduced into the tissue space at a lower force at 37°C compared to a similar polymerizable solution containing human or bovine collagen extracted from tissue at the same concentration and formulation. 71. A method for filling the tissue space beneath the epidermis, wherein a polymerizable solution containing plant-derived human collagen provides increased tissue enhancement compared to a similar polymerizable solution containing human or bovine collagen extracted from tissue, at the same concentration and formulation. 72. Use of a polymerizable solution injected into the tissue space beneath the epidermis to reduce muscle, folds, fine wrinkles, creases, or scars, wherein the polymerizable solution comprises crosslinkable plant-derived human collagen, and the use comprises the step of shaping or forming the polymerizable solution within a desired configuration for reducing muscle, folds, fine wrinkles, creases, or scars. 73. A method for inducing a cell proliferation scaffold in the tissue space beneath the epidermis, comprising the step of introducing a solution into the tissue space, wherein the solution is (a) Plant-derived human collagen, (b) A method comprising at least one growth factor or a source thereof. 74. A method for inducing a cell proliferation scaffold in the tissue space beneath the epidermis, wherein at least one source of growth factors comprises plasma or platelet-rich plasma. 75. A method for inducing a cell proliferation scaffold in a tissue space beneath the epidermis, wherein the cell proliferation scaffold promotes healing or replacement resulting from the degradation or damage of collagen-containing tissue. 76. A method for inducing a cell proliferation scaffold in a tissue space beneath the epidermis, wherein the collagen-containing tissue is selected from the group consisting of tendons, ligaments, skin, cornea, cartilage, blood vessels, intestines, intervertebral discs, muscles, bones, or teeth. 77. A method for inducing a cell proliferation scaffold in the tissue space beneath the epidermis, wherein the cell proliferation scaffold promotes the healing of tendinitis. 78. A method for inducing a cell proliferation scaffold in the tissue space beneath the epidermis, wherein the plant-derived collagen includes rh-collagen. 79. A method for inducing a cell proliferation scaffold in the tissue space beneath the epidermis, wherein the plant-derived collagen is obtained from genetically modified plants. 80. A method for inducing a cell proliferation scaffold in the tissue space beneath the epidermis, wherein the genetically modified plant is selected from the group consisting of tobacco, maize, alfalfa, rice, potato, soybean, tomato, wheat, barley, canola, carrot, and cotton. 81. A method for inducing a cell proliferation scaffold in the tissue space beneath the epidermis, wherein the genetically modified plant is a tobacco plant. 82. A method for inducing a cell proliferation scaffold in the tissue space beneath the epidermis, wherein the genetically modified plant comprises an expressible sequence of at least one gene sequence of star deoxyribonucleic acid (DNA) selected from the group consisting of COL1, COL2, P4H-α, P4H-β, and LH3. 83. A method for inducing a cell proliferation scaffold in the tissue space beneath the epidermis, wherein the plant-derived human collagen comprises at least one modified human collagen α-1 chain expressed in a genetically modified plant as shown in SEQ ID NO: 3, and at least one modified human collagen α-2 chain expressed in a genetically modified plant as shown in SEQ ID NO: 6, the genetically modified plant further expressing exogenous prolyl-4-hydroxylase (P4H). 84. A method for inducing a cell proliferation scaffold in a tissue space beneath the epidermis, further comprising the step of expressing an exogenous polypeptide selected from the group consisting of lysyl hydroxylase (LH), protease N, and protease C. 85. A method for inducing a cell proliferation scaffold in the tissue space beneath the epidermis, wherein human collagen α-1 chain is encoded by the sequence described in Sequence ID No. 1. 86. A method for inducing a cell proliferation scaffold in the tissue space beneath the epidermis, wherein human collagen α-2 chain is encoded by the sequence described in Sequence ID No. 2. 87. A method for inducing a cell proliferation scaffold in the tissue space beneath the epidermis, wherein the exogenous P4H is mammalian P4H. 88. A method for inducing a cell proliferation scaffold in the tissue space beneath the epidermis, wherein the exogenous P4H is human P4H. 89. A method for inducing a cell proliferation scaffold in a tissue space beneath the epidermis, further comprising the steps of targeting human collagen α-1 to a vacuole of a plant or genetically modified plant, and digesting the same with ficin. 90. A method for inducing a cell proliferation scaffold in a tissue space beneath the epidermis, further comprising the steps of targeting human collagen α-2 to a vacuole of a plant or genetically modified plant, and digesting the same with ficin. 91. A method for inducing a cell proliferation scaffold in the tissue space beneath the epidermis, wherein the plant-derived human collagen is atelocollagen. 92. A method for inducing a cell proliferation scaffold in the tissue space beneath the epidermis, wherein a solution containing plant-derived human collagen has a lower viscosity at room temperature compared to a similar solution containing human or bovine collagen extracted from tissue at the same concentration and formulation. 93. A method for inducing a cell proliferation scaffold in the tissue space beneath the epidermis, wherein a solution containing plant-derived human collagen has a lower viscosity at 37°C compared to a similar polymerizable solution containing human or bovine collagen extracted from tissue at the same concentration and formulation. 94. A method for inducing a cell proliferation scaffold in the tissue space beneath the epidermis, wherein a solution containing plant-derived human collagen is introduced into the tissue space with less force at room temperature compared to a similar solution containing human or bovine collagen extracted from tissue at the same concentration and formulation. 95. A method for inducing a cell proliferation scaffold in the tissue space beneath the epidermis, wherein a solution containing plant-derived human collagen is introduced into the tissue space at a lower force at 37°C compared to a similar solution containing human or bovine collagen extracted from tissue at the same concentration and formulation. 96. A method for inducing a cell proliferation scaffold in the tissue space beneath the epidermis, wherein a solution containing plant-derived human collagen increases scaffold formation or promotes an increase in cell proliferation compared to a similar solution containing human or bovine collagen extracted from tissue at the same concentration and formulation. 97. Use of a solution injected into a subcutaneous tissue space to induce a cell proliferation scaffold, the solution comprising plant-derived human collagen and at least one growth factor or its source, and the use of a solution that promotes healing or replacement resulting from the degradation or damage of collagen-containing tissue. 98. Use of a solution injected into a subcutaneous tissue space for inducing a cell proliferation scaffold, wherein at least one source of growth factors comprises plasma or platelet-rich plasma.

[0276] definition

[0277] As used herein, the singular forms "a," "an," and "the" include multiple references unless the context explicitly indicates otherwise. For example, the term "a molecule" also includes multiple molecules.

[0278] As used herein, the term “approximately” refers to ±10% or ±5%.

[0279] The terms "comprises," "comprising," "includes," "including," and "having," and their conjugations, all mean "not limited to including."

[0280] The term "consisting of" means "including and limited to".

[0281] The term “essentially consisting of” means that the configuration, method, or structure may include additional components, processes, and / or parts only if the additional components, processes, and / or parts do not substantially alter the fundamental and novel properties of the claimed configuration, method, or structure.

[0282] As used herein, the term “method” refers to methods, means, techniques, and procedures for accomplishing a given task, including, but not limited to, methods, means, techniques, and procedures known to practitioners of chemistry, pharmacology, biology, biochemistry, and medicine, or methods, means, techniques, and procedures readily developed from methods, means, techniques, and procedures known to practitioners of chemistry, pharmacology, biology, biochemistry, and medicine.

[0283] As used herein, the phrase “genetically modified plant” encompasses any lower (e.g., moss) or higher (e.g., vascular) plant, or its tissue or isolated cells (e.g., cell suspensions), that are stably or transiently transformed with an exogenous polynucleotide sequence. Examples of plants include, but are not limited to, tobacco, maize, alfalfa, rice, potato, soybean, tomato, wheat, barley, canola, cotton, carrot, and lower plants such as moss.

[0284] As used herein, the phrase “collagen chain” includes collagen subunits such as the α1 or α2 chain of a collagen fiber, preferably a type I fiber. As used herein, the phrase “collagen” refers to an assembled collagen trimer, which in the case of type I collagen includes two α1 chains and one α2 chain. A collagen fiber is collagen lacking terminal propeptides C and N.

[0285] As used herein, the phrase "telopeptide-containing collagen" encompasses soluble collagen molecules containing telopeptides longer than the telopeptide remnants found in atelocollagen. Therefore, telopeptide-containing collagen may also be procollagen containing full-length propeptides. Alternatively, telopeptide-containing collagen may be procollagen molecules containing partially digested propeptides. Furthermore, telopeptide-containing collagen may also be telocollagen.

[0286] As used herein, the term “procollagen” encompasses collagen molecules (e.g., human) that contain either an N-terminal propeptide, a C-terminal propeptide, or both. Exemplary human procollagen amino acid sequences are shown by SEQ ID NOs: 1, 2, 7, and 8.

[0287] As used herein, the term “telocollagen” encompasses collagen molecules that lack both the N-terminal and C-terminal propeptides typically found in procollagen, but still contain telopeptides. The telopeptides of fibrous collagen are remnants of the N-terminal and C-terminal propeptides after digestion with native N / C proteinases. Recombinant human telocollagen can be produced in cells transformed to express both exogenous human procollagen and their respective proteases (i.e., C or N, or both). Polynucleotide sequences encoding such proteases are exemplified by SEQ ID NOs: 10 (protease C) and 11 (protease N). Such proteases can be expressed so that they accumulate in the same intracellular compartment as the collagen chain, as further described below herein.

[0288] As used herein, the term “atelocollagen” encompasses collagen molecules that typically lack both the N-terminal and C-terminal propeptides present in at least a portion of procollagen and its telopeptides, but contain a sufficient portion of its telopeptide such that it can form profibrils under possible preferred conditions. According to the methods of the present invention, any type of atelocollagen can be produced. Examples include profibril-forming collagen (types I, II, III, V, and XI), reticular-forming collagen (types IV, VIII, and X), fibril-associating collagen (types IX, XII, and XIV), transmembrane proteins (types XIII and XVII), or 11 nm periodic spherical filaments (type VI). According to one embodiment, the atelocollagen comprises the α1 and / or α2 chains of type I collagen.

[0289] It will be understood that in some embodiments, the skin fillers disclosed herein may include genetically modified forms of collagen / atelocollagen, such as collagenase-resistant collagen.

[0290] As used herein, the phrase “plant promoter” or “promoter” includes promoters capable of directing gene expression in plant, fungal, and yeast cells (including DNA-containing organelles). Such promoters may be of plant, bacterial, viral, fungal, or animal origin. Such promoters may be constitutive (i.e., capable of directing high levels of gene expression in multiple tissues), tissue-specific (i.e., capable of directing gene expression in a specific tissue or multiple tissues), inducible (i.e., capable of directing gene expression by stimulation), or chimeric (i.e., formed from at least two different promoter parts).

[0291] As used herein, the phrase “intracellular compartment lacking endogenous P4H activity” refers to any compartmentalized region of a cell that does not contain enzymes having plant P4H or plant-like P4H activity. Examples of such intracellular compartments include vacuoles, apoplasts, and cytoplasm, as well as organelles such as chloroplasts and mitochondria.

[0292] Throughout this specification, the phrase “construction material” encompasses the phrase “uncured construction material” or “uncured construction material formulation” and comprehensively describes the material used to sequentially form layers as described herein. This phrase encompasses the uncured material that forms the final object, i.e., one or more uncured modeling material formulations, and optionally, the uncured material used to form the support, i.e., an uncured support material formulation. The uncured construction material may comprise one or more modeling formulations, which can be distributed so that different parts of the object are made when different modeling formulations are cured, and thus may be made of different cured modeling materials or different mixtures of cured modeling materials.

[0293] As used herein, “bioprinting” means carrying out an additive manufacturing process using one or more bioink formulations containing biological components, by a methodology compatible with an automated or semi-automated computer-aided additive manufacturing system (e.g., a bioprinter or bioprinting system) as described herein.

