Dermal filler and use thereof

Abstract

The present application relates to a polymerizable solution comprising a plant-derived recombinant human collagen type 1, a kit, a bi-crosslinked dermal filler, and a method for preparing the same. Furthermore, the present application relates to a photo-initiated dermal filler, a hyaluronic acid-rhCollagen bi-crosslinked dermal filler, and a hyaluronic acid-rhCollagen semi-interpenetrating network, each of which comprises human collagen of plant origin, as well as methods of using the dermal filler and semi-interpenetrating network.

Description

[0001] This application is a divisional application of an application filed on May 2, 2019, with application number 201980040285.2 and invention name “Dermal fillers and their applications”. Technical Field

[0002] Disclosed herein are photoinitiated and dual-crosslinked dermal fillers comprising plant-derived human collagen and a cell growth promoting scaffold for soft tissue augmentation, and in some cases methods of using the same. Background Art

[0003] Collagen is the major protein responsible for the structural integrity of vertebrates and many other multicellular organisms. Collagen comprises the major component of connective tissue and is the most abundant protein in mammals, accounting for approximately 30% of human protein. Loss or deterioration of collagen may occur due to aging or injury (Olsen et al., Adv Drug Deliv Rev. 2003 Nov 28; 55(12): 1547-67).

[0004] A common aspect of aging is the appearance of lines, fine lines, or wrinkles. Treatments involving the use of tissue-derived collagen have been used to reduce or eliminate lines, fine lines, or wrinkles. Similar treatments have been used to reduce scarring.

[0005] Collagen is also a component of tendons. Tendinopathy is a common injury usually associated with sports and physical activity, which is related to the degeneration and irregular arrangement of tendon collagen fibers. Repairing damaged tendons requires the coordinated activity of specific cells and the long-term presence of relevant growth factors (GF) near the injury. Today, tendinopathy is the main cause of consultation for musculoskeletal diseases (Kaux et al. (January 2011) "Journal of Sports Science and Medicine (J.Sport.Sci.Med.)" January: 238-253). Tendinopathy refers to a variety of painful conditions that develop in and around tendons and ligaments, which may be due to an imbalance between pathological changes caused by overuse of the tendon and the resulting regenerative response (Andres et al. (2008) "Clin.Orthop.Relat.Res. 466: 1539-1554). Tendinopathy is associated with degeneration and irregular arrangement of collagen (Maffulli et al. (2003) Clin. Sport. Med. 22:675-692), sometimes with microtears in the fibers, increased vascularity, and the presence of mild inflammation (Khan et al. (1999) Sport. Med. 27(6):393-408). Clinically, it is characterized by the onset of tendon stiffness, pain associated with activity, decreased function, and sometimes localized swelling (Kaux 2011; Andres 2008). Rather than the normal tight, parallel, bundled appearance, the collagen fibers exhibit uneven and irregular curling, laxity, and increased waviness (Mafulli 2003). As the aging population remains active, the incidence of tendon injuries is expected to rise in the coming decades. Treatments for tendinopathy vary, including physical therapy, medication, and combinations thereof; however, clinical outcomes are unsatisfactory and symptom recurrence is common (Kaux 2011). In the past few decades, injections of autologous platelet-rich plasma (PRP) have gained widespread attention for the treatment of tendinopathy (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 the plasma portion of the blood that contains 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 GFs 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). Many in vitro studies and in vivo models have shown that PRP treatment can enhance collagen expression and extracellular matrix production, stimulate angiogenesis, and increase cell migration, differentiation, and proliferation, thereby supporting tendon injury healing (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 on the efficacy of PRP treatment is limited (Delong 2016; Yuan 2013; Moraes et al. (2014)).

[0006] Collagen is the major component and major 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 is typically composed of three left-handed helices of procollagen (usually two identical helices and a third different helix) that are linked to form right-handed triple-helical tropocollagen to form fibrils.

[0007] The conformation and most of the properties of native collagen are determined by the triple helical domain, which makes up more than 95% of the molecule. The domain consists of three alpha chains, each containing about 1,000 amino acids, wrapped in a rope-like manner to form a compact triple helical structure. The triple helix is ​​wound in such a way that the peptide bonds connecting adjacent amino acids are buried inside the molecule, making the collagen molecule resistant to attack by proteases such as pepsin.

[0008] Type I collagen represents the prototype fibrillar collagen and is the major type of collagen in most tissues, including bone, tendon, skin, aorta, and lung. Type I collagen fibrils have excellent tensile strength and limited elongation. The most abundant molecular form of type I collagen is composed of two different α chains [α1(I) 2 and α2(I) (Inkinen, Connective Tissue Formation in Wound Healing an Experimental Study, Academic Dissertation, University of Helsinki, Faculty of Science, Department of Biological Sciences, Institute of Biochemistry, September 2003).

[0009] In all fibrous collagen molecules, three polypeptide chains are composed of repeated Gly-XY triplets, where X and Y can be any amino acid, but are usually 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 usually has a repeated amino acid sequence. Procollagen is modified by adding hydroxyl groups on proline and lysine residues. These hydroxylation reactions are catalyzed by prolyl-4-hydroxylase and lysyl-hydroxylase, respectively. Then, the hydroxyl groups on the lysine residues are glycosylated and subsequently form triple helices.

[0010] An important feature of fibril-forming collagen is that it is synthesized as a precursor procollagen containing globular N-terminal and C-terminal extended propeptides. The biosynthesis of procollagen is a complex process that involves many different post-translational modifications, including hydroxylation of proline and lysine, N-linked and O-linked glycosylation, and formation of intra- and inter-chain disulfide bonds. The enzymes that carry out these modifications act in a coordinated manner to ensure the folding and assembly of correctly aligned and thermally stable triple helical molecules.

[0011] The three-component polypeptide chain is assembled in the rough endoplasmic reticulum (RER) to form procollagen.Due to the co-translational transfer of polypeptide chains on the membrane of the endoplasmic reticulum (ER), the prolyl-4-hydroxylase (P4H) dependent hydroxylation of proline and lysine residues occurs in the Gly-XY repeat region.The stability of the final triple helical structure of collagen is highly dependent on the hydroxylation of the collagen chain mediated by P4H.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 the post-translational modification of collagen.These enzymes modify the lysyl residues in the specific position to hydroxylysyl, galactosylhydroxylysyl and glucosylgalactosylhydroxylysyl residues in sequence. These structures are unique to collagen and are essential for its functional activity (Wang et al. (2002) Matrix Biology, 21(7):559-566). A single human enzyme, lysyl hydroxylase 3 (LH3), catalyzes all three sequential steps in the formation of hydroxylysine-linked carbohydrates (Wang et al. (2002) Matrix Biology, 21(7):559-566). Once the polypeptide chain is fully transferred into the lumen of the endoplasmic reticulum, the three pro-α chains associate via their C-propeptides to form a trimer molecule, with the Gly-XY repeat region forming a nucleation point at its C-terminus to ensure proper alignment of the chains. The Gly-XY region then folds in the C-to-N direction to form a triple helix (Khoshnoodi et al. (2006) J. Biol. Chem. 281:38117-38121).

[0012] The temporal relationship between polypeptide chain modification and triple helix formation is crucial, because the hydroxylation of proline residues is necessary to ensure the stability of triple helix at body temperature, and once formed, triple helix no longer serves as a substrate for hydroxylase. C-propeptide (and to a lesser extent, N-propeptide) keeps procollagen soluble during its departure from cells (Bulleid et al. (2000) Biochem. Socy. Transact., 28 (4): 350-353). After or during the secretion of procollagen molecules into the extracellular matrix, the propeptide is removed by procollagen N-proteinase and C-proteinase, thereby triggering the collagen molecules to spontaneously self-assemble into fibrils (Hulmes, 2002, J. Struct. Biol., January-February; 137 (1-2): 2-10). Removal of the propeptides by procollagen N-protease and C-protease reduces the solubility of procollagen by more than 10,000 times and is necessary to initiate the self-assembly of collagen into fibers at 37°C. Crucial to this assembly process are the short telopeptides, which are the non-triple helical residues of the N-terminal and C-terminal propeptides remaining after digestion with N / C proteases. These peptides serve to ensure the correct covalent registration of collagen molecules within the fibril structure and reduce the critical concentration for self-assembly through their cross-linkable aldehydes (Bulleid et al. (2000) Journal of the Biochemical Society, 28(4): 350-353).

[0013] Natural collagen is usually present in connective tissue in the form of telopeptide-containing collagen molecules that are packed side by side in the form of fibrils. Each longitudinal course consists of molecules aligned end to end with a small longitudinal space that is staggered relative to the next consecutive transversely adjacent longitudinal course. In this way, in a given longitudinal course, gaps are created between the opposite end regions of consecutive molecules, and in parallel longitudinal courses that are transversely adjacent to the longitudinal course, the gaps are limited by the staggered sides of the molecules.

[0014] Various proteolytic enzymes can be used to achieve dispersion and dissolution of native animal collagen, which break intermolecular bonds and remove immunogenic non-helical terminal peptides without affecting the basic rigid triple helical structure that confers the desired properties of collagen (see U.S. Pat. Nos. 3,934,852; 3,121,049; 3,131,130; 3,314,861; 3,530,037; 3,949,073; 4,233,360 and 4,488,911 for general methods for preparing purified soluble collagen). The resulting soluble terminal collagen can then be purified by repeated precipitation at low pH and high ionic strength, followed by washing and re-dissolving at low pH. However, soluble preparations are often contaminated by cross-linked collagen chains, which reduces the homogeneity of the protein preparation.

[0015] Due to its unique properties and diverse functions in the human body, collagen has been selected from a variety of biocompatible materials for use in tissue repair to support structural integrity, induce cell infiltration, and promote tissue regeneration. Of the five major types of collagen, type I collagen is the most abundant form of collagen in the human body.

[0016] Type I collagen can be self-assembled into a fibrous hydrogel that can support tissue cells through bioactive adhesion sites. Methacrylate groups are added to collagen to produce collagen methacrylate (CMA), which is more resistant to degradation (Gaudet et al. "Biointerphases" (2012) 7: 25-33). The thiolation of collagen can improve cohesion and mucoadhesion, and affect swelling capacity (Duggan et al., "European Journal of Pharmaceutics and Biopharmaceutics (Eur.J.Pharm.Biopharm.)" (April 2015) 91: 75-81).

[0017] The unique properties of collagen contribute to its use in regenerative medicine products. Collagen provides biomaterials with desirable properties for a variety of applications including pharmacology (hemostatic dressings, sponges, healing dressings), medicine (prostheses such as heart valves, tendons and ligaments, skin substitutes, fillers), dentistry (gingival implants / gum disease) and cosmetology (additives, anti-wrinkle agents, microcontainers for flavoring substances). Collagen-based products produced in all of the above markets require large amounts of collagen raw materials for production.

[0018] Human and animal derived collagen, such as collagen products from cadaveric or animal sources (cow, pig or horse) have been used for administration, injection, implantation and oral ingestion. Uses include preforming into desired shapes to repair or partially replace damaged bone or cartilage structures, injection into damaged joints and injection as dermal fillers.

[0019] The use of collagen of animal origin (including human origin) is problematic because of the risk of contamination with unconventional infectious agents. While the risk from bacterial or viral contamination can be fully controlled, prions are less controllable and present a considerable health risk. These infectious agents, which appear to be proteinaceous in nature, are involved in the development of degenerative animal encephalopathy (shaking disease in sheep, bovine spongiform encephalopathy) and human encephalopathy (Creutzfeld-Jacob disease, Gerstmann-Straussler syndrome, and kuru disease). Other diseases (e.g., acquired immunodeficiency syndrome [AIDS], hepatitis, rabies, some cancers) may also be transmitted to the recipient. Because of the long onset of encephalopathy and some other diseases, formal control is difficult. (See generally, Castrow et al. (1983) J. Am. Acad. Dermatol. 9(6):889-93; Siegle et al. (1984) Arch. Dermatol. 120(2):183-187).

[0020] In addition, in some patients, treatment with human or animal collagen can trigger cellular or humoral immune responses, including allergies. In addition, the quality of collagen generally decreases with the age of the source corpse or organism, or is affected by other factors. In addition, the extraction process can also cause severe structural damage, thereby compromising its biological and mechanical functions (Stein et al. (2009) Biomacromolecules 10(9):2640-2645; Shilo et al. (2013) Tissue Engineering Part A 19(13-14):1519-1526; Shoseyov et al. (2013) Tissue Engineering Part A 19(13-14):1527).

[0021] Plants expressing collagen chains are known in the art (see, e.g., WO 2005 / 035442; U.S. Pat. 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. (Mar. 3, 2000) FEBS Letters 469(1): 132-6). Although such plants can be used to produce collagen chains and collagen, these chains are incorrectly hydroxylated, so their self-assembly (whether in planta or not) results in collagen that is inherently unstable. For example, although plants are able to synthesize hydroxyproline-containing proteins, the prolyl hydroxylases responsible for hydroxyproline synthesis in plant cells exhibit relatively relaxed substrate sequence specificity compared to mammalian P4H, so that plant co-expression of collagen and P4H genes is required to produce collagen containing hydroxyproline only in the Y position of the Gly-XY triplet (Olsen et al. (2003) Adv. Drug Deliv. Rev., 55(12):1547-1567).

[0022] Processing of "insoluble collagen" of animal origin with proteases of plant origin (such as ficin and / or papain) is also known in the art (U.S. Pat. Nos. 4,597,762, 5,670,369, 5,316,942, 5,997,895, and 5,814,328).

[0023] Attempts to produce human collagen that relied on the hydroxylation machinery naturally present in plants resulted in collagen that was poorly proline hydroxylable (Merle et al. (2002) FEBS Letters 515: 114-118). This collagen melts or loses its triple helical structure at temperatures below 30°C. Co-expression of collagen and prolyl hydroxylases produced stable hydroxylated collagen at body temperature that is relevant for applied biology (Merle et al. (2002) FEBS Letters 515: 114-118).

[0024] The hydroxylysine formation of human collagen expressed in tobacco is less than 2% of the hydroxylysine found in bovine collagen (0.04% of residues / 1.88% of residues). This suggests that the plant endogenous lysyl hydroxylases are not able to adequately hydroxylate the lysine in collagen.

[0025] Recent advances in technology have led to the development of a system for purifying pure human type I collagen (rhCollagen) by introducing five human genes encoding heterotrimeric type I collagen into tobacco plants (COLLPLANT, Israel TM; SIGMA- (also available) [see, e.g., Stein H. (2009) Biomacromolecules 10:2640-5; Yaari et al. (2013) Tissue Engineering Part A 19(13 / 14):1502-1506; Willard et al. (2013) Tissue Engineering Part A 19(13 / 14):1507-1518; Shilo et al. (2013) Tissue Engineering Part A 19(13 / 14):1519-1526; Shoseyov et al. (2013) Tissue Engineering Part A 19:1527-1533; and Shoseyov et al. (Jan / Feb 2014) Bioengineered 5:1, 1-4]. The protein is purified to homogeneity by a cost-effective industrial process that exploits the unique properties of collagen. See also WO 2006 / 035442, WO 2009 / 053985, and patents and patent applications derived therefrom, all of which are incorporated herein by reference as if fully set forth herein.

[0026] Plant-derived human type I collagen has a more consistent structure and more cell-binding domains than tissue-derived collagen, which can be partially denatured and deprived of cell-binding domains (Shoseyov et al. (Jan / Feb 2014) Bioengineering 5:1, 1-4; Majumdar et al. (2015) J. Biomed. Mater. Res. Part B: Appl. Biomater. 104B: 300-307). rhCollagen can form functional three-dimensional (3D) matrices and scaffolds and is used in additive manufacturing (AM) processes, in which 3D objects are fabricated in a layered manner by 3D bioprinting using a computer model of the object. Moreover, rhCollagen generally lacks the immunogenicity and disease transfer issues of tissue-derived collagen.

[0027] Methods for producing collagen in plants by expressing at least one type of collagen α chain and enabling its accumulation in subcellular compartments lacking endogenous P4H activity are available (U.S. Pat. No. 8,455,717), as are methods for producing atelocollagen from human telopeptide-containing collagen of non-animal cell origin by protease treatment (U.S. Pat. No. 8,759,487).

[0028] Type I collagen and rhCollagen are considered candidates for use as the main components of building materials in 3D bioprinting. Various types of scaffolds have been used in cosmetic and other reconstructive applications.

[0029] In addition, the use of dermal fillers for soft tissue augmentation (e.g., wrinkle reduction) has increased. One possible method for using dermal fillers involves injecting a polymerizable dermal filler material into a desired area and then contouring or molding the filler into a desired conformation. Polymerizing and crosslinking the material by one of a variety of methods can convert monomers in the injected material into polymers and chains that can form a network, thereby retaining the desired molded conformation. There are many methods for forming polymers and crosslinking polymers. One method involves a photoreactive agent and a light-induced reaction that produces a reactive species in a monomer solution. See, for example, U.S. Patent 9,795,711; U.S. Publication 8,945,624; U.S. Publication 6,352,710; and U.S. Publication 2009 / 0324722, and Elisseeff et al. (March 1999) Proc. Natl. Acad. Sci. USA 96:3104-3107.

[0030] However, at least some of these methods continue to focus on tissue-derived collagen or non-collagen polymers (e.g., polyvinyl alcohol, hyaluronic acid, or polyethylene glycol). In addition, due to the sensitivity of tissue-extracted collagen to temperature and ionic strength, its use is limited, and the collagen drives spontaneous 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 hinders precise fluidity. Keeping collagen at low temperatures until use is a possible solution to this phenomenon, but this means serious technical limitations. Another solution is to use gelatin, which is a denatured form of collagen that does not become gelatinous under these conditions. However, gelatin lacks the true tissue and cell interactions of natural collagen, and therefore loses key biological functions. Moreover, the viscosity of gelatin makes it more difficult to inject it into the subdermis using a fine-gauge needle, and also makes it more difficult to spread and mold into smaller cavities.

[0031] Therefore, improved injectable dermal fillers having highly tunable rheological and mechanical properties, as well as methods and uses thereof, are needed and would be highly desirable and advantageous. Summary of the invention

[0032] In one aspect, disclosed herein is a dual cross-linked dermal filler comprising:

[0033] (a) human collagen of plant origin; and

[0034] (b) cross-linked hyaluronic acid;

[0035] wherein the plant-derived human collagen is cross-linked with the cross-linked hyaluronic acid.

[0036] In a related aspect, the plant-derived human collagen comprises

[0037] (a) recombinant human collagen type 1 (rhCollagen); or

[0038] (b) the cross-linked hyaluronic acid comprises cross-linked and non-cross-linked hyaluronic acid; or

[0039] (c) any combination thereof.

[0040] In a related aspect, the cross-linking agent connecting the cross-linked hyaluronic acid is different from the cross-linking agent connecting the plant-derived human collagen to the cross-linked hyaluronic acid; or the ratio of the cross-linked hyaluronic acid to the plant-derived human collagen includes a range between 4:1 and 1:2; or a combination thereof. In another related aspect, the cross-linking agent that cross-links the hyaluronic acid and the cross-linking agent that cross-links the plant-derived human collagen are independently selected from 1,4-butanediol diglycidyl ether (BBDE), 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide methyl iodide (EDC), N,N'-dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide (DIC).

[0041] In one aspect, disclosed herein is a method of preparing a double cross-linked dermal filler comprising plant-derived human collagen cross-linked with cross-linked hyaluronic acid, the method comprising the steps of

[0042] (a) cross-linking hyaluronic acid;

[0043] (b) neutralizing the cross-linked hyaluronic acid;

[0044] (c) neutralizing the plant-derived human collagen;

[0045] (d) mixing the neutralized cross-linked hyaluronic acid with the neutralized plant-derived human collagen;

[0046] (e) adding lower molecular weight hyaluronic acid (MW HA);

[0047] (f) cross-linking a mixture of cross-linked hyaluronic acid and plant-derived human collagen; and

[0048] (g) Dialysis of double cross-linked hyaluronic acid-plant-derived human collagen dermal filler.

[0049] In a related aspect, the plant-derived human collagen comprises recombinant human collagen type 1 (rhCollagen); or the cross-linking agent connecting the cross-linked hyaluronic acid of step (a) is different from the cross-linking agent connecting the plant-derived human collagen to the cross-linked hyaluronic acid of step (e); or a combination thereof. In a related aspect, the ratio of cross-linked hyaluronic acid to the plant-derived human collagen comprises a range between 4:1 and 1:2; or the cross-linking agent that cross-links the hyaluronic acid and the cross-linking agent that cross-links the plant-derived human collagen are independently selected from 1,4-butanediol diglycidyl ether (BBDE), 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide methyl iodide (EDC), N,N'-dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide (DIC); or a combination thereof.

[0050] Additionally, in one aspect, disclosed herein is a method of filling a subepidermal tissue space, the method comprising:

[0051] (a) introducing a polymerizable solution into the tissue space, wherein the polymerizable solution comprises:

[0052] (i) cross-linkable plant-derived human collagen;

[0053] (ii) hyaluronic acid (HA) or a modified derivative thereof, polyvinyl alcohol (PVA) or a modified derivative thereof, polyethylene glycol (PEG) or a modified derivative thereof, oxidized cellulose (OC) or a modified derivative thereof, polymethyl methacrylate (PMMA) microspheres or a modified derivative thereof, tricalcium phosphate (TCP) or a modified derivative thereof, calcium hydroxyapatite (CaHA) or a modified derivative thereof, carboxymethyl cellulose or a modified derivative thereof, crystalline nanocellulose (CNC) or a modified derivative thereof, or a combination thereof; and

[0054] (iii) a photoinitiator; and

[0055] (b) applying light to the surface of the spatial superficial epidermis to induce polymerization.

[0056] In a related aspect, the polymerizable solution components are independently introduced into the tissue space at approximately the same location and approximately the same time, wherein the cross-linkable plant-derived human collagen and the photoinitiator are introduced together and independently of the hyaluronic acid (HA) or its modified derivatives, the polyvinyl alcohol (PVA) or its modified derivatives, the 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 hydroxyapatite (CaHA) or its modified derivatives, carboxymethyl cellulose or its modified derivatives, crystalline nanocellulose (CNC) or its modified derivatives, or its combination, at approximately the same time, are independently introduced into the tissue space. In another related aspect, the method further comprises a step of molding or carving the polymerizable solution or the components of the polymerizable solution into a desired configuration in the tissue space, wherein the step is performed simultaneously with or after the step of applying light.

[0057] In another related aspect, the polymerizable solution components are introduced into the tissue space together as a mixture, wherein the cross-linkable plant-derived human collagen and the photoinitiator are introduced together with the hyaluronic acid (HA) or a modified derivative thereof, or the polyvinyl alcohol (PVA) or a modified derivative thereof, or the polyethylene glycol (PEG) or a modified derivative thereof, or the oxidized cellulose (OC) or a modified derivative thereof, or the polymethyl methacrylate (PMMA) microspheres or a modified derivative thereof, or the tricalcium phosphate (TCP) or a modified derivative thereof, or the calcium hydroxyapatite (CaHA) or a modified derivative thereof, or the carboxymethyl cellulose or a modified derivative thereof, or the crystalline nanocellulose (CNC) or a modified derivative thereof, or a combination thereof.

[0058] In another related aspect, the polymerizable solution components are introduced into the tissue space independently of each other, wherein the cross-linkable plant-derived human collagen and the photoinitiator are introduced together and independently of the hyaluronic acid (HA) or its modified derivatives, or the polyvinyl alcohol (PVA) or its modified derivatives, or the polyethylene glycol (PEG) or its modified derivatives, or the oxidized cellulose (OC) or its modified derivatives, or the polymethyl methacrylate (PMMA) microspheres or its modified derivatives, or the tricalcium phosphate (TCP) or its modified derivatives, or the calcium hydroxyapatite (CaHA) or its modified derivatives, or the carboxymethyl cellulose or its modified derivatives, or the crystalline nanocellulose (CNC) or its modified derivatives, or a combination thereof.