[0294] Throughout this specification, in the context of bioprinting, the term “object” refers to the final product of an additive manufacturing process that contains a biological component in at least part of it. If the support material is used as part of the uncured construct, the term refers to the product obtained by the bioprinting method described herein after the removal of the support material. In some embodiments, the biological component includes, for example, recombinant human collagen as described in WO2006 / 035442, WO2009 / 053985, and the patents and patent applications derived therefrom (all of which are incorporated by reference as if fully described herein).

[0295] As used throughout this specification, the term “object” refers to the entire object or a part thereof.

[0296] Throughout this specification, “curable material” refers to a compound (monomer or oligomer or polymer compound) that solidifies or hardens when exposed to curing conditions as defined herein, forming a curable modeling material as defined herein. Curable materials are typically polymerizable materials that undergo polymerization and / or crosslinking when exposed to a suitable energy source. Alternatively, curable materials are thermoresponsive materials that solidify or harden when exposed to temperature changes (e.g., heating or cooling). In some cases, curable materials are biological materials that undergo reactions to form a cured or solidified material during biological reactions (e.g., enzyme-catalyzed reactions).

[0297] "Curing conditions" include curing energy (e.g., temperature, radiation) and / or materials or reagents that facilitate curing.

[0298] In some of the embodiments described herein, the curable material is a photopolymerizable material that polymerizes or crosslinks upon exposure to radiation as described herein, and in some embodiments, the curable material is a UV-curable or visible light-curable material that polymerizes or crosslinks upon exposure to UV-vis radiation as described herein.

[0299] In any part of the embodiments described herein, the curable material may be a monomer, oligomer or short-chain polymer, each of which is polymerizable as described herein.

[0300] In this specification, the term "curable" includes the terms "polymerizable" and "crosslinkable."

[0301] As used herein, “aeroponics” refers to the process of growing plants in an air or fog environment without the use of soil or agglomerating medium (also known as “geoponics”).

[0302] As used herein, "hydroponics" refers to the process of growing plants without soil using an inorganic nutrient solution in an aqueous solvent ("geoponics").

[0303] As used herein, “endophytes” include all endophytes of plants.

[0304] As used herein, “exudate” is a fluid released by an organism through a pore or wound. “Exudation” is the process of releasing “exudate.”

[0305] As used herein, "hydroponics" refers to the process of growing plants without soil using an inorganic nutrient solution in an aqueous solvent ("geoponics").

[0306] As used herein, “integration” or “integration hybridization” is the transfer of genes (i.e., “gene flow”) from one gene pool to another species via repeated backcrossing with one of the interspecific hybrids. Unlike simple crosses, the parent species result in a complex mixture of parental genes.

[0307] As used herein, “metabolome” is the complete set of low molecular weight chemicals found within a “biological sample” (including, but not limited to, cells, organelles, organs, tissues, tissue extracts, biological fluids, or organisms). The low molecular weight chemicals of the metabolome may be “endogenous metabolites” or “exogenous chemicals.” “Endogenous metabolites” are naturally produced by organisms and include, but are not limited to, amino acids, organic acids, nucleic acids, fatty acids, amines, sugars, vitamins, cofactors, pigments, and antibiotics. “Exogenous chemicals” are not naturally produced by organisms and include, but are not limited to, drugs, environmental pollutants, food additives, toxins, and other xenobiotics. “Endogenous metabolome” consists of endogenous metabolites, and “exogenous metabolome” consists of “exogenous chemicals.” “Endogenous metabolome” consists of “primary metabolome” and “secondary metabolome,” particularly with respect to plants, fungi, and prokaryotes. The "primary metabolome" consists of "primary metabolites" (i.e., metabolites directly involved in the normal growth, development, and reproduction of an organism), while the "secondary metabolome" consists of "secondary metabolites" (i.e., metabolites not directly involved in the normal growth, development, and reproduction of an organism). Secondary metabolites often have important ecological functions.

[0308] As used herein, “metabolites” are typically small molecules with a molecular weight of less than 1500 Da. Examples of “metabolites” include, but are not limited to, glycolipids, polysaccharides, short peptides, small oligonucleotides, organic acids, taxanes, alkaloids, and strigolactones, while very large macromolecules (e.g., proteins, mRNA, rRNA, and DNA) are generally not metabolites and are not part of the metabolome.

[0309] As used herein, "SILVA database" refers to the SILVA ribosomal RNA database.

[0310] All samples obtained from living organisms, including those that have undergone any kind of further processing, are considered to be derived from living organisms.

[0311] Methods for isolating, sequencing, amplifying, and / or cloning DNA are known to those skilled in the art. The most commonly used method for DNA amplification is PCR (polymerase chain reaction, see, e.g., PCR Basics: from background to Bench, Springer Verlag, 2000; Eckert et al., 1991. PCR Methods and Applications 1:17). Further preferred amplification methods include ligase chain reaction (LCR), transcriptional amplification and autologous sequence replication, and nucleic acid-based sequence amplification (NASBA). Similarly, methods for isolating and characterizing RNA and proteins, as well as for protein expression, are known to those skilled in the art.

[0312] The following examples are presented to more fully illustrate some embodiments of the dermal fillers disclosed herein and their uses. However, they should not be construed as limiting the dermal fillers disclosed herein or their broad range of uses. Those skilled in the art can readily devise many variations and modifications of the principles disclosed herein without departing from the scope of the invention. [Examples]

[0313] Example 1. Structure and transformation scheme.

[0314] The construction of the expression cassette and vector used in this study is shown in Figures 1A–1D (see also U.S. Patent No. 8,455,717). All coding sequences in this study were optimized for expression in tobacco and chemically synthesized in the desired flanking region (SEQ ID NOs. 1, 4, 7, 12, 14, 16, 18, 20, 22). Figure 1A: Synthetic genes encoding Col1 and Col2 (SEQ ID NOs. 1, 4) that are either fused to either a vacuolar signal or an apoplast signal (encoded by SEQ ID NO. 7), or whose signals are not cloned within an expression cassette consisting of the Chrysanthemum rbcS1 promoter and 5'UTR (SEQ ID NO. 10), Chrysanthemum rbcS1 3'UTR and terminator (SEQ ID NO. 11). The complete expression cassette was cloned within multiple cloning sites of the pBINPLUS plant transformation vector (van Engelen et al., 1995, Transgenic Res 4:288–290). Figure 1B: Synthetic genes encoding P4Hβ-human, P4Hα-human, and P4H-plant (SEQ ID NOs. 12, 14, and 16) that are either fused to either a vacuolar signal or an apoplast signal (encoded by SEQ ID NO 7), or whose signals are not cloned within an expression cassette consisting of a CaMV 35S promoter and TMV omega sequence and Agrobacterium nopaline synthetase (NOS) terminator held by vector pJD330 (Galili et al., 1987, Nucleic Acids Res 15:3257-3273). Complete expression cassettes were cloned within multiple cloning sites of the pBINPLUS vector holding either a Col1 or Col2 expression cassette. Figure 1C: Synthetic genes encoding proteinases C and N (SEQ ID NOs. 18, 20), fused to either vacuolar or apoplastic signals (encoded by SEQ ID NO: 7), were cloned within an expression cassette consisting of the Chrysanthemum rbcS1 promoter and 5'UTR (SEQ ID NO: 10), and the Chrysanthemum rbcS1 3'UTR and terminator (SEQ ID NO: 11).The complete expression cassette was cloned within multiple cloning sites of the pBINPLUS plant transformation vector. Figure 1D: A synthetic gene encoding LH3 (SEQ ID NO: 22) having an adjacent strawberry vein banding virus (SVBV) promoter (NCBI deposit AF331666 REGION:623.950 ver. AF331666.1 GI:13345788) and terminated by an Agrobacterium octopin synthase (OCS) terminator (NCBI deposit Z37515 REGION:1344.1538 ver. Z37515.1 GI:886843), either fused to either a vacuolar signal or an apoplast signal (encoded by SEQ ID NO: 7), or the signal not cloned at multiple cloning sites of the pBINPLUS vector possessing the Col1 and P4Hβ expression cassettes.

[0315] Figure 2 shows the cotransformation schemes for host plants using the expression cassettes described in Figures 1A-1D. Each expression cassette insert is represented by the short name of its coding sequence. The coding sequences and associated sequence numbers are listed in Table 1. Each cotransformation is performed by two pBINPLUS binary vectors. Each rectangle represents a single pBINPLUS vector containing one, two, or three expression cassettes. Promoters and terminators are indicated in Figures 1A-1D.

[0316] Example 2. Plant collagen expression.

[0317] The synthetic polynucleotide sequences encoding the proteins listed in Table 1 below were designed and optimized for expression in tobacco plants.

[0318] [Table 1]

[0319] Signal peptide

[0320] 1. Vacuolar signal sequence of the barley gene for the thiol protease allurein precursor (NCBI deposit P05167 GI:113603) MAHARVLLLALAVLATAAVAVASSSSFADSNPIRPVTDRAASTLA (SEQ ID NO: 24). 2. Apoplast signal of endo-1,4-β-glucanase of Arabidopsis thaliana (Cell, NCBI deposit CAA67156.1 GI:2440033); encoded by SEQ ID NO: 9, SEQ ID NO: 7.

[0321] Plasmid construction

[0322] Plant expression vectors were constructed as taught in Example 1, and the composition of each constructed expression vector was confirmed by restriction analysis and sequencing.

[0323] An expression vector containing the following expression cassette was constructed.

[0324] 1. Collagen α1 2. Collagen α1 + Human P4Hβ subunit 3. Collagen α1 + Human P4Hβ subunit + Human LH3 4. Collagen α2 5. Contains collagen α2 + human P4Hα subunit 6. Contains collagen α2 + Arabidopsis P4H 7. P4Hβ subunit + human LH3 8. Human P4Hα subunit

[0325] Each of the coding sequences described above is either translated and fused to a vacuolar transit peptide or an apoplast transit peptide, or it lacks any transit peptide sequence, in which case cytoplasmic accumulation is expected.

[0326] Plant transformation and PCR screening

[0327] Tobacco plants (Nicotiana tabacum, Samsun NN) were transformed using the expression vector described above, according to the transformation scheme taught in Figure 2.

[0328] The resulting transgenic plants were screened by multiplex PCR using four primers designed to amplify the 324 bp fragment of collagen α1 and the 537 bp fragment of collagen α2 (Table 2). Figure 3 shows the results of one multiplex PCR screen.

[0329] [Table 2]

[0330] Example 3. Detection of human collagen in transgenic tobacco plants.

[0331] Total soluble proteins were extracted from tobacco transformants 2, 3, and 4 by grinding 500 mg of leaves in 0.5 ml of 50 mM Tris-HCl (pH=7.5) with a "complete" protease inhibitor cocktail (product no. 1836145, Roche Diagnostics GmbH, one tablet per 50 ml buffer). The crude extract was mixed with 250 μl of 4X. The sample was boiled for 7 minutes in a sample application buffer containing 10% β-mercaptoethanol and 8% SDS, and then centrifuged at 13000 rpm for 8 minutes. 20 ml of the supernatant was loaded onto a 10% polyacrylamide gel and tested using anti-collagen type I (denatured) antibody (product no. AB745, Chemicon Inc.) in a standard Western blotting procedure (Figure 4). WT represents wild-type tobacco. A positive collagen band can be seen in plants that are PCR positive for collagen type I α1 or α2, or both. A 500 ng positive control band of type I collagen derived from human placenta (Category CC050, manufactured by Chemicon Inc.) represents approximately 0.3% (approximately 150 μg) of total soluble protein in the transgenic plant-derived sample.

[0332] When collagen was targeted to vacuoles, plants expressing collagen at expected molecular weights up to approximately 1% of total soluble protein were detected (Figure 4). Intracellular targeting of full-length collagen to apoplasts was successfully achieved (Figure 5). Plants expressing collagen in the cytoplasm (i.e., without the target peptide) did not accumulate collagen to detectable levels, indicating that intracellular targeting of collagen in plants is crucial for this success.