[0059] In another related aspect, after introduction into the tissue space, the method further comprises a step of molding or sculpting the polymerizable solution or a component of the polymerizable solution into a desired configuration in the tissue space, wherein the step is performed simultaneously with or after the step of applying light.

[0060] In another related aspect, the method is non-therapeutic and the molding or sculpting step reduces lines, folds, fine lines, wrinkles or scars, or a combination thereof.

[0061] In another related aspect,

[0062] (a) the cross-linkable plant-derived human collagen is methacrylated or thiolated type 1 human recombinant collagen (rhcollagen); or

[0063] (b) the modified derivatives of hyaluronic acid (HA), polyvinyl alcohol (PVA), polyethylene glycol (PEG), oxidized cellulose (OC), polymethyl methacrylate (PMMA) microspheres, tricalcium phosphate (TCP), calcium hydroxyapatite (CaHA), carboxymethyl cellulose or crystalline nanocellulose (CNC) include methacrylated or thiolated derivatives; or

[0064] (c) the hyaluronic acid (HA), polyvinyl alcohol (PVA), polyethylene glycol (PEG), oxidized cellulose (OC), polymethyl methacrylate (PMMA) microspheres, tricalcium phosphate (TCP), calcium hydroxyapatite (CaHA), carboxymethyl cellulose or crystalline nanocellulose (CNC) includes cross-linked hyaluronic acid (HA), cross-linked polyvinyl alcohol (PVA), cross-linked polyethylene glycol (PEG), cross-linked oxidized cellulose (OC), cross-linked polymethyl methacrylate (PMMA) microspheres, cross-linked tricalcium phosphate (TCP), cross-linked calcium hydroxyapatite (CaHA), cross-linked carboxymethyl cellulose or cross-linked crystalline nanocellulose (CNC); or

[0065] (d) A combination of (a) and (b) or (a) and (c).

[0066] In further related aspects, when MA-rhCollagen is selected and hyaluronic acid or a derivative thereof or cross-linked hyaluronic acid is selected, the ratio of HA to MA-rhCollagen 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.

[0067] In one aspect, disclosed herein is a method of filling a subepidermal tissue space, the method comprising introducing a dual cross-linked dermal filler into the tissue space, wherein the dual cross-linked dermal filler comprises:

[0068] (a) human collagen of plant origin; and

[0069] (b) cross-linked hyaluronic acid (HA) or a modified cross-linked derivative thereof, cross-linked polyvinyl alcohol (PVA) or a modified cross-linked derivative thereof, cross-linked polyethylene glycol (PEG) or a modified cross-linked derivative thereof, cross-linked oxidized cellulose (OC) or a modified cross-linked derivative thereof, cross-linked polymethyl methacrylate (PMMA) microspheres or a modified cross-linked derivative thereof, cross-linked tricalcium phosphate (TCP) or a modified cross-linked derivative thereof, cross-linked calcium hydroxyapatite (CaHA) or a modified cross-linked derivative thereof, cross-linked carboxymethyl cellulose or a modified cross-linked derivative thereof, cross-linked crystalline nanocellulose (CNC) or a modified cross-linked derivative thereof, or a combination thereof;

[0070] Wherein the plant-derived human collagen is cross-linked with the following: the cross-linked cross-linked hyaluronic acid (HA) or a modified cross-linked derivative thereof, cross-linked polyvinyl alcohol (PVA) or a modified cross-linked derivative thereof, cross-linked polyethylene glycol (PEG) or a modified cross-linked derivative thereof, cross-linked oxidized cellulose (OC) or a modified cross-linked derivative thereof, cross-linked polymethyl methacrylate (PMMA) microspheres or a modified cross-linked derivative thereof, cross-linked tricalcium phosphate (TCP) or a modified cross-linked derivative thereof, cross-linked calcium hydroxyapatite (CaHA) or a modified cross-linked derivative thereof, cross-linked carboxymethyl cellulose or a modified cross-linked derivative thereof, cross-linked crystalline nanocellulose (CNC) or a modified cross-linked derivative thereof.

[0071] In a related aspect, the plant-derived human collagen is type 1 human recombinant collagen (rhcollagen) or its MA or thiolated derivatives; or the modified derivatives of hyaluronic acid (HA), polyvinyl alcohol (PVA), polyethylene glycol (PEG), oxidized cellulose (OC), polymethyl methacrylate (PMMA) microspheres, tricalcium phosphate (TCP), calcium hydroxyapatite (CaHA), carboxymethyl cellulose or crystalline nanocellulose (CNC) include methacrylated or thiolated derivatives; or a combination thereof.

[0072] In another related aspect, when cross-linked HA is selected, the ratio of cross-linked HA to the 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.

[0073] In a related aspect, the method is non-therapeutic and reduces lines, folds, fine lines, wrinkles or scars, or a combination thereof.

[0074] In one aspect, disclosed herein is a polymerizable or non-polymerizable solution for tissue augmentation, wherein

[0075] (a) the polymerizable solution comprises cross-linkable plant-derived human collagen and a photoinitiator that induces polymerization prior to or simultaneously with the application of visible light, or

[0076] (b) the non-polymerizable solution comprises a dual cross-linked dermal filler, the dermal filler comprising plant-derived human collagen and cross-linked hyaluronic acid or cross-linked PVA, or cross-linked PGE or cross-linked OC, wherein the plant-derived human collagen is cross-linked with the cross-linked hyaluronic acid or cross-linked PVA, or cross-linked PGE or cross-linked OC;

[0077] And the use includes injecting the polymerizable or non-polymerizable solution into the subepidermal tissue space, and then molding or sculpting the polymerizable or non-polymerizable solution into a desired configuration to reduce lines, folds, fine lines, wrinkles or scars.

[0078] In a related aspect, the cross-linkable plant-derived human collagen is methacrylated or thiolated; or the polymerizable solution further comprises hyaluronic acid (HA) or a modified derivative thereof or a photopolymerizable modified derivative, polyvinyl alcohol (PVA) or a modified derivative thereof or a photopolymerizable modified derivative, polyethylene glycol (PEG) or a modified derivative thereof or a photopolymerizable modified derivative, polymethyl methacrylate (PMMA) microspheres or a modified derivative thereof or a photopolymerizable modified derivative, tricalcium phosphate (TCP) or a modified derivative thereof or a photopolymerizable modified derivative, calcium hydroxyapatite (CaHA) or a modified derivative thereof or a photopolymerizable modified derivative, carboxymethyl cellulose or a modified derivative thereof or a photopolymerizable modified derivative, crystalline nanocellulose (CNC) or a modified derivative thereof or a photopolymerizable modified derivative, or a combination thereof, wherein the derivatives thereof optionally include methacrylated or thiolated derivatives; or a combination thereof.

[0079] In another related aspect, tissue augmentation is required due to any medical or dental (gum implant / gum disease) condition.In a further related aspect, the tissue augmentation reduces lines, folds, fine lines, wrinkles or scars or a combination thereof.

[0080] In one aspect, disclosed herein is a method of inducing a cell growth promoting scaffold in a subepidermal tissue space, the method comprising introducing a solution into the tissue space, the solution comprising:

[0081] (a) human collagen of plant origin; and

[0082] (b) at least one growth factor or a source thereof;

[0083] The methods described herein promote healing or replacement of collagen-containing tissue.

[0084] In a related aspect, the plant-derived collagen comprises type 1 recombinant human collagen (rhCollagen); or the source of the at least one growth factor comprises plasma or platelet-rich plasma; or the collagen-containing tissue comprises skin; or any combination thereof.

[0085] In another aspect, the method is non-therapeutic and the cell growth promoting scaffold fills the tissue spaces, thereby reducing lines, folds, fine lines, wrinkles or scars, or a combination thereof.

[0086] In one aspect, disclosed herein is a solution for inducing a cell growth promoting scaffold, the solution comprising plant-derived human collagen and at least one growth factor or a source thereof, wherein the use comprises injecting the solution into a tissue space beneath the epidermis, and wherein the use is used to promote healing or replacement due to degradation or damage of collagen-containing skin tissue.

[0087] In a related aspect, the plant-derived collagen comprises type 1 recombinant human collagen (rhCollagen); or the source of the at least one growth factor comprises plasma or platelet-rich plasma; or the collagen-containing tissue comprises skin; or any combination thereof.

[0088] In another related aspect, the rhCollagen comprises methacrylate or a thiol derivative thereof.

[0089] In a related aspect, the solution used in the method further includes hyaluronic acid (HA) or a modified derivative thereof, polyvinyl alcohol (PVA) or a modified derivative thereof, polyethylene glycol (PEG) or a modified derivative thereof, oxidized cellulose (OC) or a modified derivative thereof, polymethyl methacrylate (PMMA) microspheres or a modified derivative thereof, tricalcium phosphate (TCP) or a modified derivative thereof, calcium hydroxyapatite (CaHA) or a modified derivative thereof, carboxymethyl cellulose or a modified derivative thereof, crystalline nanocellulose (CNC) or a modified derivative thereof, or a combination thereof, and a photoinitiator that induces polymerization before or simultaneously with the application of visible light; or cross-linked hyaluronic acid or cross-linked PVA, or cross-linked PGE or cross-linked OC, wherein the plant-derived human collagen is cross-linked to the cross-linked hyaluronic acid or cross-linked PVA, or cross-linked PGE or cross-linked OC.

[0090] In a related aspect, the method is non-therapeutic and the cell growth promoting scaffold fills the tissue spaces, thereby reducing lines, folds, fine lines, wrinkles or scars, or a combination thereof.

[0091] Disclosed herein is a solution for inducing a cell growth promoting scaffold, the solution comprising plant-derived human collagen and at least one growth factor or a source thereof, wherein the use comprises injecting the solution into a tissue space beneath the epidermis, and wherein the use is used to promote healing or replacement due to degradation or damage of collagen-containing tissue.

[0092] In a related aspect, the source of the at least one growth factor comprises plasma or platelet-rich plasma; or the plant-derived collagen comprises type 1 recombinant human collagen (rhCollagen); or the collagen-containing tissue comprises skin; or a combination thereof.

[0093] In another related aspect, the solution used further includes hyaluronic acid (HA) or a modified derivative thereof, polyvinyl alcohol (PVA) or a modified derivative thereof, polyethylene glycol (PEG) or a modified derivative thereof, oxidized cellulose (OC) or a modified derivative thereof, polymethyl methacrylate (PMMA) microspheres or a modified derivative thereof, tricalcium phosphate (TCP) or a modified derivative thereof, calcium hydroxyapatite (CaHA) or a modified derivative thereof, carboxymethyl cellulose or a modified derivative thereof, crystalline nanocellulose (CNC) or a modified derivative thereof, or a combination thereof, and a photoinitiator that induces polymerization before or simultaneously with the application of visible light; or cross-linked hyaluronic acid or cross-linked PVA, or cross-linked PGE or cross-linked OC, wherein the plant-derived human collagen is cross-linked to the cross-linked hyaluronic acid or cross-linked PVA, or cross-linked PGE or cross-linked OC.

[0094] In one aspect, disclosed herein is a method for filling a subepidermal tissue space, the method comprising: (a) introducing a polymerizable solution into the tissue space, wherein the polymerizable solution comprises: (i) cross-linkable plant-derived human collagen; and (ii) a photoinitiator; and applying light to the surface of the superficial epidermis of the space to induce polymerization.

[0095] In a related aspect, the polymerizable solution further comprises a step of molding or sculpting the polymerizable solution into a desired configuration in the tissue space, wherein the step is performed simultaneously with or after the step of applying light.

[0096] In another related aspect, the method is non-therapeutic and the molding or sculpting step reduces lines, folds, fine lines, wrinkles or scars, or a combination thereof.

[0097] In another related aspect, the cross-linkable plant-derived human collagen is methacrylated or thiolated type 1 human recombinant collagen (rhcollagen).