[0333] In addition, in contrast to the studies by Ruggiero et al., 2000 and Merle et al., 2002, which showed that collagen lacking N-propeptide underwent significant proteolysis using this approach, full-length collagen proteins containing both C-propeptide and N-propeptide accumulated at high levels in intracellular compartments.

[0334] This data also clearly demonstrates that crossing two plants, each expressing different types of collagen chains, is advantageous in that it allows for the selection of plants expressing optimal levels of each type of chain, and subsequent plant crosses enable the achievement of desired collagen-producing plants.

[0335] The collagen produced by the plant of the present invention is expected to contain natural propeptides and therefore form larger proteins than human controls purified by proteolysis. The calculated molecular weights of the unhydroxylated or unglycosylated collagen α1 and α2 chains are as follows: Col1 with propeptide -- 136 kDa, Col1 without propeptide -- 95 kDa, Col2 with propeptide -- 127 kDa, and Col2 without propeptide -- 92 kDa.

[0336] As can be seen in Figure 4, the Col1 band in transformants 3-5 and 3-49 appears larger than the Col1 band in the other plants. This band indicates proline hydroxylation in the collagen chain by a human proline-4-hydroxylase holoenzyme, which is co-expressed in these plants and consists of α and β subunits targeted to the same intracellular compartment (e.g., vacuole) as the human collagen chain.

[0337] Example 4. Collagen triple helix organization and thermal stability in transgenic plants

[0338] Collagen triple helix organization and helix thermal stability in transgenic plants were tested by thermal denaturation of whole crude protein extracts of transgenic plants, followed by digestion with trypsin or pepsin (Figures 6A-6B).

[0339] In the first experiment, total soluble proteins from tobacco plants 2-9 (expressing only col α1 and not P4H) and 3-5 (expressing both col α1+2 and human P4H) were extracted by grinding 500 mg of leaves in 0.5 ml of 50 mM Tris-HCl (pH=7.5), centrifuging at 13000 rpm for 10 minutes, and collecting the supernatant. 0 μl of the supernatant was heat-treated (33°C or 43°C for 15 minutes) and immediately placed on ice. Trypsin digestion was initiated by adding 6 μl of each sample to 1 mg / ml trypsin in 50 mM Tris-HCl (pH=7.5). The samples were incubated at room temperature (approximately 22°C) for 20 minutes. Digestion was stopped by adding 20 μl of 4X sample application buffer containing 10% β-mercaptoethanol and 8% SDS. The sample was boiled for 7 minutes and centrifuged at 13000 rpm for 7 minutes. 50 μl of the supernatant was loaded onto a 10% polyacrylamide gel and tested using anti-collagen type I (denatured) antibody (Chemicon Inc., no. AB745) in a standard Western blotting procedure. The positive control was a sample of 500 ng of human collagen type I (Chemicon Inc., no. CC050, extracted from human placenta by pepsin digestion) added to 50 μl of total soluble protein extracted from wt tobacco.

[0340] As shown in Figure 6A, collagen triple helices formed in plants 3–5 and control human collagen were resistant to denaturation at 33°C. In contrast, collagen formed by plants 2–9 denatured at 33°C. This difference in thermal stability indicates the success of triple helix organization and post-translational proline hydroxylation in transformants 3–5, which express both the β and α subunits of collagen α1 and collagen α2 and P4H.

[0341] The two bands in transformants 2-9 may represent stable dimers or trimers after boiling in SDS and mercaptoethanol for 7 minutes. Similar bands are observed in human collagen (upper panel) and transformants 3-5. A possible explanation is a covalent bond between two peptides within different triple helices (crosslinks) formed after oxidative deamination of two lysines by lysine oxidase.

[0342] In the second experiment, total soluble protein from transgenic tobacco 13-6 (expressing collagen type I α1 and α2 chains (indicated by arrows), human P4Hα and β subunits, and human LH3) was extracted by grinding 500 mg of leaves in 0.5 ml of 100 mM Tris-HCl (pH=7.5) and 300 mM NaCl, centrifuging at 10000 rpm for 10 minutes, and collecting the supernatant. 50 μl of the supernatant was heat-treated (33°C, 38°C, or 42°C for 20 minutes) and immediately placed on ice. Pepsin digestion was initiated in each sample by adding 4.5 μl of 0.1 M HCl and 4 μl of 2.5 mg / ml pepsin in 10 mM acetic acid. The samples were incubated at room temperature (approximately 22°C) for 30 minutes. Digestion was terminated by adding 5 μl of unbuffered 1 M Tris. Each sample was mixed with 22 μl of 4X sample application buffer containing 10% β-mercaptoethanol and 8% SDS, boiled for 7 minutes, and centrifuged at 13000 rpm for 7 minutes. 40 μl of the supernatant was loaded onto a 10% polyacrylamide gel and tested using a standard Western blotting procedure with anti-collagen type I antibody (Chemicon Inc., no. AB745). The positive control was a sample of approximately 50 ng of human collagen type I (Chemicon Inc., no. CC050, extracted from human placenta by pepsin digestion) added to total soluble protein from wt tobacco.

[0343] As shown in Figure 6B, the collagen triple helix formed in plant number 13-6 was resistant to denaturation at 42°C. Propeptide cleavage was first observed at 33°C, and the efficiency gradually improved as the temperature rose to 38°C and then again to 42°C. The cleaved collagen triple helix domains exhibited similar migration on the gel to that of pepsin-treated human collagen. The human collagen used in this experiment lacked propeptides and some telopeptides because it was extracted from human placenta by pepsin proteolysis.

[0344] Example 5. Plant P4H expression.

[0345] Induction of natural plant P4H

[0346] Tobacco P4H cDNA was cloned and used as a probe to determine the conditions and treatments that induce endogenous P4H expression. Northern blot analysis (Figure 7) clearly shows that P4H is expressed at relatively high levels in the shoot apex and at low levels in the leaves. P4H levels were significantly induced in the leaves 4 hours after abrasion treatment ("injured" in the lower panel). Similar results were obtained using other stress conditions (not shown in the figure).

[0347] Detection of human P4Hα and β subunits and collagen α1 and α2 chains in transgenic tobacco plants.

[0348] Detection of human P4Hα and β subunits and type I collagen α1 and α2 chains in transgenic tobacco plants was performed using anti-human P4Hα subunit antibodies (ICN Biomedicals Inc., no. 63-163), anti-human P4Hβ subunit antibodies (Chemicon Inc., no. NMAB2701), and anti-collagen type I antibodies (Chemicon Inc., no. AB745). The results of Western blots probed with these antibodies are shown in Figure 8.

[0349] Expression of P4Hα, P4Hβ, and collagen type 1 α1 and α2 bands was confirmed in plant 13-6 (also transformed with human LH3). The calculated molecular weights of P4Hα and β, including vacuolar signal peptides, are 65.5 kDa and 53.4 kDa, respectively. The calculated molecular weights of collagen α1 and α2 chains, including propeptides, which are neither hydroxylated nor glycosylated, are 136 kDa and 127 kDa, respectively.

[0350] Example 6. Vacuole-targeting collagen is stably expressed in plants grown in the dark.

[0351] Plants that express collagen:

[0352] The 20-279 parent tobacco plant lines were generated by cotransformation using an expression vector expressing P4Hβ+LH3 and another expression vector expressing P4Hα. Prior to each gene are vacuolar target determinants of allurein, a plant vacuolar thiol protease.

[0353] 2-300 parent tobacco plant lines were generated by co-transformation using an expression vector expressing col1 and another expression vector expressing col2. Prior to each gene are vacuolar target determinants of allurein, a plant vacuolar thiol protease.

[0354] Plants 13-652 were generated by co-transformation of tobacco plants using expression vectors encoding Col1, P4Hβ, and LH3 and a second expression vector encoding Col2 and P4Hα. Prior to each gene are vacuolar target determinants of allurein, a plant vacuolar thiol protease, and the cassette sequences contained in the vectors are described in Example 1 above.

[0355] Light and dark trial

[0356] Analysis of six 13-6 / 52 homozygous plants. Samples were taken simultaneously (12:30) daily over eight days from leaf numbers 4+5 / 6, from three plants grown under normal conditions (16 hours in light, 8 hours in darkness) and three plants grown only in darkness.

[0357] Total protein extraction and Western blot analysis.

[0358] 90 mg of tobacco leaves were homogenized at 4°C in an extraction buffer (100 mM Tris HCl (pH=7.5), a protease inhibitor cocktail available from Roche catalog no. 04-693-116-001) using a Retsch MM301 mixer mill. After centrifugation for 30 minutes (20,000Xg at 4°C), the supernatant was collected. The protein sample was fractionated by 8% SDS-PAGE (Laemmli 1970) and transferred to a nitrocellulose membrane using a BIO-RAD® Protein TRANS-BLOT® instrument. The membrane was blocked in 3% (g / v) skim milk (Difco) at room temperature for 30 minutes, and then reacted overnight at room temperature with any commercially available rabbit anti-human collagen type I polyclonal antibody (Chemicon). The membrane was rinsed 3-5 times with water and then washed with TBS for 30 minutes. After incubation with a secondary antibody [goat anti-rabbit IgG antibody conjugated to alkaline phosphatase (AP) (Chemicon)] at room temperature for 2 hours, the membrane was rinsed 3-5 times with water and then washed with TBS for 30 minutes. Immunodetection was performed at room temperature for 2 hours to overnight using nitrotetrazolium blue chloride (NBT, Sigma) and 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt (BCIP, Sigma).

[0359] result

[0360] As shown in Figure 9, tobacco plants transgenic for vacuolar target collagen express pro-α-1 and pro-α-2 (lane 1). Collagen from vacuolar target plants grown under dark conditions exhibits similar stability (lane 2), demonstrating the exceptional stability of collagen produced according to the teachings of this invention.

[0361] Examples 7-13. General materials and methods

[0362] Collagen Extraction and Enzymatic Reaction: In a blender, 300 g of tobacco leaves were mixed in a chilled extraction buffer (containing 360 mg of potassium metabisulfite, 530 mg of L-cysteine, and 1 g of EDTA, in 600 ml of 100 mM Tris-HCl, pH 7.5) with 5 g of PVPP and 2 g of activated carbon added (see U.S. Patent No. 8,759,487). Mixing was performed five times at 1-minute intervals, maintaining the temperature below 15°C. The crude extract was filtered through a gauze pad and centrifuged at 25000 g at 5°C for 30 minutes. The supernatant was collected. CaCl2 was added until the final concentration reached 10 mM. The supernatant was divided into 10 ml samples. The desired enzyme was added to each 10 ml sample according to the conditions described in Table 3 below.

[0363] [Table 3]

[0364] Enzyme description: Ficin from fig 'Cratex' (Sigma, catalog number F4125), subtilisin from Bacillus licheniformis (Sigma, catalog number P5459-5gr), bromelain from pineapple stem (Sigma, catalog number B4882-10gr), papain from Carica papaya (Fluka, catalog number 76220-25gr), Savinase 6.0 t type W from the alkaliphilic bacterium Bacillus lentus (Novozymes, catalog number PX92500501), Neutrase 1.5 MG from the bacterium Bacillus amyloliquefaciens (Novozymes, catalog number PW201041), Protamex, commercially available Bacillus proteinase complex (Novozymes, catalog number PW2A1021), Alcalase 3.0 T, Bacillus Subtilis alkaline proteinase (Novozymes, catalog number PJ90000901), Esperase 6.0 T, alkaliphilic bacterium Bacillus lentus (Novozymes, catalog number PE90110401), Alcalase 2.4 L FG, Bacillus subtilis alkaline proteinase (Novozymes, catalog number PLN05330), Esperase 8.0 L, alkaliphilic bacterium Bacillus lentus (Novozymes, catalog number PE00077) were all donated by Novozymes. Pancreatic trypsin 6.0 S unsalted type (Novozymes, catalog number P245-D20), a trypsin derived from animal pancreas, was also used. TRYPZEAN®, a recombinant trypsin expressed in maize, was purchased from Sigma Chemical Co. (catalog number: T3449).

[0365] Determination of atelocollagen concentration: The concentration of atelocollagen produced according to Examples 9-10 was assayed using the following two methods.

[0366] SIRCOL® Assay: The SIRCOL® Collagen Assay Kit was purchased from Biocolor Ltd. (catalog number 85000). This assay is based on the interaction between Sirius Red dye and the collagen triple helix. The analysis was performed according to the supplier's instructions for use (4th edition, 2002). A calibration curve was created using bovine collagen standards (0–50 μg collagen). Three samples of 10–50 μl of collagen in 10 mM HCl solution were placed in 1.5 ml Eppendorf tubes, and the volume was diluted to 100 μl with 0.5 M acetic acid. 1 ml of SIRCOL® dye reagent was added to each tube, and the tubes were shaken at room temperature for 30 minutes. The tubes were centrifuged at 12,000 rpm for 10 minutes at room temperature, the supernatant was aspirated, and the tubes were inverted onto absorbent paper to remove the remaining supernatant. Access drops were removed from the tube walls using a cotton swab. 1 ml of alkaline reagent was added to each tube, mixed thoroughly, and incubated at room temperature for 10 minutes. Absorbance at 540 nm was measured using a spectrophotometer, and the collagen concentration was calculated relative to the calibration curve using 10 mM HCl as a blank sample.

[0367] SDS-PAGE Instant Blue Assay: Samples were boiled in SAB buffer (reducing conditions) for 5 minutes, centrifuged at 12,000 rpm for 5 minutes, and then loaded onto SDS-PAGE, 8% acrylamide. The gels were run on Mini Protean 3 units (BioRad numbers 165-3301, 165-3302). Instant Blue reagent (Novexin number ISB01L) was applied to the gel until the protein was visualized as a blue band on the gel. The gel was rinsed with water and dried. The concentration of the collagen band was calculated by concentration assay against a human standard loaded on the same gel.

[0368] Coomassie Analysis: A collagen sample (in 10 mM HCl) was titrated with 1 M Tris until pH 7.5. A sample application buffer containing 10% β-mercaptoethanol and 8% SDS was added to 30 μl of the pH-titrated sample, diluted fourfold. The sample was boiled for 7 minutes. 30 μl of the supernatant was loaded onto a 10% polyacrylamide gel and separated over 100 volts for 2 hours. The gel was transferred to a Coomassie solution with shaking over 1 hour. The Coomassie dye was removed using a standard decolorizing solution.

[0369] SDS-PAGE and Western blot analysis of α-1 and α-2 collagen chains: Samples were boiled for 7 minutes in reducing sample application buffer (2.5% β-mercaptoethanol and 2% SDS), and then centrifuged at 13,000 rpm for 15 minutes. 30 μl of supernatant was separated on a 10% polyacrylamide gel. After separation, the samples were blotted onto nitrocellulose membranes using a standard Western blot protocol. After transfer, the membranes were incubated with anti-collagen type I antibody (Chemicon Inc. catalog #AB745) for immunodetection of α-1 and α-2 collagen chains. Molecular weight markers were purchased from Fermentas Inc. (catalog number SM0671).

[0370] Control: A positive control of human cutaneous collagen type I, purchased from Calbiochem (number 234138), was used as a marker for Western blot analysis. The pulverized control sample reflects a pellet derived from tobacco leaves immediately before resuspending in extraction buffer. The "D" control sample reflects the same pellet after resuspending in extraction buffer. The "K" control sample contains ficin-digested procollagen in 10 mM HCl. To monitor background ficin-independent protease activity, ficin-free cleavage samples were always prepared in parallel with all ficin digestion tests.

[0371] Purification of collagen from transgenic plants: Digestion of propeptides in collagen-containing extracts was initiated by the addition of 30 mg / L trypsin, 5 mg / L (50 μl / L) subtilisin (Sigma, catalog no. P5459), or 5 mg / L ficin (Sigma, catalog no. F4125). Proteolysis was carried out at 15°C for 4 hours. Removal of insoluble contaminants was performed by centrifugation at 22,000 g at 15°C for 30 minutes. The supernatant was collected, and collagen was precipitated by slowly adding crystalline NaCl with constant stirring at room temperature for 20 minutes until a final concentration of 3.13 M was reached. This solution was incubated overnight in a refrigerator without stirring. Collagen collection was performed by centrifugation at 25,000 g at 5°C for 2 hours.

[0372] The supernatant was carefully poured onto a 4-layer gauze pad. Using a magnetic stirrer, the pellet was resuspended in 200 ml of 250 mM acetic acid and 2 M NaCl over 5 minutes. The suspension was centrifuged at 25,000 g at 5°C for 40 minutes. A small amount of supernatant was removed from the glass vial. The pellet was redissolved in 200 ml of 0.5 M acetic acid at room temperature over 1 hour. Insoluble material was removed by centrifugation at 16,000 g for 30 minutes at 15°C. The supernatant was poured onto a 12-layer gauze pad. Collagen was precipitated by slowly adding NaCl to a final concentration of 3 M while constantly stirring at room temperature for 20 minutes. This solution was incubated at 4°C for 8 hours to overnight. Collagen was collected by centrifugation at 25,000 g at 5°C for 2 hours. After aspirating the supernatant, the pellet was redissolved in 200 ml of 0.5 M acetic acid using a magnetic stirrer at room temperature for 1 hour. Insoluble material was removed by centrifugation at 16,000 g for 30 minutes at 15°C. The supernatant was poured onto a 12-layer gauze pad. Collagen was precipitated by slowly adding NaCl to a final concentration of 3 M while continuously stirring at room temperature for 20 minutes. This solution was incubated at 4°C for 8 hours. Collagen was collected by centrifugation at 2,000 g for 2 hours at 5°C. The supernatant was aspirated. The pellet was redissolved in 40 ml of 10 mM HCl by pipetting and vortexing at room temperature for 5 minutes. This solution was transferred to a dialysis bag (MWCO 14,000 Da) and dialyzed against 4 L of 10 mM HCl at 4°C for 4 hours. This dialysis was repeated overnight.

[0373] Sterilization of the collagen was performed by first filtering the solution through a 0.45 μm filter, and then filtering it through a 0.2 μm filter using a 30 ml syringe. The collagen was further concentrated by ultrafiltration using a Vivaspin PES 20 ml filter tube (Vivascience, No. VS2041, MWCO 100000). Centrifugation was performed at 5000 g, 5°C for 45 minutes until the volume was reduced to 0.75 ml.

[0374] Optimization of digestive kinetics and conditions for procollagen cleavage with food-grade ficin: The pellet (collected as described in Example 10 and saturated with 25% ammonium sulfate (AMS)) was resuspended in buffer (Buffer A: 4.5 mM potassium metabisulfite, 12.5 mM L-cysteine, and 7.5 mM EDTA dissolved in 0.1 M sodium phosphate buffer and titrated with 10 M NaOH or 6N HCl to pH 7.5) at a ratio of 4.36 g pellet to 200 mL of ice-cooled buffer. The sample was then stirred at 15°C for 20 minutes. Aliquots of 10 mL were then prepared per 15 mL test tube, followed by administration of increasing ficin concentration (5–15 mg / L) (Fig Cratex, Biochem Europe food-grade ficin). The samples were incubated at 15°C for 1–3 hours, separated by SDS-PAGE, and then analyzed by Western blotting for the presence of collagen migrating at a molecular weight lower than procollagen.

[0375] Tobacco leaf pellets were resuspended in phosphate buffer A (27.2 g: 800 mL buffer) at various pH values ​​(5.5, 7.5, or 8.5) and treated with 10 mg / L ficin in the presence of 0-3 M NaCl at 15°C. The reaction was stopped by centrifugation of 1 mL of sample from each reaction mixture (10 minutes, 15000 g, 4°C). The pellets were resuspended in 1 mL of buffer A (pH 7.5), separated by SDS-PAGE, and analyzed by Western blotting.

[0376] Optimization of digestive kinetics and conditions for procollagen cleavage with pharmaceutical-grade ficin: Tobacco leaf pellets were resuspended for 5–45 minutes in extraction buffer (10 mg / L) containing pharmaceutical-grade (Biochem-Europe Pharma grade) ficin at various pH values ​​(7.5, 8.5, 9.5) while increasing the NaCl concentration (0–3 M). Further experiments investigated the necessity, optimal conditions, and concentrations of EDTA and L-cysteine ​​as additives to the extraction buffer. Samples were incubated in the digestion mixture in the presence of 0–100 mM EDTA and 0–80 mM L-cysteine ​​at 15°C, pH 7.5, without NaCl, for 1–3 hours.

[0377] Fibrillar formation: Fibrillar formation is considered a functional test of collagen. Therefore, the ability of purified collagen digested by ficin to form fibrils is an essential characteristic of the obtained product. Test method: The pH of collagen-containing solutions (multiple samples) was neutralized to pH 6.7 using sodium phosphate (pH 11.2), and then incubated at 27 ± 2 μC for 6 hours. The samples were centrifuged to allow the formed hydrogels to settle. Protein concentrations in both pre-neutralization and post-neutralization (supernatant) samples were determined by the Lowry method. PURECOL (trademark) (purchased from NUTACON, catalog number 5409) was used as a positive control, and gelatin was used as a negative control.

[0378] Example 7. Extraction and purification of collagen from transgenic plants in the presence of trypsin and pepsin.

[0379] The use of mammalian proteins for human cosmetic or medical purposes may pose risks to human health due to the relative relative nature of their evolutionary proximity. Therefore, the production of human collagen in plants has been initiated to avoid the use of collagen from mammalian sources. Creutzfeldt-Jakob disease (CJD), a known disease, is one example of a disease caused by the consumption of infected mammalian proteins by humans.

[0380] Initially, the purification of collagen from transgenic plants was performed using bovine pancreatic trypsin and the digestive protease pepsin, both of which catalyze the hydrolysis of proteins in the animal digestive system. The following examples illustrate the identification of non-animal source proteases suitable for use in the collagen purification process.

[0381] result

[0382] Propeptide digestion during collagen purification was initially performed by the pancreatic enzyme trypsin. Trypsin digested collagen propeptides at 300 mg / L, but the collagen yield was very low at the end of the purification process (Figure 10). Lowering the trypsin concentration to 20 mg / L or 30 mg / L increased the yield, but procollagen digestion occurred only partially and was inconsistent across the same sample (Figure 11).

[0383] In attempts to overcome this problem, various incubation temperatures and times were tried. However, the results did not lead to a change in yield (data not shown). Adding pepsin enzyme after the purification process resolved the partial digestion problem (Figure 12), yielding α-1 and α-2 collagen that could be transported together with the porcine collagen control sample.

[0384] Example 8. Collagen extraction and its enzymatically induced digestion.

[0385] However, the trypsin-pepsin solution was not optimal because it required two different enzymes and the purification process was lengthy. Furthermore, both enzymes are of animal origin. To overcome these challenges, we screened for different non-animal protease enzymes. The various enzymes screened yielded different digestion patterns. Digestion of polypeptides obtained by incubation of collagen with sabinase (Figure 15) and esperase (Figure 17) enzymes was little to no. Incubation with papain (Figure 14), bromelain (Figure 13), 2.4 L of alcalase, and 8.0 L of esperase (Figure 18) resulted in either over- or under-digestion of the propeptide. Alcalase and protamex enzymes (Figure 16) produced the desired digestion pattern and level (25 mg / L, 6 hours), with α1 and α2 chains migrating similarly to porcine collagen samples. However, not all molecules were completely digested, and longer incubation times may be required. Optimal results were obtained by incubating procollagen with ficin (5 mg / L and 25 mg / L) (Figure 15), where the α1 and α2 chain bands migrated with the porcine collagen control sample, and there was no apparent overdigestion. Similar results were shown with subtilisin 5 mg / L for 3 hours (Figure 13) and neutrase 25 mg / L for 6 hours (Figure 17).