[0098] Other objects, features and advantages of the present invention will become apparent from the following description and accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0099] Figure 1a-1d The construction of various expression cassettes and vectors previously used to transform test plants is shown. All coding sequences synthesized as part of the study were optimized for expression in tobacco. Figure 1a A scheme for cloning type I collagen αI chain or type II collagen α2 chain into a plant expression vector according to some embodiments of the present invention is shown; Figure 1bShown is a scheme for cloning prolyl-4-hydroxylase (P4H) into a plant expression vector according to some embodiments of the present invention; Figure 1c Shown is a scheme for cloning proteinase C or proteinase N into a plant expression vector according to some embodiments of the present invention; Figure 1d A scheme for cloning lysyl hydroxylase 3 (LH3) into a plant expression vector according to some embodiments of the present invention is shown.

[0100] Figure 2 Various co-transformation methods used previously are shown. Each expression cassette is represented by the abbreviation of the coding sequence. The coding sequences are specified in Table 1. Each co-transformation is performed by two pBINPLUS binary vectors. Each rectangle represents a single pBINPLUS vector carrying one, two or three expression cassettes. The promoter and terminator are specified in Example 1.

[0101] Figure 3 was a previous multiplex PCR screen of transformants which showed plants positive for either collagen alpha 1 (324 bp fragment) or collagen alpha 2 (537 bp fragment) or both.

[0102] Figure 4 It is the previous western blot analysis of the transgenic plants produced by co-transformation 2, 3 and 4.Total soluble protein was extracted from tobacco co-transformants #2, #3 and #4 and tested with anti-collagen I antibody (#AB745, from Chemicon Inc.).Size marker is #SM0671 from Fermentas Inc..WT is wild-type tobacco.Positive collagen bands are visible in plants that are PCR positive for type I collagen α1 or α2 or both.The positive control band of 500ng type I collagen from human placenta (#CC050 from Chemicon Inc., extracted from human placenta by pepsin digestion) accounts for about 0.3% of the total soluble protein (about 150 μg) in the transgenic plant sample.As detected by the anti-carboxyl terminal propeptide detection of type I collagen antibody (#MAB1913 from Chemicon Inc.), the larger band of about 140kDa in the human collagen sample is the procollagen with C-propeptide. The smaller band of about 120 kDa in human collagen samples is collagen without propeptide. Due to their unusual composition, proline-rich proteins (including collagen) always migrate on polyacrylamide gels as bands with higher molecular weights than expected. Therefore, collagen chains without propeptide with a molecular weight of about 95 kDa migrate as a band of about 120 kDa.

[0103] Figure 5This is a previous Western blot analysis of transgenic plants generated by co-transformation #8 (carrying apoplast signal translationally fused to collagen chains). Total soluble protein was extracted from transgenic tobacco leaves and tested with anti-collagen I antibody (#AB745 from Chemical Reagents International). A positive collagen α2 band was seen in plant 8-141. Type I collagen from human placenta (#CC050 from Chemical Reagents International) was used as a control.

[0104] Figure 6a and 6b The triple helical assembly and thermal stability of collagen are shown, as previously described by heat treatment and trypsin or pepsin digestion. Figure 6a Total soluble protein from tobacco strains 2-9 (expressing colα1 only without P4H) and 3-5 (expressing colα1+2 and human P4Hα and β subunits) was heat treated (15 min at 38°C or 43°C) followed by trypsin digestion (20 min at room temperature [RT]) and tested in a Western blotting procedure with an anti-collagen I antibody. A positive control was a sample of 500 ng of total soluble protein from human collagen I + wild-type tobacco. Figure 6b Total soluble protein was extracted from transgenic tobacco 13-6 (expressing collagen I α1 and α2 chains - indicated by arrows, human P4H α and β subunits, and human LH3) and heat treated (20 min at 33°C, 38°C, or 42°C), immediately cooled on ice to prevent triple helical reassembly, and incubated with pepsin for 30 min at room temperature (approximately 22°C) and then tested with anti-collagen I antibody (#AB745 from Chemical Reagents International) in a standard Western blot procedure. The positive control was a 50 ng human collagen I sample (#CC050 from Chemical Reagents International, extracted from human placenta by pepsin digestion) added to the total soluble protein extracted from wild-type (wt, wt) tobacco.

[0105] Figure 7 A Northern blot analysis previously performed on wild-type tobacco is shown. The blot was probed with tobacco P4H cDNA.

[0106] Figure 8 is a previous Western blot analysis of transgenic plants generated by cotransformation 2, 3, and 13. Total soluble protein was extracted from tobacco cotransformants and tested with anti-human P4H α and β antibodies and anti-collagen I antibodies.

[0107] Fig. 9Previous Western blot analysis of cross-bred vacuolar-targeted plant A (2-300+20-279) grown under normal light regime (lane 1); previous Western blot analysis of vacuolar-targeted plants 13-652 grown in darkness for 8 days. All plants expressed exogenous col1, col2, P4H-α and P4H-β, and LH3 (confirmed by PCR).

[0108] Fig.10 Shown is the collagen of the purification from tobacco leaf after trypsin digestion.As described in " Materials and Methods " chapters and sections, from the 13-6 tobacco plant transgenic leaf system ground in 100mM Tris buffer, purified collagen is centrifuged, proteolyzed and precipitated in high salt concentration buffer.After resuspension, the precipitate containing collagen is washed, dialyzed and condensed into the final product.This gel has described the Coomassie (Coomassie) staining analysis of the collagen sample collected, and wherein channel 1 and 2 are the collagens obtained after 300mg / L trypsin digestion procollagen.Top sample does not contain the collagen (0.5mg / ml) of the pig source of propeptide, and runs as the positive control of type 1 collagen α1 and α2 chain.

[0109] Fig.11 Shown is purified collagen from tobacco leaves after digestion with different concentrations of trypsin. Fig.10 Collagen was extracted and purified as described in , and then digested with 20 mg / L trypsin (lanes 1-7) or 30 mg / L (lanes 8-10). Products were separated on 10% SDS PAGE and analyzed with a Coomassie-based staining solution. Collagen from porcine origin (0.5 mg / ml) without propeptide was loaded and run as a positive control for type 1 collagen α1 and α2 chains.

[0110] Fig.12 Shown is purified collagen from tobacco leaves after digestion with trypsin and pepsin. Fig.10 Collagen was extracted and purified as described in , and then digested with 30 mg / L trypsin and 1 μg / 200 ml pepsin (lanes 1-2). Products were separated on 10% SDS PAGE and analyzed with a Coomassie-based staining solution. Collagen from porcine origin (0.5 mg / ml) without propeptide was loaded and run as a positive control for type 1 collagen α1 and α2 chains.

[0111] Fig.13The collagen chains obtained when procollagen is digested with subtilisin or bromelain are shown.Collagen is purified from the transgenic leaf line of tobacco plant No. 13-361, which is ground in 100mM Tris buffer, by centrifugation and proteolysis with subtilisin (1-25mg / L) or bromelain (1-25mg / L), and incubated for 3 hours or 6 hours.Samples are separated on 10% SDS PAGE, and blotted on nitrocellulose membrane.Use anti-collagen I to carry out immunodetection to collagen chains.The untreated supernatant collected after homogenization and centrifugation is used as the negative control (channel 3-4) that does not contain collagen.Collagen (2.5 μ g) in the pig source that does not contain propeptide is used as the positive control (channel 1) of α1 and α2 chain.

[0112] Fig.14 The collagen chains obtained when procollagen is digested with papain are shown. Collagen is purified from the transgenic leaf line of tobacco plant No. 13-361, which is ground in 100mM Tris buffer, by centrifugation and proteolysis with papain (1-25mg / L) in the incubation period of 3 hours or 6 hours. Samples are separated on 10% SDS PAGE, and blotted on nitrocellulose membrane. Anti-collagen I is used to carry out immunodetection of collagen chains. Homogenization, centrifugation and untreated supernatant (enzyme-free) collected after hatching 3 hours (channel 3) or 6 hours (channel 2) at 15 ℃ are used as the negative control that does not contain collagen. Collagen (2.5 μ g) in the pig source that does not contain propeptide is used as the positive control (channel 1) of α1 and α2 chain.

[0113] Fig.15 The collagen chains obtained when procollagen is digested with ficin or Savinase are shown. Collagen is purified from the transgenic leaf line of tobacco plant No. 13-361 ground in 100mMTris buffer, centrifuged and proteolyzed with ficin (1-25mg / L) or Savinase (1-25mg / L) during the incubation period of 3 hours or 6 hours. Samples are separated on 10% SDS PAGE and blotted on nitrocellulose membrane. Anti-collagen I is used to carry out immunodetection of collagen chains. Untreated supernatant collected before proteolysis is used as a control sample (channel 3) that does not contain collagen. Collagen (2.5 μg) from pig sources that does not contain propeptide is used as a positive control (channel 1) for α1 and α2 chains.

[0114] Fig.16The collagen chains obtained when procollagen is digested with compound protease or alkaline protease are shown.Collagen is purified from the transgenic leaf system of tobacco plant No. 13-361 ground in 100mMTris buffer, centrifuged and carried out proteolysis with compound protease (1-25mg / L) or alkaline protease (1-25mg / L) in the incubation period of 3 hours or 6 hours.Samples are separated on 10%SDS PAGE, and blotted on nitrocellulose membrane.Use anti-collagen I to carry out immunodetection to collagen chains.Untreated supernatant collected before proteolysis is used as the control sample (channel 14) that does not contain collagen.Collagen (2.5 μ g) in the pig source that does not contain propeptide is used as the positive control (channel 1) of α1 and α2 chain.

[0115] Fig.17 The collagen chains obtained when procollagen is digested with Esperase or neutral protease are shown. Collagen is purified from the transgenic leaf system of tobacco plant No. 13-361, which is ground in 100mMTris buffer, by centrifugation and after the incubation period of 3 hours or 6 hours, Esperase (1-25mg / L) or neutral protease (1-25mg / L) is used for proteolysis. Samples are separated on 10% SDS PAGE, and are blotted on the nitrocellulose membrane. Anti-collagen I is used to carry out immunodetection of collagen chains. Collagen (2.5 μ g) in the pig source that does not contain propeptide is used as the positive control (channel 1) of α1 and α2 chain.

[0116] Fig.18 The collagen chains obtained when procollagen is digested with Esperase 8.0L or alkaline protease are shown. Collagen is purified from the transgenic leaf system of tobacco plant No. 13-361, which is ground in 100mM Tris buffer, by centrifugation and after the incubation period of 3 hours or 6 hours, Esperase (1-25mg / L) or neutral protease (1-25mg / L) is used for proteolysis. Samples are separated on 10% SDS PAGE, and are blotted on nitrocellulose membrane. Anti-collagen I is used to carry out immunodetection of collagen chains. Homogenization, centrifugation and the untreated supernatant (no proteolytic enzyme) collected after hatching 3 hours (channel 3) or 6 hours (channel 2) at 15 ℃ are used as the negative control that does not contain collagen. Collagen (2.5 μ g) in the pig source that does not contain propeptide is used as the positive control (channel 1) of α1 and α2 chain.

[0117] Fig.19The collagen chains obtained at various purification stages after digestion of procollagen with ficin are shown. Collagen was purified from the transgenic leaf line of tobacco plant No. 13-361 ground in 100mM Tris buffer, centrifuged and incubated at 15 ℃ for 3 hours and then proteolyzed with ficin (5mg / L). Samples were separated on 10% SDS PAGE and blotted onto nitrocellulose membrane. Immunodetection of collagen chains was performed using anti-collagen I. Samples collected after grinding, centrifugation and incubation of supernatant with ficin were loaded onto channel 5. Lanes 6-14 depict samples of collagen treated with ficin at different stages of the purification process: Lane 6: Sample after ficin incubation and centrifugation; Lane 7: After salt precipitation and resuspension in 0.5 M acetic acid; Lane 8: Same sample as Lane 7 with an additional centrifugation step; Lane 9: Same sample as Lane 8, after resuspension in 0.5 M acetic acid and centrifugation; Lane 10: Mature collagen after resuspension in 10 mM HCl and dialysis; Lane 11: Same sample as Lane 10, with an additional filtration step; Lane 12: Same sample as Lane 11, with an additional 5-fold concentration step; Lane 13: Same sample as Lane 11, with an additional 20-fold concentration step; Lane 14: Same sample as Lane 13, with an additional 5-fold concentration. Untreated procollagen samples (lanes 3-4) were used as negative controls. Porcine-derived collagen (2.5 μg) without propeptide was used as a positive control for α1 and α2 chains (lane 1).