[0386] Example 9. Extraction and purification of collagen from post-digestion transgenic plants using subtilisin or ficin.

[0387] Collagen purification was performed from 450 grams of leaves of transgenic plants (13-361 or 13-6-52) after procollagen digestion using ficin (Figure 19) or subtilisin (Figure 20). Collagen samples at various stages of the purification process were analyzed by Western blotting. Propeptide digestion with ficin and subtilisin yielded the desired degree of processing for collagen 1 and collagen 2. Low molecular weight bands were observed in Western blotting throughout the purification process, but these bands appeared in plant extracts before enzymatic incubation (lanes 3-4) and also in a porcine-derived collagen control sample (positive control) (Figure 19).

[0388] Example 10. Extraction and purification of scaled-up collagen from post-digestion transgenic plants using ficin.

[0389] 1 kg of transgenic tobacco leaves were ground for 20 minutes in a 4 L reactor (ESCO Model EL-3) using 2 L of pre-cooled extraction buffer (100 mM sodium phosphate buffer pH 7.5, 4.5 mM potassium metabisulfite, 12.23 mM L-cysteine, and 7.5 mM EDTA) (5°C, 50% scraper speed, and 100% homogenizer blade rpm). 6.68 g of charcoal and 16.67 g of PVPP were added to the extract and stirred continuously for 20 minutes (5°C and 50% scraper speed). The extract was centrifuged (11000 rpm, 5°C, 0.5 H), and the supernatant was saturated with 15% ammonium sulfate (stirred for 1 hour, 5°C). After 30 minutes at 6880 rpm, 5°C, the supernatant was saturated to 25% ammonium sulfate and stirred for 1 hour (5°C). After recentrifugation, the pellet (6880 rpm, 5°C, 30 min) was resuspended in 15% of the volume collected after the first centrifugation step (in extraction buffer). Propeptide removal was achieved by digestion with 5 mg / L ficin (Biochem Europe) at 15°C for 3 hours. The sample was centrifuged (11,000 rpm, 15°C, 30 min), and mature collagen was precipitated using 3M NaCl (NaCl was slowly added with agitation and left overnight at 4°C). After precipitation (13,000 rpm, 5°C, 2 hours), the supernatant was discarded, and the pellet was resuspended in 0.5M acetic acid. After further 3M salting-out (overnight) and centrifugation, the pellet was resuspended in 40 ml of 10 mM HCl. The sample was transferred to a dialysis bag (12-14 kDa) and dialyzed in 4 L of 10 mM HCl at 4°C for 4 hours. Dialysis was repeated overnight using 4 L of fresh 10 mM HCl. The dialyzed solution was filtered through a 0.45 micron filter (pre-washed with 10 mM HCl), and then filtered through a 0.25 micron filter. Finally, the sample was concentrated in a Vivaspin (Vivascience) filter tube (100 kDa).

[0390] Example 11. Solubility of atelocollagen produced as recombinant human procollagen in transgenic tobacco plants.

[0391] The concentrations of atelocollagen produced according to Examples 9-10 were assayed by the following two methods, as described in the Methods section. The concentration results obtained for several typical preparations digested with ficin are listed in Table 4 below.

[0392] [Table 4]

[0393] Example 12. Ficin-dependent proteolysis of tobacco leaf-derived procollagen.

[0394] Digestive kinetics of procollagen by food-grade ficin: To calibrate the appropriate ficin concentration and incubation time to enable the best collagen yield, procollagen-expressing tobacco leaf pellets were incubated at 15°C for 1–3 hours with increasing concentrations of food-grade ficin (5–15 mg / L). Samples were then analyzed by immunodetection of α-1 and α-2 collagen chains by Western blotting. Increasing the ficin concentration improved collagen chain yield after 1 hour of incubation (Figure 22, lanes 5 vs 6). However, extending the reaction time resulted in overdigestion of collagen with increasing ficin concentration (Figure 22, lanes 11 vs 12–14 and lanes 17 vs 18–20). Therefore, the optimal conditions for procollagen-to-collagen digestion were determined to be the addition of 10 mg / L of food-grade ficin at 15°C for 1 hour.

[0395] Digestive kinetics of procollagen by pharmaceutical-grade ficin: Similar experiments were performed on tobacco leaf pellets expressing procollagen to determine optimal conditions for procollagen digestion by pharmaceutical-grade ficin. The pellets were resuspended and incubated at 15°C for 0.5–3 hours while increasing the concentration of pharmaceutical-grade ficin (2.5–10 mg / L). Digestion efficiency was determined by immunodetection of collagen chains by Western blotting. As shown in Figures 23A–23C, increasing the ficin concentration increased collagen yield and decreased procollagen levels. The most effective digestion of procollagen by pharmaceutical-grade ficin was observed at 10 mg / L after 1 hour of reaction.

[0396] Optimization of pH and salt concentration for ficin-dependent procollagen cleavage: Next, the contributions of both pH and salt concentration of the digestion buffer were evaluated. Similar tobacco leaf pellets after AMS were resuspended in extraction buffer titrated to pH 5.5, 7.5, 8.5, or 9.5 with salt content ranging from 0.5 to 3 M NaCl. The samples were then incubated with 10 mg / L of pharmaceutical-grade ficin at 15°C for 1 hour, followed by immunoassay by Western blotting. Acidic assay conditions (pH 5.5) resulted in insufficient collagen yield (Figure 24A, lanes 2-6), and increasing pH value showed a correlated increase in ficin-dependent collagen content, with the peak value observed at pH 8.5 in the presence of 2 M NaCl (Figure 24B, lane 10). These results were further supported by scaled-up extraction and purification experiments performed on two 15 kg pellets pooled for ficin-induced procollagen digestion. Aside from the increased collagen chain yield observed by immunoblotting, samples digested in buffer at pH 8.5 in the presence of 2M NaCl were as efficiently fibrillated as those digested in buffer A (pH 7.5, 0mM NaCl) (see Table 5 below, batches YC1 and YC2). Therefore, higher pH and salt concentration improved collagen yield after ficin-induced procollagen digestion.

[0397] Measurement of the vitality of EDTA and L-cysteine ​​in digestion reaction mixtures: Both EDTA and L-cysteine ​​are additives present in the extraction buffer during the initial stages of the collagen purification process. Herein, it was determined that these two components are essential for effective ficin-dependent collagen cleavage. Procollagen pellets after AMS were resuspended in extraction buffer containing increasing concentrations of EDTA (8–80 mM) and L-cysteine ​​(10–100 mM), and incubated with ficin (10 mg / L) at 15°C for 1 hour at pH 7.5. A significant improvement in digestion efficiency was observed in the presence of 10 mM L-cysteine ​​(Figure 25, lanes 7–10), and there was no clear contribution of EDTA to ficin-dependent collagen output (Figure 25, lanes 7 vs 8–10).

[0398] Optimization of temperature conditions for ficin-induced procollagen digestion: Tobacco leaf pellets expressing procollagen were incubated with ficin at 15°C for 1.5 hours, and then transferred to a 30°C bath for a further 1.5 hours. Western blotting and fibrillation assays did not show any improvement in collagen yield or sample purity associated with increased reaction temperature.

[0399] Fibrillation of collagen extracted from ficin-induced procollagen cleavage: After ficin-induced digestion, a fibrillation assay was performed to determine the ultimate method for determining the ability of the obtained collagen to form fibrils and the function of the collagen. Table 5 below summarizes the results of fibrillation determined after ficin cleavage of procollagen using two variable protocols. Protocols A and B differed in reaction buffer pH and salt content, and yielded a considerable proportion of collagen fibrils. Therefore, the proteolysis reaction parameters developed and optimized herein produce functional collagen in high yield.

[0400] [Table 5]

[0401] Example 13. Determination of the effectiveness of TRYPZEAN® protease in procollagen cleavage.

[0402] Tobacco leaf pellets expressing procollagen, resuspended in EDTA (7.5 mM) and L-cysteine ​​(12.5 mM) rich extraction buffer (pH 7.5), were incubated with TRYPZEAN® (30–100 mg / L) at 15°C for 1–3 hours. Within 1 hour, doses of 60 and 100 mg / L of TRYPZEAN® efficiently cleaved the procollagen, generating two distinct α-collagen chains, with no detectable overdigestion (Figure 26). Therefore, procollagen treatment with TRYPZEAN® at pH 7.5 induces effective digestion into collagen chains α-1 and α-2.

[0403] Consideration

[0404] Examples 7–13 above describe the identification of non-mammalian proteases suitable for use in the purification process of plant-derived collagen. Examination of proteases from bacterial and plant sources revealed three enzymes suitable for digesting collagen propeptides: neutrase, subtilisin, TRYPZEAN®, and ficin.

[0405] Neutralase and subtilisin are both secreted by the bacterium Bacillus sp. Subtilisin is primarily used (more than 90%) in detergents and household cleaning products. Approximately 10% of subtilisin's use is for industrial applications such as protein hydrolysis, leather processing, and the textile and cosmetics industries. Standard use of subtilisin in collagen purification processes at high concentrations presents problems due to over-digestion of collagen. Neutralase is primarily used in the beverage and alcohol industry and in cheese aging. In Examples 7–13, as described above herein, neutrase was effective only when digesting propeptides at high concentrations, and at least 6 hours were required for the desired digestion results.

[0406] Under the experimental conditions described here, recombinant trypsin and ficin were found to be the most suitable of the four, as they did not exhibit excessive collagen digestion after high enzyme concentrations or prolonged incubation. Furthermore, these enzymes clearly did not digest the helical regions of collagen, as determined by SDS-PAGE analysis. Ficin, a natural enzyme extracted from the fig plant (Ficus carica), is commercially available at low cost in several grades, including pharmaceutical grade, from several suppliers. It is used in the food industry, including the alcohol and beer industry, protein hydrolysis, meat processing, the baking industry, and the preparation of pet and health foods. It is also applied in the pharmaceutical industry, as well as in the cosmetics and textile industries, for contact lens cleaning agents, cancer treatments, anti-arthritis treatments, and digestive aids.

[0407] Example 14. Further analysis of the properties of rh collagen.

[0408] material and method

[0409] material

[0410] Human recombinant collagen (rh collagen) type I, expressed and isolated from transgenic tobacco plants, was manufactured and supplied by CollPlant Ltd (Israel). Type I bovine collagen (PureCol) was purchased from Advanced Biomatrix, USA. Methacrylic anhydride, glycidyl methacrylate, triethylamine, tetrabutylammonium bromide, 2,4,6-trinitrobenzenesulfonic acid (TNBS), 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (Irgacure 2959), sodium dihydrogen phosphate anhydride, HCl 1N, HCl >37%, sodium bicarbonate, and NaOH were purchased from Sigma Aldrich Ltd (Israel). Phosphate-buffered saline (PBS), x10 PBS, fetal bovine serum (FBS), DMEM high glucose, and penicillin / streptomycin were purchased from Biological Industries Ltd (Israel). Disodium hydrogen phosphate anhydride was purchased from Canton, India. Anhydrous ethanol and acetone were purchased from Bio-Lab Ltd (Israel). Hyaluronic acid was purchased from Lifecore, USA.

[0411] Preparation of buffer and photoinitiator stock solutions

[0412] Fibrillation buffer (FB): Dissolve disodium hydrogen phosphate in redistilled water (DDW) to a final concentration of 162 mM. The solution was titrated with 10N NaOH until the pH reached 11.2.

[0413] Culture medium preparation: 50 ml of fetal bovine serum and 5 ml of penicillin / streptomycin (10,000 units / mL and 10 mg / mL, respectively) were added to 500 ml of DMEM high-glucose medium under sterile conditions. The medium was gently mixed and stored in a refrigerator.