[0118] Fig. 20The collagen chains obtained at various purification stages after digestion of procollagen with ficin are shown. Collagen was purified from the transgenic leaf line of tobacco plant No. 13-361 ground in 100mM Tris buffer, centrifuged and incubated at 15 ℃ for 3 hours and then proteolyzed with ficin (5mg / L). Samples were separated on 10% SDS PAGE and blotted onto nitrocellulose membrane. Immunodetection of collagen chains was performed using anti-collagen I. Samples collected after grinding, centrifugation and incubation of supernatant with ficin were loaded onto channel 5. Lanes 6-14 depict samples of collagen treated with ficin at different stages of the purification process: Lane 6: Sample after ficin incubation and centrifugation; Lane 7: After salt precipitation and resuspension in 0.5 M acetic acid; Lane 8: Same sample as Lane 7 with an additional centrifugation step; Lane 9: Same sample as Lane 8, after resuspension in 0.5 M acetic acid and centrifugation; Lane 10: Mature collagen after resuspension in 10 mM HCl and dialysis; Lane 11: Same sample as Lane 10, with an additional filtration step; Lane 12: Same sample as Lane 11, with an additional 5-fold concentration step; Lane 13: Same sample as Lane 11, with an additional 20-fold concentration step; Lane 14: Same sample as Lane 13, with an additional 5-fold concentration. Untreated procollagen samples (lanes 3-4) were used as negative controls. Porcine-derived collagen (2.5 μg) without propeptide was used as a positive control for α1 and α2 chains (lane 1).

[0119] Fig.21 The collagen content of the samples treated with ficin at various stages of purification is shown. At each extraction and purification stage of the purification procedure based on reactor size AMS described in the "Materials and Methods" section, samples containing collagen were collected. The samples were treated with ficin (5 mg / L, 15° C., 3 hours) to remove the propeptide, separated on 10% SDS PAGE and stained with Coomassie stain.

[0120] Fig. 22 Optimization of procollagen cleavage by food grade ficin is shown: Optimization of ficin concentration and reaction time. Tobacco leaf extract expressing procollagen precipitated by AMS was resuspended in extraction buffer and then incubated with increasing concentrations of food ficin (5-15 mg / L). The reaction mixture was then incubated at 15°C for 1-3 hours. The cleavage was terminated by centrifugation and the protein samples were separated on 8% SDS-PAGE, transferred to a nitrocellulose membrane, and immunoblotted for alpha-1 and alpha-2 collagen chains with anti-collagen I. The procollagen band is indicated by a white arrow, while the red arrow indicates the cleaved collagen band.

[0121] Figures 23A-23COptimization of procollagen cleavage by pharmaceutical grade ficin is shown: Optimization of ficin concentration and reaction time. Tobacco leaf extract expressing procollagen precipitated by AMS was resuspended in extraction buffer and then incubated with increasing concentrations of pharmaceutical ficin (2.5-10 mg / L). The reaction mixture was then incubated at 15°C for 0.5-3 hours. The cleavage was terminated by centrifugation and the protein samples were separated on 8% SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted for alpha-1 and alpha-2 collagen chains with anti-collagen I. Arrows indicate procollagen bands and collagen bands.

[0122] Figures 24A-24B Optimization of procollagen cleavage by pharmaceutical grade ficin is shown: Optimization of pH and salt concentration in the reaction buffer. Tobacco leaf extract expressing procollagen precipitated by AMS was resuspended in extraction buffer containing 10 mg / L pharmaceutical grade ficin at different pH values ​​(5.5-9.5) and increasing NaCl concentrations (0.5-3M). The reaction mixture was then incubated at 15°C for 1 hour. The cleavage was terminated by centrifugation and protein samples of both the resulting precipitate and the supernatant were separated on 8% SDS-PAGE, transferred to a nitrocellulose membrane, and immunoblotted for alpha-1 and alpha-2 collagen chains with anti-collagen I. Arrows indicate collagen bands.

[0123] Fig.25 Optimization of procollagen cleavage by pharmaceutical grade ficin is shown: Optimization of EDTA and L-cysteine ​​concentrations in the reaction buffer. Tobacco leaf extract expressing procollagen precipitated by AMS was resuspended in extraction buffer (pH 7.5) containing different concentrations of L-cysteine ​​(10-100 mM - concentrations above) and EDTA (8-80 mM - concentrations below). The samples were then incubated at 15°C for 1 hour with 1 mg / L pharmaceutical grade ficin. The cleavage was terminated by centrifugation and the protein samples were separated on 8% SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted for alpha-1 and alpha-2 collagen chains with anti-collagen I.

[0124] Fig.26 Effective procollagen digestion at pH 7.5 by recombinant trypsin was shown. Tobacco leaf extract expressing procollagen precipitated by AMS was resuspended in extraction buffer (pH 7.5) containing L-cysteine ​​and EDTA. The samples were then incubated at 15°C for 1-3 hours with 30-100 mg / L pharmaceutical grade ficin. The cleavage was terminated by centrifugation and the protein samples were separated on 8% SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted for alpha-1 and alpha-2 collagen chains with anti-collagen I.

[0125] Fig. 27 Viscosity (eta [η], cP) as a function of shear rate is shown, solid line - 2.7 mg / mL bovine collagen in phosphate buffered saline (PBS), dashed line - 2.79 mg / mL rhCollagen in PBS. ▼: measured values ​​at 4°C, ▲: measured values ​​at 37°C.

[0126] Fig.28 Viscosity as a function of shear rate is shown, 3.4 mg / mL bovine collagen in FB. ▼: values ​​measured at 4°C, ▲: values ​​measured at 37°C.

[0127] Fig.29 Viscosity as a function of shear rate is shown, 10 mg / mL rhCollagen-MA in PBS. ▲: values ​​measured at 4°C, ▼: values ​​measured at 37°C.

[0128] Fig.30 Shown are viscosity measurements of rhCollagen-MA in DMEM with and without added HA / HAMA.

[0129] Fig.31 Shown are the storage and loss moduli and the tan phase shift angle of rhCollagen-MA formulations at different concentrations before (upper panel) and after (lower panel) photocrosslinking.

[0130] Fig.32 Shown are the G' and G" values ​​at 37°C recorded in a frequency sweep test and plotted at 1 Hz.

[0131] Figures 33A-33C Flow charts for processing rhCollagen and rhCollagen methacrylate are provided. Fig.33A The upstream separation and processing of procollagen and collagen (steps AH) are shown. Figures 33B-33C Two stages of downstream processing are shown (step IM and steps NP and Z, respectively).

[0132] Fig.34 The viscosity (eta [η], cP) of 5 mg / ml rhCollagen methacrylate (CollMA) (solid black curve) and 5 mg / ml Collagen MA + polyvinyl alcohol methacrylate (PVMA) (light grey curve) at a ratio of collMA:PVAMA of 5:1 (solid line), 2:1 (dashed line), 1:2 (dash-dotted line) is shown. The viscosity of 5 mg / ml rhCollagen methacrylate is reported for comparison (black curve).

[0133] Fig.35 The viscosity of 5mg / ml CollMA (solid black curve) and 5mg / ml Collagen MA+ hyaluronic acid methacrylate (HAMA) (grey curve) at a ratio of collMA:HAMA of 5:1 (solid line), 2:1 (dashed line) is shown. The viscosity of 5mg / ml rhCollagen methacrylate is reported for comparison (solid black curve). These materials are not cross-linked yet, but will be cross-linked after injection. The viscosity represents the injectability of the material. (HAMA-HA methacrylate; CollagenMA (ColMA)-rhCollagen methacrylate).

[0134] Fig.36 The viscosity of 5 mg / ml CollMA (solid black curve) and 5 mg / ml Collagen MA + oxidized cellulose (OC) (grey curve) at a ratio of collMA:OC of 5:1 (solid line), 2:1 (dashed line), 1:2 (dash-dotted line) is shown. The viscosity of 5 mg / ml rhCollagen methacrylate is reported for comparison (solid black curve).

[0135] Fig.37 Provided by Figure 33A-Figure 35 Comparison of data. It shows the viscosity of 5mg / ml CollMA (solid black curve) and 5mg / ml Collagen MA + different additives (light grey to dark grey curves as shown) at a ratio of 5:1 (solid line), 2:1 (dashed line), 1:2 (dotted line). The viscosity of 5mg / ml rhCollagen methacrylate is reported for comparison (solid black curve).

[0136] Fig.38 Shown are polymerized scaffolds of rhCollagen methacrylate (ColMA) + additives at a collMA:additive ratio of 2:1. ColMA alone was compared to ColMA in combination with polyvinyl methacrylate (PVMA), hyaluronic acid methacrylate (HAMA), or oxidized cellulose (OC). The solution was mixed with the photoinitiator 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylacetophenone (0.1%) and irradiated with ultraviolet (uv) light (365nm) for 20 seconds.

[0137] Fig.39Feasibility studies are shown. Top: Comparison of normal human fibroblast (nHDF) viability (and proliferation) when cultured in the presence of GFs released from rhCollagen-PRP matrix (black), in the presence of GFs released from activated PRP (grey), and under starvation conditions (white). Data are the average of two different fibroblast proliferation assays performed using PRP extracted from 2 different donors. * Significant difference (p<0.0002). Bottom: Microscope images of nHDF cells proliferating in the presence of GFs released from rhCollagen matrix combined with PRP (A), in the presence of GFs released from activated PRP (b) and cultured under starvation conditions (C). Images were taken 7 days after seeding the cells.

[0138] Fig.40 Scaffold weight is shown as a function of time (each point is the average of 6 scaffolds, 2 rats per time point, 3 injections per rat).

[0139] Figure 41A-41B Two studies using a subcutaneous rat model are shown. (A) Changes in PDGF levels over time (B) Changes in VEGF levels over time in a subcutaneous rat model. * There was a significant difference between the rhCollagen matrix combined with PRP and PRP alone (p<0.038), and between the rhCollagen matrix alone and PRP alone (p<0.004). ** There were significant differences between the rhCollagen matrix alone and PRP alone, and between the rhCollagen matrix alone and the rhCollagen matrix combined with PRP (p<0.021) (A), and between the rhCollagen matrix combined with PRP and PRP alone, and between the rhCollagen matrix combined with PRP and the rhCollagen matrix alone (p<0.007) (B).

[0140] Fig.42 Shown is the integration of nominal PDGF and VEGF levels in the injected matrix over 45 (or 30) days in the rat model.

[0141] Figures 43A-43C Shown are histopathological scores of Achilles tendons in a rat model of tendinopathy treated with PRP or rhCollagen / PRP matrix: (A) mature fibrosis, (B) presence of mononuclear inflammatory cells and (C) presence of immature granulation tissue.

[0142] Fig.44A comparison of the force (Newton, N) required to inject cross-linked hyaluronic acid (HA) (black curve -□), cross-linked hyaluronic acid (HA) + monomeric collagen (▼), or cross-linked hyaluronic acid (HA) + fibrillated collagen (▲) by a 32-gauge needle and 1 ml syringe (Becton Dickinson [BD], reference number 309628) is shown. Cross-linked HA + monomeric collagen and cross-linked HA + fibrillated collagen are semi-interpenetrating networks in which collagen is not cross-linked to any substance.

[0143] Fig.45 Shown is a comparison of the expressed force (Newtons, N) required to inject cross-linked HA (black) or a double cross-linked network of cross-linked HA and collagen (gray) via a 32-gauge needle and 1 ml syringe (BD, reference number 309628).