[0414] Preparation of phosphate-buffered saline: 39 ml of 0.1 M sodium dihydrogen phosphate solution was mixed with 61 ml of 0.1 M disodium hydrogen phosphate solution, and the final volume was adjusted to 200 ml using DDW. If necessary, the final pH was adjusted to 7 using concentrated NaOH or HCl. NaCl was added to bring the final concentration to 150 mM.

[0415] Washing buffer: HCl was added to the fibrillation buffer to achieve a final concentration of 16.2 mM disodium hydrogen phosphate and 10 mM HCl. The pH was adjusted to 7.2-7.4 using 10N NaOH.

[0416] A 10% (v / v) stock solution of photoinitiator, Irgacure 2959, was dissolved in an anhydrous ethanol / PBS 1:1 solution to a final concentration of 100 mg / mL.

[0417] Methacrylate of rh collagen.

[0418] Fibrous rh-collagen-methacrylamide and monomeric rh-collagen-methacrylamide were prepared by the reaction of lysine and hydroxylysine collagen residues with methacrylic anhydride in an aqueous medium as described below, and were then stored protected from light at 4°C until use.

[0419] Fibrous rh collagen-methacrylamide

[0420] Fibrous rh-collagen-methacrylamide at concentrations of 3–10 mg / mL was synthesized at room temperature (RT) or 12°C in either a washing buffer, fibril-forming buffer, or DDW. For example, in short, fibrous rh-collagen-MA was synthesized in DDW as follows: A 3–4 mg / mL solution of monomeric rh-collagen (COLLAGE®) in 10 Mm HCl was mixed with fibril-forming buffer in a 9:1 (v:v) ratio and stirred at room temperature for 1 hour to obtain fibrils. The solution was centrifuged at 7500 rpm at 4°C for 30 minutes, and the supernatant was discarded. The pellet was resuspended in an equal volume of washing buffer and centrifuged under the same conditions. The deposited fibrils were then resuspended in DDW to a concentration of 10 mg / mL. The concentration was confirmed by measuring the percentage of solids. Methacrylic anhydride (MA) was added dropwise to collagen lysine at a ratio of 10-20 moles under nitrogen flow at room temperature. The pH of the reaction solution was monitored over time and adjusted to pH 7 using 10N NaOH. After 24 hours of reaction, the mixture was dialyzed at 4°C for 3 days against a wash buffer (pH 7) using a 10kDa cutoff dialysis tube (Spectrum Laboratories Inc, CA, US). The dialysate (wash buffer in this case) was changed at least 6 times to remove reaction byproducts, and the mixture was finally freeze-dried for 3-4 days.

[0421] Monomer rh collagen-methacrylamide

[0422] 200 mM MOPS, phosphate, or Tris buffer with 150 mM NaCl added was used. For example, 200 mM MOPS and 150 mM NaCl were added to 3–4 mg / mL of COLLAGE® and stirred at room temperature until a clear solution was obtained. Then, 10–20 times excess methacrylic anhydride was added dropwise at 12°C under a nitrogen stream, and the pH was adjusted over time to pH 7 using 10N NaOH. After 24 hours of reaction, the mixture was dialyzed at 4°C for 3 days against 10 mM HCl and 20 mM NaCl (pH 2) using a 10 kDa cutoff dialysis tube, with the dialysate changed at least 6 times, and then lyophilized for 3–4 days.

[0423] Methacrylate of hyaluronic acid (HA)

[0424] 500 mg of HA was functionalized as described by Leach et al. [Leach et al., 2002, Biotechnology and Bioengineering, vol. 82, no. 5]. Briefly, 1.8 ml of triethylamine, 1.8 ml of glycidyl methacrylate, and 1.8 g of tetrabutylammonium bromide were separately added to 50 ml of a 10 mg / mL HA solution in DDW and thoroughly mixed before the next components were added. The reaction mixture was mixed overnight at room temperature, and HAMA was precipitated in 20 times its volume of acetone and redissolved in DDW. This precipitation process was repeated twice to remove all reaction residue. The material was finally lyophilized.

[0425] Preparation of solutions for viscosity measurement

[0426] PureCol and Collage® in PBS: An 8 ml monomer collagen solution (3 mg / mL in 10 mM HCl) of either rh collagen (COLLAGE®) or bovine collagen (PureColl) was neutralized by adding 1 ml of PBSX10. The solution was then adjusted to pH 7–7.5 by titration with 0.1 N NaOH. Finally, double distilled water was added to bring the final volume to 10 ml. The samples were incubated at 37°C for at least 90 minutes before measurement (either 37°C or 4°C).

[0427] COLLAGE® in fibrillation buffer: 9 ml of monomer rh-collagen (COLLAGE®) solution (3.79 mg / mL in 10 mM HCl) was neutralized by adding 1 ml of fibrillation buffer. Samples were incubated at 37°C for at least 90 minutes before measurement (either 37°C or 4°C).

[0428] Fibrous rh-collagen-methacrylamide in PBS: Lipo-dried fibrous rh-collagen-MA prepared in DDW was dialyzed, and the resulting solution and washing buffer (using a 10-fold excess of MA as described above) were dissolved in PBS to a concentration of 10 mg / mL. Before measurement (either 37°C or 4°C), the samples were incubated at 37°C for at least 90 minutes.

[0429] Rh collagen-methacrylamide in DMEM: Lyophilized fibrous rh collagen-methacrylamide (15-fold excess MA, prepared in wash buffer as described above and dialyzed) was dissolved in DMEM medium to final concentrations of 20 and 26 mg / mL.

[0430] Rh collagen-methacrylamide / hyaluronic acid in DMEM was added to a solution of fibrous rh collagen-MA to obtain final concentrations of 10 mg / mL HA and 20 mg / mL rh collagen-MA in DMEM medium.

[0431] Rh collagen-methacrylamide / hyaluronic acid methacrylate (HA-MA) in DMEM: Hyaluronic acid methacrylate (see above) was added to a solution of fibrous rh collagen-MA to obtain final concentrations of 10 mg / mL and 20 mg / mL of rh collagen-MA in DMEM medium.

[0432] Photocrosslinking of rh-collagen-MA for measurement of loss modulus and storage modulus

[0433] The rh-collagen-MA crosslinked scaffold was formed in two different preparations for investigation in two separate experiments. In the first preparation, 1–2 wt% fibrous rh-collagen-MA synthesized with a 10-fold excess of methacrylic acid reagent was dissolved in 0.1 M PBS at room temperature, then 0.1% Irgacure 2959 was added, and a final volume of 1 mL of the solution was poured into a disc-shaped mold. Subsequently, a curing process was performed using a mercury light source at an average intensity of 670 mW / cm2 for 7 seconds and 10 seconds from a distance of 1.5 cm to finally obtain a crosslinked scaffold. The second preparation contained two different batches of fibrous rh-collagen-MA synthesized with 15-fold and 20-fold excess of methacrylic acid reagent. 1–2 wt% was dissolved in 0.1 M PBS, 0.1% Irgacure 2959 was added, and a final volume of 1.5 mL was obtained. To obtain a highly bridged scaffold, the hardening process was carried out from a distance of 2 cm for 60 seconds, with an average strength of 420 mW / cm². 2 It was implemented there.

[0434] TNBS assay

[0435] The assay protocol was similar to that reported by Sashidhar et al. [Sashidhar RB, Capoor, AK, Ramana, D, Journal of Immunological Methods. 1994, 167, 121-127] and based on Habeeb [Habeeb AFSA, Analytical Biochemistry. 1966, 14, 328-336]. Briefly, 0.4 mL of freshly prepared 0.01% (v / v) TNBS was added to 0.4 mL of fibrous rh-collagen-MA at 0.1-2 mg / mL in 4% sodium bicarbonate. After reacting at 40°C for 2 hours, 0.2 mL of 1N HCl and 0.4 mL of 10% (v / v) SDS were added. Absorbance was measured at 335 nm using a spectrophotometer in a 1 mL polystyrene cuvette. A control (blank) was prepared using the same procedure, except that sodium bicarbonate buffer was added instead of rh-collagen-MA solution. The absorbance of 1-2 mg / mL fibrous rh-collagen prepared under the same conditions was recorded for calibration.

[0436] Rheological property determination

[0437] Viscosity: Viscosity measurements were performed using a HAAKE RHEOSTRESS600™ rheometer (Thermo Electron Corporation) with a temperature-controlled cell chamber, using a C60 / 1° Ti cone-plate setting. Viscosity was measured for 1 mL samples in rotational gradient mode at shear rates ranging from 0.0001 to 1000 sec⁻¹, at 4°C, 25°C, and 37°C.

[0438] Storage modulus and loss modulus of scaffolding: The rheological behavior of rh-collagen crosslinked disks was investigated using a parallel plate system with a PP20 serrated spindle and a 20 mm serrated plate setup. To characterize uncrosslinked rh-collagen-MA, a C60 / 1° Ti cone-plate element was used. Two sets of experiments were performed separately to evaluate the rheological behavior of rh-collagen-MA. In the first experiment, a 1 mL sample was subjected to an oscillatory force in a controlled stress mode, and the values ​​of the storage modulus G' and loss modulus G'' were recorded while applying a shear stress of 5 Pa for 300 seconds at a frequency of 1 Hz and 37°C. The gap was adjusted to 90% of the original sample height, and the values ​​of G' and G'' were averaged over a range of 150–300 seconds. In the second experiment, a 1.5 mL crosslinked disk was tested under frequency sweep oscillatory conditions at 37°C, and G' was recorded over a shear stress of 1 Pa and a frequency range of 0.01–100 Hz. To begin the measurements, the spindle was lowered to contact the hydrogel surface, and then further lowered until the instrument's axial force equaled 0.4 N. Before all measurements, the samples were held on a plate covered with a humidity lid for 1 minute to reach thermal equilibrium.

[0439] result

[0440] TNBS assay

[0441] The degree of modification of rh-collagen was quantified using TNBS colorimetric analysis. This assay quantifies the molar content of free, unreacted ε-amino groups derived from lysine and hydroxyllysine, and subsequently the degree of functionalization. As shown in Table 6, the degree of functionalization of 10-fold, 15-fold, and 20-fold different batches of fibrous rh-collagen was determined by the TNBS assay.

[0442] [Table 6]

[0443] These results demonstrate the high modification ability of fibrous rh collagen, suggesting that adding methacrylic acid reagent at a molar ratio of 10 may be preferable for obtaining maximum functionalization of fibrous collagen.

[0444] Rheology

[0445] 1. Viscosity

[0446] Temperature dependence of viscosity of rh collagen / bovine collagen

[0447] Figure 27 shows the viscosity of rh collagen (COLLAGE®) and bovine collagen (PureCol) in PBS, expressed as a function of shear rate, at T=4°C (blue, dashed and solid lines, respectively) and T=37°C (red, dashed and solid lines, respectively). Bovine collagen (solid line) shows a clear temperature dependence of zero shear rate viscosity (η0). That is, a flat state of viscosity at low shear rate values, with an η0 value at 37°C (red) being more than an order of magnitude higher than the 4°C (blue) value. In contrast, rh collagen (dashed line) shows no significant difference between the η0 values ​​at 4°C and 37°C. rh collagen neutralized with FB (see Methods) exhibits very similar behavior (Figure 28), i.e., the viscosity at 4°C and 37°C is nearly identical. Figure 29 shows the viscosity of fibrous rh collagen-MA at 4°C (blue line) and 37°C (red line). Although the profiles are not identical, the zero shear rate value is approximately 1000 cP at both temperatures.

[0448] Viscosity of rh collagen-methacrylamide

[0449] Figure 30 shows the viscosity of rh-collagen-MA dissolved in DMEM at 25°C. The typical shear-thickening behavior of rh-collagen, as seen in Figures 27 and 28, is maintained in rh-collagen-MA with or without the addition of HA / HA-MA. Increasing the rh-collagen concentration from 2026 mg / mL to 26 mg / mL (green and red lines, respectively) increases the zero-shear viscosity by subsequently adding 10 mg / mL of HA or HAMA to bring the final polymer concentration to 30 mg / mL.

[0450] Those skilled in the art will recognize that rh-collagen-MA is not crosslinked and that a photoinitiator and light must be added to achieve crosslinking.

[0451] 2. Loss modulus and storage modulus of scaffolding

[0452] Figure 31 shows the rheological analysis of a 1 mL disk over time at 37°C, performed in the first experiment. The upper graph reports the loss modulus, storage modulus, and tan(delta) before UV curing, while the lower graph reports the values ​​after UV curing (when the photoinitiator was added). This data shows that the storage modulus of rh-collagen-MA doubles when irradiated in the presence of a photoinitiator. Furthermore, this result demonstrates the ability to control the scaffold properties by changing the rh-collagen-MA concentration. The large difference between the G' and G'' values, and the near-zero tan(delta) value of the crosslinked disc, indicate their elastic-like behavior. (G' - storage modulus; G'' - loss modulus; G', "storage / modulus," represents the energy fraction of G* stored by the gel during deformation and used later to return to its original shape. G' measures the elastic behavior of the gel, or how well the gel can recover its shape after shear deformation. For example, vulcanized rubber is a purely elastic material because it deforms instantaneously when stressed and completely recovers its shape when the stress is removed (i.e., G* ~ G') (~ represents an approximation). G'', "loss / viscosity coefficient," represents the energy fraction of G* lost due to shear deformation caused by internal friction. Since HA fillers are not purely viscous, G'' is not directly related to viscosity. Instead, this term reflects the fact that the gel cannot completely recover its shape after the shear stress is removed.)

[0453] In the second experiment, as shown in Figure 32, the 1.5 mL disc irradiated for 60 seconds showed a higher G' value. This data indicates that G' increases with rh-collagen-MA concentration and degree of methacrylate, demonstrating the ability to control the scaffold properties.

[0454] Example 15. Procedure for obtaining and processing rh collagen from tobacco plants.

[0455] As described above, genetically modified tobacco plants are grown, leaves are harvested and prepared for initial upstream extraction and purification (Figures 33A-33C). As shown in Figure 33A, the leaves are mechanically shredded (Step A) to remove the pulp from the slurry, while the portion containing procollagen is retained and subjected to enzymatic digestion to convert the procollagen to collagen (Steps B-C). The pulp is discarded again, and the portion containing collagen is retained from the slurry (Step C). After the acidification step, the sample undergoes initial centrifugation for the first washing step, after which the pellet is discarded (Steps D-F). After AMS precipitation and a second centrifugation, the protein precipitates (H pellet), and the supernatant is discarded (Steps G-H). The H pellet can be stored frozen at -20°C.

[0456] As shown in Figure 33B, the H pellet is resuspended to produce a protein suspension, followed by a third centrifugation, after which the pellet is discarded (steps I-J). The suspension is washed using a depth filter, and collagen is precipitated by salting out with NaCl (steps K-L). A fourth centrifugation yields a collagen pellet, and the supernatant is discarded (step M).

[0457] As shown in Figure 33C, the collagen pellet is resuspended in HCl to produce solubilized collagen (step N). After filtration to 0.2-0.8 microns, ultrafiltration (UF) (concentration and dialysis filtration) is performed to produce bulk concentrated collagen (steps O-P). After filtration to 0.2 microns and packing, the purified collagen is stored in a final container (step Z).

[0458] Example 16. Viscosity and polymerization of rh collagen methacrylate using additives

[0459] The viscosities of 5 mg / ml rh-collagen methacrylate rich in different additives (polyvinyl alcohol methacrylate (PVAMA) (Figures 34 and 37), hyaluronic acid methacrylate (HAMA) (Figures 35 and 37), and oxidized cellulose (OC) (Figures 36 and 37)) at collagen MA:additive ratios of 5:1, 2:1, and 1:2 are shown in Figures 31-37. For comparison, the viscosity of 5 mg / ml rh-collagen methacrylate is reported in each figure (black curve). All samples were prepared in 0.1 M phosphate buffer (pH 7.4) + 11.3 mM NaCl (physiological osmotic pressure), and measurements were performed at T=22°C. The data are compared and summarized in Figure 37.

[0460] Polymerization of rh collagen methacrylate rich in different additives is also demonstrated for a typical scaffold of 5 mg / ml collagen MA + different additives in a collMA:additive 2:1 ratio (Figure 38). ColMA alone was compared to ColMA combined with polyvinyl alcohol methacrylate (PVMA), hyaluronic acid methacrylate (HAMA), or oxidized cellulose (OC). The solutions were mixed with the photoinitiator 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (0.1%) and irradiated with ultraviolet (UV) light (365 nm) for 20 seconds.

[0461] Example 17. Injectable rh-collagen / platelet-rich plasma scaffold.

[0462] Injectable rh-collagen / platelet-rich plasma (PRP) scaffolds were investigated as scaffolds and healing devices for tendon injuries. A slow-degrading rh-collagen matrix, combined with a growth factor (GF) source, e.g., platelet-rich plasma (PRP), was injected near the injured tendon to provide the necessary support to promote healing. This treatment used a matrix of plant-derived recombinant human type I collagen (rh-collagen) mixed with PRP. This aids in the sustained release of growth factors at the injury site, thereby promoting healing. The effects of the rh-collagen-PRP matrix were compared in vitro and in vivo with PRP in assisting fibroblast proliferation, clot degradation, GF release, and tendon healing in a rat model of Achilles tendon injury induced by collagenase. rh-collagen-PRP demonstrated superior performance in vitro and in vivo compared with PRP alone. These results suggest promising potential for the use of rh-collagen matrix combined with PRP in clinical trials for the indication of tendon injury.

[0463] material and method

[0464] Rh Collagen Matrix

[0465] A monomer solution of rh collagen (CollPlant, Nessiona, Israel) in 10 mM HCl was fibrillated by pH neutralization in a phosphoric acid solution and crosslinked with 18 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (Sigma Aldrich, Israel). Next, the crosslinked collagen was washed by repeated centrifugation in redistilled water, and calcium chloride (CaCl2) (Merck, Israel) was added to calculate a final concentration of 20 mM. Syringes filled with the rh collagen slurry were freeze-dried and finally sterilized with ethylene oxide.

[0466] Preparation of platelet-rich plasma (PRP)

[0467] Granulocyte-free PRP was prepared using the Tropocell PRP Kit (ESTAR, Israel) according to the manufacturer's instructions. Human blood was collected from healthy human volunteers for in vitro cell proliferation assays (Helsinki Permit No. 2012068). Blood was collected from Hsd:Sprague DawleySD rats (Harlan) for in vivo animal studies.

[0468] Preparation of rh collagen matrix / PRP and control

[0469] Rh collagen matrix / PRP: A syringe containing freeze-dried cross-linked rh collagen was hydrated with PRP or physiological saline to obtain rh collagen with a final concentration of 20 mg / ml.

[0470] Thrombin-activated PRP (control): Human PRP was mixed with purified thrombin (Sigma Aldrich, Israel) to obtain a final concentration of 100 IU / ml.

[0471] CaCl2-activated PRP (control): Rat PRP was mixed with CaCl2 (Merck, Israel) to obtain a final concentration of 20 Mm.

[0472] In vitro cell proliferation assay

[0473] This study evaluated the effects of growth factors (GFs) on the viability and proliferation of normal human dermal fibroblasts (nHDFs). Cell viability and proliferation were compared during GF diffusion from either a matrix composed of cross-linked rh-collagen matrix combined with PRP or a clot composed of thrombin-activated PRP. The rh-collagen matrix combined with PRP or thrombin-activated PRP (200 μl each) was injected into Transwells (Thincerts® 24-well 8.0 μm, Greiner bio-one, Israel) placed on a 24-well plate (Thermo Scientific, Israel) and incubated at 37°C for 20 minutes to allow clot formation. Normal human dermal fibroblasts (nHDFs) (5,000 cells per 0.5 ml) were seeded at the bottom of each well in serum-deficient medium (including Dulbecco's Modified Eagle Medium, DMEM, 1% fetal bovine serum, FBS, Biological Industries, Israel). A transwell containing the matrix (rh collagen matrix combined with either PRP or thrombin-activated PRP) was placed over the seeded wells, and an additional 0.2 ml of culture medium was added to the top of the sample. 0.5 ml of nHDF and 1% FBS in DMEM were seeded as a control. Samples were tested three times, 7 and 10 days after seeding, using the WST-1 cell proliferation kit (Roche, Israel) according to the manufacturer's instructions.

[0474] In vivo testing

[0475] animal

[0476] Hsd:SpragueDawleySD rats weighing 230g ± 20% were selected for the animal experiment. Each animal was given a unique animal identification ear number and randomly assigned to a specific group. The animals were housed in individually ventilated (IVC) cages in a dedicated animal facility with heat, ventilation, and air conditioning (HVAC). Temperature and humidity were continuously monitored. The animals were given free access to commercially available rodent diet (Harlan Teklad TRM Ra / Mouse Diet) and autoclaved water. The facility was not exposed to ambient light and was maintained in an automated alternating cycle of 12 hours of light and 12 hours of darkness. All animals were treated according to guidelines for the treatment and care of laboratory animals, and all protocols were approved by the local Institutional Animal Care and Use Committee. No abnormalities were detected in any of the animals throughout the study period. There were no statistically significant differences in the mean body weight and weight gain of the groups. All weight gains were within the range of values ​​normally expected at the end of the study.

[0477] In vivo blood clot breakdown and growth factor release

[0478] In a subcutaneous (SC) rat model (Science in Action Ltd., Nesgiona, Israel), the degradation time and GF content over time were compared for rh collagen matrix combined with PRP, rh collagen matrix alone, and CaCl2-activated PRP.

[0479] The injection sites on the backs of 34 female Sprague Dawley rats (Harlan Laboratories, Nesgiona, Israel) were shaved and marked. Each rat was injected with 0.5 ml of the same formulation into four separate locations on the dorsal plane: two on the anterior side of the rat's back and two on the posterior side. The animals were euthanized at 1, 7, 14, 21, 30, and 45 days after treatment (10 or 12 rats per group, 2 rats per time point). At each time point, the injection sites were exposed and evaluated visually. The skin at the injection site was gently separated from the muscle using scissors, and the site was washed with 0.25 ml of DMEM, 1% FBS (Biological Industries, Israel). Blood clots were extracted and weighed. The extracted blood clots were transferred to 6-well or 12-well plates while the washing medium was transferred to Eppendorf tubes (1.5–2 ml). After weighing, the blood clots were combined with their respective washing media, cut with scissors, and finely chopped with a pestle to facilitate the release of GF from the blood clots into the surrounding media. Next, the Eppendorf tubes were centrifuged for at least 5 minutes to separate the blood clot pellet from the media. The supernatant was collected and stored at -80°C until assay. Without injecting into animals, a control (T0) containing approximately 0.5 ml of each formulation was formed in vitro using the same procedure as above. At the end of the study, the PDGF and VEGF content in the stored supernatant was evaluated by ELISA (Quantikine ELISA Mouse / rat PDGF and Quantikine ELISA Rat VEGF, R&D Systems, Israel).

[0480] In vivo tendon injury induced in rats.

[0481] The healing properties of rh collagen matrix combined with PRP and PRP alone were compared in a collagenase-induced tendon injury model using 36 male Sprague Dawley rats (18 rats per group, 6 animals per time point). The experiment was conducted at Harlan Laboratories Israel Ltd. (Nesgiona, Israel).

[0482] A skin incision was made in the proximal portion of the right hind limb of a rat over the common calcaneal tendon. At appropriate magnification, the central branch of the tendon was identified and isolated, and tendon injury was induced by injecting 0.3 mg of collagenase (10 mg / ml, Sigma) under the common calcaneal tendon sac using a 0.5 ml insulin syringe. Finally, the skin was closed by interrupting the subcutaneous suture using 4 / 0 Vicryl. One week after induction of tendon injury, a puncture wound was made into the tendon sac using an ocular corneal / scleral knife. A tunnel was then created under the tendon sac using a cannula, and 50 μl of rh collagen combined with PRP or PRP alone was injected into a pre-prepared tube. Animals were euthanized 3, 7, and 14 days after the procedure. The treated tendons were excised and preserved for histopathological evaluation.