[0144] Fig.46 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) + fibrillated collagen (▲) is shown.

[0145] Fig.47 Shown is a comparison of the viscosity (eta [η], cP) of cross-linked hyaluronic acid (HA) (black) or a double cross-linked network of cross-linked hyaluronic acid (HA) and collagen (grey).

[0146] Figures 48A-48B Shown are photographs of (A) a mouse patch placed over a methacrylated collagen (collMA / rhCollagenMA) solution and (B) methacrylated collagen (collMA / rhCollagenMA) polymerized and integrated into skin tissue when irradiated through the skin with a white light emitting diode (LED) torch.

[0147] Fig.49 Two examples of dermal filler components are shown. On the left is a schematic diagram of a semi-interpenetrating dermal filler comprising cross-linked hyaluronic acid (HA) and rhCollagen. On the right is a schematic diagram of a double cross-linked dermal filler comprising cross-linked hyaluronic acid and rhCollagen, wherein the cross-linked HA is further cross-linked to rhCollagen. The light grey bars represent the HA cross-linker, the black chains represent the HA, the rhcollagen is represented as thin grey chains, and the second cross-linker (cross-linking the cross-linked HA to the rhCollagen) is a black circle.

[0148] Fig.50 The diagram depicts the use of the HAAKE-RHEO STRESS 600 TM Instruments (THERMO SCIENTIFICTM ) Plot of rheological measurements of storage modulus and loss modulus for various dual crosslinked formulations measured using cone (1 degree) versus plate configuration (C35 / 1). Frequency sweep measurements were performed at a constant deformation of 0.8% and in the frequency range of 0.02-100 Hz. Storage modulus (solid line) and loss modulus (dashed line) of representative dual cross-linked formulations (see Table 7) compared to commercially available dermal fillers (solid and dashed lines: solid black - commercial product; solid ▼ - Formulation 2; solid □ - Formulation 2A; solid pentagon with upward pointing - Formulation 3; solid pentagon with downward pointing - Formulation 1A; solid ○ - Formulation 1; dashed ▲ - commercial product G"; dashed ▼ - Formulation 2G"; dashed □ - Formulation 2A-G"; dashed ○ - Formulation 1G"; dashed pentagon with downward pointing - Formulation 1A G"; dashed pentagon with upward pointing - Formulation 3").

[0149] Fig.51 The plot is shown at f = 1 Hz Fig.50 A graph comparing the storage modulus and loss modulus of the formulations reported in (open bars G' [Pa]; grey bars G" [Pa])

[0150] Fig.52 Shown is a diagram depicting the use of MULTI-TEST 1-i MECMESIN TM Compression Tester with 1ml LUER-LOK TM Syringe (BECTON-DICKINSON TM ) and a 30G needle for Formulations 2, 2A, and 3 (Table 8) of the injectability of selected double cross-linked formulations. Commercially available dermal fillers are included for comparison with the double cross-linked formulations. The expressivity of representative double cross-linked formulations as a function of plunger displacement (12 mm / min) is compared to the expressivity of commercial dermal fillers. (Black ▲ Commercial dermal filler; Gray □ - Formulation 3; Gray upward pentagon - Formulation 2; Gray ▼ - Formulation 2A)

[0151] Fig.53 Graphs depicting rheological measurements of storage modulus and loss modulus of various combinations of highly cross-linked hyaluronic acid (HA), rhCollagen methacrylate (MA) and / or rhCollagen (see Table 10) before (dashed lines) and after (solid lines) photocuring with visible light compared to highly cross-linked HA (black intermittent line oblique triangles). TM Instruments (THERMO SCIENTIFIC TM) Storage modulus and loss modulus were measured using a cone (1 degree) to plate configuration (C35 / 1). Frequency sweep measurements were performed at a constant deformation of 0.8% in the frequency range of 0.02-100 Hz. After light curing (6 minutes of visible light exposure with a white LED flashlight), the HAAKE-RHEO STRESS 600 TM Instruments (THERMO SCIENTIFIC TM ) Storage modulus and loss modulus were measured using a sawtooth plate-to-plate configuration (PP20). Frequency sweep measurements were performed in the frequency range of 0.02-100 Hz at a constant normal load of 0.3 N and a constant shear stress of 3 Pa. (Solid line ▲-Formulation 4-after; solid line ▼-Formulation 5-after; solid line □-Formulation 6-after; dotted upward pentagon-Formulation 4-before; dotted downward pentagon-Formulation 5-before; dotted line ○-Formulation 6-before; dotted black oblique triangle-highly cross-linked HA)

[0152] Fig.54 A graph depicting a comparison of the storage modulus and loss modulus of formulations 4, 5 and 6 in Table 10 and non-curable highly cross-linked HA at a frequency of F = 1 Hz before and after photocuring is shown. (Open bars G' [Pa]; grey bars G" [Pa])

[0153] Fig.55 Shown is a diagram depicting the use of MULTI-TEST 1-i MECMESIN TM Compression Tester with 1ml LUER-LOK TM Syringe (BECTON-DICKINSON TM ) and a graph of the injectability of selected double cross-linked formulations measured with a 30G needle for all samples. The expression force of representative double cross-linked formulations as a function of plunger displacement (12 mm / min) was compared to that of highly cross-linked HA. (Black ▲-highly cross-linked HA; Gray ▼-Formulation 4; Gray □-Formulation 5; Gray ○-Formulation 6)

[0154] Fig.56 Representative histological images are shown at day 7 after subcutaneous injection of Formulations 2, 2A, and a control (commercially available dermal filler) into the back of Sprague Dawley rats. In each case, the arrows indicate an increased (not severe) inflammatory response in Formulations 2 and 2A, suggesting initiation of tissue regeneration.

[0155] Fig.57 Shows Fig.56 Histological scores of Formulation 2, 2A and control at day 7 in the tissues analyzed in (black - control; light grey Formulation 2; dark grey Formulation 2A)

[0156] Fig.58 The results of photocurable histology scoring of photocurable dermal fillers at day 7, day 14 and day 20 are shown. (Black - Control highly cross-linked HA; Grey - Formulation 4 highly cross-linked HA and rhColMA)

[0157] Fig.59 Shown are the fibrosis score results at day 7 and day 14 after injection of Formulation 4 (grey - highly cross-linked HA + rhColMA) and control (black - highly cross-linked HA). DETAILED DESCRIPTION

[0158] Disclosed herein are photoinitiated dermal fillers and dual cross-linked dermal fillers, as well as cell growth promoting scaffolds, and methods of using the dermal fillers and scaffolds, for example, but not limited to, for soft tissue augmentation.

[0159] In contrast to the plant-derived human collagen of the present application, collagen-producing plants can be used to produce collagen chains as well as collagen, but these chains are incorrectly hydroxylated and thus their self-assembly (whether in the plant or not) results in an inherently unstable collagen.

[0160] While practicing the polymerizable and double-crosslinked solutions and methods of use of the present invention, practitioners have designed a plant expression method that ensures that the collagen chains are properly hydroxylated, thereby allowing collagen to be produced in plants that closely mimics the properties of human type I collagen (e.g., molecular structure, temperature stability, cellular interactions).

[0161] In one aspect, disclosed herein is a method of filling a subepidermal tissue space, the method comprising:

[0162] (a) introducing a polymerizable solution into the tissue space, the polymerizable solution comprising:

[0163] (i) cross-linkable plant-derived human collagen; and

[0164] (ii) a photoinitiator; and

[0165] (b) applying light to the surface of the spatial superficial epidermis to induce polymerization.

[0166] In a specific embodiment, the method further comprises a step of molding or sculpting the polymerizable solution into a desired configuration in the tissue space before or simultaneously with the step of applying light. In another specific embodiment, the molding or sculpting step reduces lines, folds, fine lines, wrinkles or scars.

[0167] In yet another specific embodiment, the polymer solution further comprises a filler, the filler comprising hyaluronic acid (HA) or a modified derivative thereof, polyvinyl 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 a specific embodiment, the isolated plant-derived human collagen is optionally formulated with, for example, hyaluronic acid (HA), polyvinyl alcohol (PVA), polyethylene glycol (PEG), oxidized cellulose (OC), polymethyl methacrylate (PMMA) microspheres, tricalcium phosphate (TCP), calcium hydroxyapatite (CaHA), carboxymethyl cellulose, crystalline nanocellulose (CNC), or a combination thereof.

[0168] Modified derivatives include, but are not limited to, photopolymerizable forms of, for example, 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 a light emitting diode (LED), a laser, a xenon lamp, and the like.

[0169] In some embodiments, in the formulations disclosed herein, methacrylate rhCollagen is cross-linked only to itself under irradiation conditions. In some embodiments, in the formulations disclosed herein, methacrylate rhCollagen is cross-linked to thiolated rhCollagen under irradiation conditions. In some embodiments, in the formulations disclosed herein, methacrylate rhCollagen is cross-linked to any MA / thiolated additive under irradiation conditions. In some embodiments, in the formulations disclosed herein, methacrylate rhCollagen is cross-linked to methacrylated HA under irradiation conditions. In some embodiments, in the formulations disclosed herein, methacrylate rhCollagen is cross-linked to thiolated HA under irradiation conditions. In some embodiments, in the formulations disclosed herein, methacrylate rhCollagen is cross-linked to methacrylated PVA under irradiation conditions. In some embodiments, in the formulations disclosed herein, methacrylate rhCollagen is cross-linked to thiolated PVA under irradiation conditions. In some embodiments, in the formulations disclosed herein, methacrylate rhCollagen is cross-linked to methacrylated PEG under irradiation conditions. In some embodiments, in the formulations disclosed herein, methacrylate rhCollagen is cross-linked to thiolated PEG under irradiation conditions. In some embodiments, in the formulations disclosed herein, methacrylate rhCollagen is cross-linked to methacrylated OC under irradiation conditions. In some embodiments, in the formulations disclosed herein, methacrylate rhCollagen is cross-linked to thiolated OC under irradiation conditions.

[0170] Those skilled in the art will appreciate that a photocurable formulation is actually a semi-IPN before curing and becomes an IPN (interpenetrating network) after curing. An IPN may encompass two entangled networks, each network crosslinked to itself but not to the other network.

[0171] In some embodiments, the cross-linked formulation contains a ratio of unmodified rhCollagen to adjust the stiffness after cross-linking (with light) without reducing the final total amount of rhCollagen, because unmodified rhCollagen cannot cross-link under irradiation and therefore does not enhance the final stiffness. Methacrylated HA can also be added to the final formulation.

[0172] In some embodiments, HA or MA-HA can be cross-linked to itself using a cross-linking agent such as, but not limited to, BDDE, as described in Example 23. In some embodiments, the cross-linking agent that cross-links HA or MA-HA includes divinyl sulfone (DVS) or glutaraldehyde. In certain embodiments, BDDE cross-linked HA or MA-HA is not further cross-linked to rhCollagen or MA-rhCollagen, thereby creating a so-called interpenetrating network (IPN). Fig.49 left side).

[0173] In still another specific embodiment, the plant-derived collagen comprises rhCollagen. In another specific embodiment, the plant-derived collagen is obtained from a genetically modified plant. In another specific embodiment, the genetically modified plant is a genetically modified plant selected from the group consisting of: tobacco, corn, alfalfa, rice, potato, soybean, tomato, wheat, barley, canola, carrot and cotton. Specifically, the genetically modified plant is a tobacco plant.

[0174] In yet another specific embodiment, the genetically modified plant comprises an expressible sequence of at least one gene sequence of human deoxyribonucleic acid (DNA), the human deoxyribonucleic acid being selected from the group consisting of: COL1, COL2, P4H-α, P4H-β and LH3. In another specific embodiment, the plant-derived human collagen comprises: at least one modified human collagen α-1 chain as shown in SEQ ID NO:3 and expressed in the genetically modified plant; and at least one modified human collagen α-2 chain as shown in SEQ ID NO:6 and expressed in the genetically modified plant; and wherein the genetically modified plant further expresses an exogenous prolyl 4-hydroxylase (P4H). In another specific embodiment, the method further comprises expressing an exogenous polypeptide selected from the group consisting of: lysyl hydroxylase (LH), proteinase N and proteinase C. In yet another specific embodiment, the human collagen α-1 chain is encoded by the sequence shown in SEQ ID NO:1. In another specific embodiment, the human collagen α-2 chain is encoded by the sequence shown in SEQ ID NO:4.