[0483] histology

[0484] The tissue was embedded in paraffin and sequentially cut into samples 4-5 microns thick. The slides were stained with hematoxylin and eosin (H&E) for histopathological examination and blinded and evaluated by a pathologist.

[0485] result

[0486] In vitro cell proliferation assay

[0487] This study compared the viability and proliferation of cells seeded near a matrix composed of rh-collagen combined with PRP and a blood clot composed of thrombin-activated PRP. Cells seeded in untreated wells were used as a control. The matrix (composed of either rh-collagen combined with PRP or thrombin-activated PRP) was placed in a transwell above the seeded wells to allow diffusion of GF from the matrix to the wells without direct contact with the cell layer. The number of viable cells on days 7 and 10 is reported as the average of two different experiments (three repeats for the experiment) in which PRP was extracted from two different donors (upper side of Figure 39). As shown in Figure 39, cell viability (days 7 and 10) in the presence of GF released from the rh-collagen matrix combined with PRP is significantly higher than in the thrombin-activated PRP blood clot or control. Furthermore, in the presence of rh collagen matrix combined with PRP, cell numbers increased from day 7 to day 10, while in the presence of thrombin-activated PRP and in the control group, cell numbers decreased. Both cell viability and proliferation were significantly superior in the presence of rh collagen matrix. The data were confirmed by microscopic analysis (bottom of Figure 39). Cells cultured in the presence of rh collagen matrix combined with PRP (bottom of Figure 39, panel A) showed an elongated shape and reached full confluence as early as day 7 after seeding. On the other hand, cells cultured in the presence of thrombin-activated PRP were almost non-viable, which may indicate the toxic effect of thrombin in this experimental setting (bottom of Figure 39, panel B). Cells cultured in the presence of medium alone showed very limited viability (bottom of Figure 39, panel C).

[0488] In vivo matrix degradation profiling and growth factor release.

[0489] Matrix Decomposition Profile

[0490] The degradation profile of the injected formulation was determined by weighing the matrix at different time points after subcutaneous injection into rats.

[0491] When activated PRP was injected, the material was already gone by day 1 (Figure 40), suggesting complete degradation of the fibrin clot within the first 24 hours. On the other hand, rh collagen matrix alone or in combination with PRP had a two-phase degradation profile (Figure 40), starting with a rapid weight loss on day 1, followed by a relatively slow degradation rate, and being completely eliminated after 30–45 days (final weight < 0.5% of initial weight).

[0492] Growth factor content

[0493] The GF content at the injection site as a function of time was assessed by ELISA for PDGF and VEGF (Figures 41A-41B). The PDGF content at time 0 was similar in the rh collagen matrix combined with PRP and in the activated PRP treatment (Figure 41A), suggesting that the PDGF content on day 0 was solely due to the GF-rich platelets provided by the PRP. However, when PRP alone was injected, the PDGF content at the injection site was already below the detection limit after 1 day post-injection and remained undetectable throughout the study, consistent with rapid clot degradation (Figure 40). Different images are shown when PRP was incorporated into the rh collagen matrix (Figure 41A). The PDGF content gradually increased from day 1 to day 14 with scaffold degradation, and then decreased again until it was completely gone by day 45 (Figure 40). Interestingly, the PDGF content in the rh collagen matrix-only group increased from day 7, following the pattern shown by the matrix combined with PRP. On day 0, VEGF content was below the detection limit for all formulations and remained at baseline levels in the activated PRP group (Figures 41A-41B and 42). The VEGF profile of the rh collagen matrix combined with PRP showed a sharp increase in VEGF content around day 7, followed by a sharp decrease until day 14, and then a flat state until day 30, with VEGF finally decreasing on day 45, accompanied by scaffold degradation (Figure 41B).

[0494] The increase in GF observed in PDGF analysis from day 1 to day 14 and in VEGF analysis from day 0 to day 7 demonstrates the ability of the rh collagen scaffold to enable GF accumulation, likely reflecting cells migrating and proliferating within the scaffold. The integral of nominal PDGF and VEGF content across the entire study of each formulation is summarized in Figure 42. It is clear that the GF content at the injection site is significantly higher when rh collagen matrix is ​​injected alone or in combination with PRP compared to activated PRP alone.

[0495] In vivo tendon injury induced in rats

[0496] The healing properties of rh-collag...

Claims

1. A kit comprising a set of components for a polymerizable solution for tissue enhancement, The aforementioned set of components is (i) Methacrylic-modified plant-derived type I recombinant human collagen, (ii) Hyaluronic acid (HA) or its modified derivatives, poly(vinyl alcohol) (PVA) or its modified derivatives, polyethylene glycol (PEG) or its modified derivatives, cellulose oxide (OC) or its modified derivatives, polymethyl methacrylate (PMMA) microspheres or its modified derivatives, tricalcium phosphate (TCP) or its modified derivatives, calcium hydroxyl apatite (CaHA) or its modified derivatives, carboxymethylcellulose or its modified derivatives, crystalline nanocellulose (CNC) or its modified derivatives, or combinations thereof, (iii) photoinitiator and, The aforementioned organizational strengthening, (a) A step of introducing the components (i) to (iii) into the tissue space beneath the epidermis, (b) A step of inducing polymerization by shining light on the surface of the epidermis on the surface of the tissue space, A kit comprising methacrylated or thiolated derivatives of the modified derivatives of hyaluronic acid (HA), poly(vinyl alcohol) (PVA), polyethylene glycol (PEG), cellulose oxide (OC), polymethyl methacrylate (PMMA) microspheres, tricalcium phosphate (TCP), calcium hydroxyl apatite (CaHA), carboxymethyl cellulose, or crystalline nanocellulose (CNC).

2. The kit according to claim 1, Each component of the polymerizable solution is introduced independently, at approximately the same location, and almost simultaneously. (a) The methacrylated plant-derived type I recombinant human collagen and the photoinitiator are as follows: (b) The hyaluronic acid (HA) or its modified derivative, the poly(vinyl alcohol) (PVA) or its modified derivative, the polyethylene glycol (PEG) or its modified derivative, the oxidized cellulose (OC) or its modified derivative, the polymethyl methacrylate (PMMA) microspheres or its modified derivative, the tricalcium phosphate (TCP) or its modified derivative, the calcium hydroxyl apatite (CaHA) or its modified derivative, the carboxymethyl cellulose or its modified derivative, the crystalline nanocellulose (CNC) or its modified derivative, or any combination thereof. A kit that is introduced independently of the epidermis.

3. The kit according to claim 1, The set of components is introduced together as a mixture into the tissue space beneath the epidermis, in a kit.

4. A kit according to any one of claims 1 to 3, The aforementioned organizational strengthening, A kit further comprising molding or shaping the polymerizable solution or components of the polymerizable solution within a desired configuration in the tissue space, wherein the molding or shaping occurs simultaneously with or following the exposure to light, and the molding or shaping reduces muscles, folds, fine lines, wrinkles, or scars, or a combination thereof.

5. A kit according to any one of claims 1 to 4, A kit comprising the hyaluronic acid (HA), the poly(vinyl alcohol) (PVA), the polyethylene glycol (PEG), the oxidized cellulose (OC), the polymethyl methacrylate (PMMA) microspheres, the tricalcium phosphate (TCP), the calcium hydroxyl apatite (CaHA), the carboxymethyl cellulose, or the crystalline nanocellulose (CNC).

6. A polymerizable solution for tissue strengthening, (i) Methacrylic-modified plant-derived type I recombinant human collagen, (ii) comprising a photoinitiator, The aforementioned organizational strengthening, (a) Introducing the polymerizable solution into the tissue space beneath the epidermis, (b) A polymerizable solution comprising irradiating the surface of the epidermis on the surface of the tissue space with light to induce polymerization.

7. A polymerizable solution according to claim 6, The tissue enhancement further includes molding or shaping the polymerizable solution within a desired configuration in the tissue space, wherein the molding or shaping occurs simultaneously with or following the application of light. A polymerizable solution that, when molded or shaped, reduces muscles, folds, wrinkles, scars, or combinations thereof.

8. A polymerizable solution according to claim 6, This includes hyaluronic acid (HA) or its modified derivatives, poly(vinyl alcohol) (PVA) or its modified derivatives, polyethylene glycol (PEG) or its modified derivatives, cellulose oxide (OC) or its modified derivatives, polymethyl methacrylate (PMMA) microspheres or their modified derivatives, tricalcium phosphate (TCP) or its modified derivatives, calcium hydroxyl apatite (CaHA) or its modified derivatives, carboxymethylcellulose or its modified derivatives, crystalline nanocellulose (CNC) or its modified derivatives, or combinations thereof. A polymerizable solution comprising methacrylated or thiolated derivatives of the modified derivatives of hyaluronic acid (HA), poly(vinyl alcohol) (PVA), polyethylene glycol (PEG), cellulose oxide (OC), polymethyl methacrylate (PMMA) microspheres, tricalcium phosphate (TCP), calcium hydroxyl apatite (CaHA), carboxymethyl cellulose, or crystalline nanocellulose (CNC).

9. A polymerizable solution for inducing a cell proliferation-promoting scaffold in the tissue space beneath the epidermis, (a) Methacrylic-modified plant-derived type I recombinant human collagen, (b) Hyaluronic acid (HA) or its modified derivatives, poly(vinyl alcohol) (PVA) or its modified derivatives, polyethylene glycol (PEG) or its modified derivatives, cellulose oxide (OC) or its modified derivatives, polymethyl methacrylate (PMMA) microspheres or its modified derivatives, tricalcium phosphate (TCP) or its modified derivatives, calcium hydroxyl apatite (CaHA) or its modified derivatives, carboxymethylcellulose or its modified derivatives, crystalline nanocellulose (CNC) or its modified derivatives, or combinations thereof (c) Photoinitiator and (d) comprising at least one growth factor or a source thereof, Inducing the cell proliferation-promoting scaffold involves introducing the solution into the tissue space to promote the healing or replacement of collagen-containing tissue, wherein the cell proliferation-promoting scaffold fills the tissue space and reduces muscle, folds, fine lines, wrinkles, or scars, or combinations thereof. A polymerizable solution comprising methacrylated or thiolated derivatives of the modified derivatives of hyaluronic acid (HA), poly(vinyl alcohol) (PVA), polyethylene glycol (PEG), cellulose oxide (OC), polymethyl methacrylate (PMMA) microspheres, tricalcium phosphate (TCP), calcium hydroxyl apatite (CaHA), carboxymethyl cellulose, or crystalline nanocellulose (CNC).

10. A polymerizable solution according to claim 9, The source of the at least one growth factor is a polymerizable solution comprising plasma or platelet-rich plasma.

11. A polymerizable solution according to claim 9 or 10, A polymerizable solution containing the aforementioned collagen-containing tissue, including skin.

12. A polymerizable solution according to any one of claims 8 to 11, A polymerizable solution comprising the hyaluronic acid (HA), the poly(vinyl alcohol) (PVA), the polyethylene glycol (PEG), the oxidized cellulose (OC), the polymethyl methacrylate (PMMA) microspheres, the tricalcium phosphate (TCP), the calcium hydroxyl apatite (CaHA), the carboxymethyl cellulose, or the crystalline nanocellulose (CNC), wherein the hyaluronic acid (HA), the poly(vinyl alcohol) (PVA), the cross-linked polyethylene glycol (PEG), the cross-linked oxidized cellulose (OC), the cross-linked polymethyl methacrylate (PMMA) microspheres, the cross-linked tricalcium phosphate (TCP), the cross-linked calcium hydroxyl apatite (CaHA), the cross-linked carboxymethyl cellulose, or the cross-linked crystalline nanocellulose (CNC).

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