[0175] In yet another embodiment, the exogenous P4H is a mammalian P4H. Specifically, the exogenous P4H is a human P4H. In yet another embodiment, the method further comprises targeting human collagen alpha-1 to the vacuole of the plant or genetically modified plant and digesting it with ficin. In yet another embodiment, the method further comprises targeting human collagen alpha-2 to the vacuole of the plant or genetically modified plant and digesting it with ficin.

[0176] In yet another embodiment, the plant-derived human collagen is atelocollagen. In another embodiment, the plant-derived human collagen is atelocollagen having an amino acid (AA) sequence derived from SEQ ID NO: 1 and SEQ ID NO: 4. Atelocollagen is derived by enzymatic digestion (e.g., with ficin) of procollagen, which is the product of SEQ ID NO: 1 and SEQ ID NO: 4.

[0177] In yet another embodiment, the photoinitiator induces polymerization of the polymerizable solution in response to visible light. Specifically, the visible light has a wavelength of 390-800 nm. Specifically, the photoinitiator is selected from the group consisting of: Eosin Y+triethanolamine, riboflavin, etc.

[0178] In another embodiment, the photoinitiator induces polymerization of the polymerizable solution in response to ultraviolet (UV) light. Specifically, the photoinitiator is selected from the group consisting of lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) or 1-[4-(2-hydroxyethoxy)phenyl]-2-methylpropan-1-one ( 2959).

[0179] In another embodiment, the photoinitiator induces polymerization of the polymerizable solution in response to infrared light.

[0180] In yet another embodiment, the polymerizable solution is introduced into the tissue space through a hollow needle or cannula ranging from 27 gauge to 33 gauge.

[0181] In yet another embodiment, the polymerizable solution in the tissue space is molded or sculpted into a desired configuration by manual massaging.In another embodiment, the polymerizable solution in the tissue space is molded or sculpted into a desired configuration using a molding or sculpting tool.

[0182] In still another embodiment, the polymerizable solution in the tissue space is substantially non-gelling at room temperature. In another embodiment, the polymerizable solution in the tissue space is substantially non-gelling at 37°C. In yet another embodiment, the polymerizable solution comprising plant-derived human collagen has a reduced viscosity at room temperature compared to a similar polymerizable solution comprising tissue-extracted human or animal-derived collagen (such as but not limited to bovine or porcine or horse collagen) at the same concentration and formulation. In another embodiment, the polymerizable solution comprising plant-derived human collagen has a reduced viscosity at 37°C compared to a similar polymerizable solution comprising tissue-extracted human or animal-derived collagen at the same concentration and formulation.

[0183] The term "collagen of animal origin" as used throughout may encompass bovine or porcine or horse collagen or rat tail collagen, and is in contrast to collagen of human origin.

[0184] In still another embodiment, a polymerizable solution comprising plant-derived human collagen is introduced into a tissue space with reduced force at room temperature compared to a similar polymerizable solution comprising tissue-extracted human or animal-derived collagen at the same concentration and formulation. In still another embodiment, a polymerizable solution comprising plant-derived human collagen is introduced into a tissue space with reduced force at 37° C. compared to a similar polymerizable solution comprising tissue-extracted human or animal-derived collagen at the same concentration and formulation.

[0185] On the other hand, disclosed herein is a method of injecting a polymerizable solution into a subepidermal tissue space to reduce lines, folds, fine lines, wrinkles or scars, the polymerizable solution comprising methacrylated or thiolated cross-linkable plant-derived human collagen and a photoinitiator that induces polymerization before or simultaneously with the application of visible light, and molding or carving the polymerizable solution into a desired configuration to reduce lines, folds, fine lines, wrinkles or scars. In a specific embodiment, the polymer solution further comprises a filler, the filler comprising hyaluronic acid (HA) or a modified derivative thereof, polyvinyl alcohol (PVA) or a modified derivative thereof, polyethylene glycol (PEG) or a modified derivative thereof, oxidized cellulose (OC) or a modified derivative thereof, polymethyl methacrylate (PMMA) microspheres or a modified derivative thereof, tricalcium phosphate (TCP) or a modified derivative thereof, calcium hydroxyapatite (CaHA) or a modified derivative thereof, carboxymethyl cellulose or a modified derivative thereof, crystalline nanocellulose (CNC) or a modified derivative thereof, or a combination of any one of these.

[0186] Modified derivatives include, but are not limited to, photopolymerizable forms of, for example, HA, PVA, PEG, OC, PMMA, TCP, CaHA, carboxymethylcellulose, or CNC. Modifications include, but are not limited to, methacrylate or thiolation.

[0187] In another aspect, disclosed herein is a method of filling a subepidermal tissue space, the method comprising:

[0188] (a) introducing a polymerizable solution into the tissue space, the polymerizable solution comprising cross-linkable plant-derived human collagen.

[0189] The present technology relates in part to cosmetic and medical collagen-based polymerizable fillers that form moldable compositions that can be polymerized upon photoactivation by a light source, such as a visible light source. The polymerizable filler includes crosslinkable plant-derived human collagen and a photoinitiator.

[0190] The present technology of interest has the advantage of allowing for the in situ formation of customized, contoured dermal fillers or implants, generally without the need for invasive surgical intervention or general anesthesia. Typically, a collagen-based polymerizable solution is introduced into the tissue space beneath the skin (i.e., beneath the epidermis) and polymerization is induced by exposure to visible light applied to the skin surface (i.e., from outside the body or the skin) or applied to the epidermis.

[0191] In situ polymerization methods provide cosmetic and medical corrective and / or enhancement procedures using polymerizable solutions including polymer components that are capable of forming an insoluble cross-linked network when activated by a visible light source.

[0192] In some embodiments, the dermal fillers or cell growth promoting scaffolds disclosed herein are used for cosmetic purposes. In some embodiments, the dermal fillers or cell growth promoting scaffolds disclosed herein are used for medical correction purposes. In some embodiments, the dermal fillers or cell growth promoting scaffolds disclosed herein are used for augmentation procedures, such as but not limited to tissue augmentation. In some embodiments, the double cross-linked dermal fillers disclosed herein are used for cosmetic purposes. In some embodiments, the double cross-linked dermal fillers disclosed herein are used for medical correction purposes. In some embodiments, the double cross-linked dermal fillers disclosed herein are needed due to medical or dental (gingival implants / gum disease) conditions. In some embodiments, the double cross-linked dermal fillers disclosed herein are needed due to medical conditions requiring skin augmentation. In some embodiments, the double cross-linked dermal fillers disclosed herein are used for augmentation procedures, such as but not limited to tissue augmentation. In some embodiments, the photocurable dermal fillers disclosed herein are used for cosmetic purposes. In some embodiments, the photocurable dermal fillers disclosed herein are used for medical correction purposes. In some embodiments, the photocurable dermal fillers disclosed herein are needed due to medical or dental (gingival implants / gum disease) conditions. In some embodiments, the photocurable dermal fillers disclosed herein are needed due to a medical condition requiring skin augmentation. In some embodiments, the photocurable dermal fillers disclosed herein are used for augmentation procedures, such as, but not limited to, tissue augmentation. In some embodiments, the cell growth promoting scaffolds disclosed herein are used for cosmetic purposes. In some embodiments, the cell growth promoting scaffolds disclosed herein are used for medical corrective purposes. In some embodiments, the cell growth promoting scaffold dermal fillers disclosed herein are needed due to a medical or dental (gum implant / gum disease) condition. In some embodiments, the cell growth promoting scaffold dermal fillers disclosed herein are needed due to a medical condition requiring skin augmentation. In some embodiments, medical corrective uses include the treatment of tendinitis. In some embodiments, the cell growth promoting scaffolds disclosed herein are used for augmentation procedures, such as, but not limited to, tissue augmentation.

[0193] In some embodiments, the tissue augmentation is an augmentation of skin tissue.

[0194] In some embodiments, the use of a dermal filler comprising a cell growth promoting scaffold disclosed herein is used in a human. In some embodiments, the use of a dermal filler comprising a cell growth promoting scaffold disclosed herein in a human may reduce lines, folds, fine lines, wrinkles, or scars, or any combination thereof. In some embodiments, the reduction of lines, folds, fine lines, wrinkles, or scars, or any combination thereof, is for cosmetic purposes. In some embodiments, the reduction of lines, folds, fine lines, wrinkles, or scars, or any combination thereof, is for cosmetic purposes. In some embodiments, the use of a dermal filler comprising a cell growth promoting scaffold disclosed herein in a human may enhance tissue, such as, but not limited to, epidermal or dermal tissue. In some embodiments, tissue augmentation is for cosmetic purposes. In some embodiments, tissue augmentation is for medical treatment. In some embodiments, tissue augmentation is part of an enhancement procedure. In some embodiments, tissue augmentation is part of a skin enhancement procedure.

[0195] In some embodiments, tissue augmentation is required due to any medical or dental (gum disease / gum implant) condition.

[0196] In certain embodiments, the dermal filler for use as described herein comprises an interpenetrating (IPN) network or a semi-interpenetrating (Semi-IPN) network, in which different components can be cross-linked to themselves but not to each other. In some embodiments, an IPN or semi-IPN dermal filler comprises rhCollagen and a filler such as hyaluronic acid (HA), polyvinyl alcohol (PVA), polyethylene glycol (PEG), oxidized cellulose (OC) or a derivative thereof or a combination thereof. In some embodiments, an IPN or semi-IPN comprises rhCollagen and cross-linked HA. In some embodiments, an IPN or semi-IPN comprises a derivative of rhCollagen, such as, but not limited to, methacrylated rhCollagen or a thiol rhCollagen and / or a derivative of a filler, such as, but not limited to, methacrylated HA, PVA, PEG or OC, or thiolated HA, PVA, PEG or OC or a combination thereof.

[0197] In some embodiments, an IPN or semi-IPN network or a dual cross-linked network comprising a dermal filler comprises a filler ratio such as, but not limited 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 of HA, PVA, PEG, or OC to rhCollagen. In some embodiments, an IPN or semi-IPN network comprising a dermal filler comprises a MA-filler ratio such as, but not limited 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 of HA, PVA, PEG, or OC to rhCollagen. In some embodiments, an IPN or semi-IPN network comprising a dermal filler comprises a filler such as, but not limited to, HA, PVA, PEG, or OC to MA-rhCollagen 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, an IPN or semi-IPN network comprising a dermal filler comprises a MA-filler to MA-rhCollagen 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.

[0198] In some embodiments, the IPN or semi-IPN or double cross-linked network comprising a dermal filler comprises a ratio of HA to rhCollagen 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 cross-linked network comprising a dermal filler comprises a ratio of MA-HA to rhCollagen 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 comprising a dermal filler comprises a ratio of HA to MA-rhCollagen 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 comprising a dermal filler comprises a ratio of MA-HA to MA-rhCollagen 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.

[0199] In some embodiments, the IPN or semi-IPN or double cross-linked network comprising a dermal filler comprises a filler to rhCollagen 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 IPN or semi-IPN or double cross-linked network comprising a dermal filler comprises MA-HA or MA-PVA or MA-PEG or MA-OC to rhCollagen 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 IPN or semi-IPN network comprising a dermal filler comprises MA-HA or MA-PVA or MA-PEG or MA-OC to MA-rhCollagen 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 IPN or semi-IPN network comprising a dermal filler comprises MA-HA or MA-PVA or MA-PEG or MA-OC to MA-rhCollagen 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.

[0200] In some embodiments, an IPN or semi-IPN network including a dermal filler or a doubly cross-linked dermal filler includes a cell growth promoting scaffold.

[0201] In certain embodiments, dermal fillers for the purposes described herein include photocurable dermal fillers, wherein at least one component (such as, but not limited to, rhCollagen) includes a methacrylate-rhCollagen derivative or a thiol-rhCollagen derivative. In some embodiments, the curable dermal filler includes rhCollagen and a filler, such as hyaluronic acid (HA), polyvinyl alcohol (PVA), polyethylene glycol (PEG), oxidized cellulose (OC) or a derivative thereof or a combination thereof. In some embodiments, the photocurable dermal filler includes MA-rhCollagen and HA or a derivative thereof. In some embodiments, the photocurable dermal filler includes a rhCollagen derivative, such as, but not limited to, methacrylated rhCollagen or a thiol rhCollagen and / or a derivative of a filler, such as, but not limited to, methacrylated HA, PVA, PEG or OC, or thiolated HA, PVA, PEG or OC or a combination thereof.

[0202] In some embodiments, the photocurable dermal filler comprises a filler ratio such as, but not limited 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 of HA, PVA, PEG, or OC, or a derivative thereof to rhCollagen. In some embodiments, the photocurable dermal filler comprises a filler ratio such as, but not limited 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 of HA, PVA, PEG, or OC, or a derivative thereof to MA-rhCollagen. In some embodiments, the photocurable dermal filler comprises a filler such as, but not limited to, HA, PVA, PEG, or OC to thiol-rhCollagen 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 a MA-filler to MA-rhCollagen 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.

[0203] In some embodiments, the photocurable dermal filler comprises a ratio of HA to MA-rhCollagen 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 comprises a ratio of MA-HA to MA-rhCollagen 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 a ratio of PVA, PEG, or OC to MA-rhCollagen 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 a ratio of MA-PVA, MA-HA- or OC to MA-rhCollagen 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 comprises cross-linked HA or cross-linked MA-HA.

[0204] Throughout this application, various embodiments of dermal fillers and uses thereof may be presented in range format. It should be understood that the description in range format is for convenience and brevity only, and should not be construed as a fixed limitation on the scope of the invention. Therefore, the description of a range should be considered to have explicitly disclosed all possible sub-ranges as well as individual numerical values ​​within the range. For example, the description of a range (such as from 1:1 to 6:1) should be considered to have explicitly disclosed sub-ranges such as from 1.1:1, 1.2:1, 1.3:1 to 5.9:1, from 1:1.1 to 1:1.9, etc., as well as individual numbers and fractions thereof within the range, such as 1, 2, 3, 4, 5, and 6. This applies regardless of the width of the range.

[0205] Whenever a numerical range is indicated herein, it is intended to include any cited numeral (fractional or integer) within the indicated range. The phrases "ranging" or "ranges" between a first indicated numeral and a second indicated numeral and "ranging from a first indicated numeral to a second indicated numeral" are used interchangeably herein and are meant to include the first indicated numeral and the second indicated numeral and all fractions and integers therebetween.

[0206] For example, the present disclosure provides a dermal filler for use in the subepidermal tissue space, comprising cross-linkable plant-derived human collagen, alone or with a filler, such as hyaluronic acid (HA), polyvinyl alcohol (PVA), polyethylene glycol (PEG), oxidized cellulose (OC), or a combination thereof, which can be cross-linked to provide a dermal filler that forms a water-insoluble cross-linked polymer formulation in situ under visible light activation in the presence of a photoinitiator. In some embodiments, the collagen is methacrylated or thiolated.

[0207] In some embodiments, the dermal fillers provided for the uses described herein form an IPN or semi-IPN network. In some embodiments, the dermal fillers provided for the uses described herein form a dual cross-linked network.

[0208] In certain embodiments, the dual cross-linked dermal fillers provided for the uses described herein include rhCollagen cross-linked with a cross-linked filler such as cross-linked hyaluronic acid (HA), cross-linked polyvinyl alcohol (PVA), cross-linked polyethylene glycol (PEG), or cross-linked oxidized cellulose (OC), or a cross-linked derivative thereof, or a combination thereof. In certain embodiments, the dual cross-linked dermal fillers provided for the uses described herein include rhCollagen further cross-linked with a methacrylated or thiolated cross-linked filler such as HA, PVA, PEG, or OC.

[0209] In certain embodiments, the ratio of cross-linked filler to rhCollagen in a double cross-linked 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 MA-filler to rhCollagen in a double cross-linked 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 filler to MA-rhCollagen in a double cross-linked 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 MA-filler to MA-rhCollagen in the double cross-linked 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 rhCollagen in the double cross-linked 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 filler to thiolated rhCollagen in the double cross-linked 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 rhCollagen in the dual cross-linked 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.

[0210] In certain embodiments, in a double cross-linked dermal filler, the cross-linked HA or cross-linked MA-HA is further cross-linked to rhCollagen or methacrylated rhCollagen or thiol rhCollagen, thereby producing a double cross-linked dermal filler. In certain embodiments, in a double cross-linked dermal filler, the ratio of cross-linked HA to rhCollagen or methacrylated rhCollagen or thiol rhCollagen 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 cross-linked dermal filler, the ratio of cross-linked MA-HA to rhCollagen or methacrylated rhCollagen or thiol rhCollagen 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 rhCollagen or methacrylated rhCollagen or thiol rhCollagen in the dual cross-linked 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.

[0211] In certain embodiments, in a double cross-linked dermal filler, cross-linked PVA, PEG or OC or cross-linked MA-PVA, MA-PEG or MA-OC is further cross-linked to rhCollagen or methacrylated rhCollagen, thereby producing a double cross-linked dermal filler. In certain embodiments, in a double cross-linked dermal filler, the ratio of cross-linked PVA, PEG or OC to rhCollagen 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 cross-linked dermal filler, the ratio of cross-linked MA-PVA, MA-PEG or MA-OC to rhCollagen 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-rhCollagen in the double cross-linked 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 MArhCollagen or thiol rhCollagen in the double cross-linked 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.

[0212] In certain embodiments, in a double cross-linked dermal filler, the cross-linked thiol-PVA, thiol-PEG or thiol-OC is further cross-linked to rhCollagen or methacrylated rhCollagen, thereby producing a double cross-linked dermal filler. In certain embodiments, in a double cross-linked dermal filler, the ratio of cross-linked thiol-PVA, thiol-PEG or thiol-OC to rhCollagen 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 cross-linked dermal filler, the ratio of cross-linked thiol-PVA, thiol-PEG or thiol-OC to MArhCollagen or thiol rhCollagen 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.

[0213] In certain embodiments, any water-soluble coupling agent that can crosslink 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) etc. Carbodiimide coupling agents can promote the formation of ester or amide bonds and will not become a part of the connection. In other words, ester bonds or amide bonds can include atoms from a carboxylate group in hyaluronic acid or collagen, and atoms from another hydroxyl or amine group. However, other coupling agents that become a part of the crosslinking group can be used. The concentration of the coupling agent can vary. In certain embodiments, the coupling agent can exist at about 2mM to about 150mM, about 2mM to about 50mM, about 20mM to about 100mM or about 50mM. In certain embodiments, coupling agent is EDC, and it exists with the concentration of about 20mM to about 100mM, about 2mM to about 50mM or about 50mM.In certain embodiments, coupling agent is EDC, and it exists with the amount of 10 to 100 times of EDC equaling the quantity of the free amines in rhcollagen.In certain embodiments, coupling agent is EDC, and it exists with the amount of 50 times of EDC equaling the quantity of the free amines in rhcollagen.Increasing carbodiimide concentration to about 50mM may cause crosslinked macromolecular matrix to have higher hydrogel rigidity and / or less swelling.

[0214] Those skilled in the art will appreciate that a dermal filler comprising dual crosslinking (where the filler is crosslinked to itself and then crosslinked to rhCollagen) is different from a dermal filler comprising direct crosslinking of collagen and HA in a single reaction using a single type of crosslinker. The properties of such a dermal filler are different.

[0215] For example, the polymerizable solutions of the present invention can be used to block or fill various cavities and voids just below the surface of the skin. Thus, the present technology provides methods for tissue augmentation in a host such as a human patient, wherein the polymerizable solution of interest is introduced to the site of interest using methods known in the art, such as injecting the polymerizable solution at or into the tissue site in need of augmentation, and after application, the covered body surface is exposed to visible light to cause polymerization of the deposited polymerizable solution.

[0216] "Augmentation" means repairing, preventing or alleviating defects, especially those caused by the absence or deficiency of tissue, by providing, supplementing or replacing such tissue with a polymer or network of interest. Augmentation is also intended to encompass the supplementation of natural structures or features, i.e., adding structures to existing body parts, for example, to increase the size of said existing body parts, such as lips, nose, chest, ears, organ parts, chin, cheeks, etc. Thus, tissue augmentation may include: filling or reducing lines, folds, wrinkles, scars, minor facial hollows, cleft lip, superficial wrinkles, etc., such as in or on the face, neck, hands, feet, fingers and toes; correcting minor deformities due to aging or disease, including deformities of the hands, feet, fingers and toes; augmentation of the vocal cords or glottis to restore speech; dermal augmentation of sleep lines and expression lines; replacing dermis and subcutaneous tissue lost due to aging; lip augmentation; filling wrinkles and orbital grooves around the eyes; breast augmentation; chin augmentation; cheek and / or nose augmentation; filling indentations in the soft tissue, dermis or subcutaneous tissue due to excessive liposuction or other trauma; filling acne or traumatic scars and folds; filling the nasolabial line, nasal bridge line and jawline, etc.

[0217] In some embodiments, the polymerizable solutions of interest encompass polymerizable solutions having a viscosity suitable for ready extrusion through a delivery device such as a fine surgical needle (needle gauge of at least 27 gauge, at least 33 gauge or finer) at the temperature of use. Thus, an "injectable" solution is one whose texture and viscosity permit flow through a suitable delivery device such as a surgical needle, other surgical instrument, or other delivery device such as that used in endoscopic or percutaneous discectomy. Thus, the polymerizable solutions of interest can be injected through a suitable applicator known in the art such as a catheter, cannula, needle, syringe, tubular device, etc.

[0218] Once injected into the tissue space, typically after light initiation to trigger polymerization, the polymerizable solution can be manipulated, massaged, molded, or sculpted into the desired contour in the tissue space. In one embodiment, manipulation, massage, molding, or sculpting is performed during the gelation process. The polymerizable, polymerized, or partially polymerized solution can be shaped by external manipulation using, for example, a shaping device (such as a surgical pressure plate or other tool or instrument with a flat or curved surface, fingers, palms, knuckles, etc.).

[0219] Surprisingly, the genetically modified, cross-linkable, plant-derived human collagen of the present method provides for improved collagen-containing dermal fillers and improved skin augmentation methods by enabling the use of smaller gauge needles and reduced injection forces as well as its ability to fill smaller tissue spaces.

[0220] The "expression force" (Newtons, N) of an injection encompasses the force required to inject from a needle or cannula.

[0221] "Absolute viscosity" ("dynamic viscosity") is the resistance of a fluid to flow when a force is applied. It is proportional to the force-to-speed ratio. The Greek letter η (eta) represents absolute viscosity in calculations. It is usually measured in cP because many common fluids have viscosities between 0.5 cP and 1000 cP.

[0222] "Gel" is a semi-rigid slab or cylinder of an organic polymer used as a medium for separating macromolecules. Gels are essentially dilute cross-linked systems that do not exhibit fluidity when in a steady state. Gels are primarily liquids by weight, but due to the three-dimensional cross-linked network within the liquid, some of the properties of the gel are also the same as those of a solid, while still retaining certain properties of the liquid (such as deformability). It is the cross-linking within the fluid that gives the gel its structure (hardness) and contributes to adhesion (viscosity). Therefore, a gel can be viewed as a dispersion of liquid molecules in a solid, that is, liquid particles are dispersed in a solid medium. "Gel time" is the time it takes for a polymerizable solution to form a gel.

[0223] A "hydrogel" is a network of hydrophilic polymer chains, sometimes in the form of a colloidal gel, in which water is the dispersion medium. A hydrogel is a highly absorbent (e.g., can contain more than 90% water) polymer network and has a flexibility very similar to natural tissue due to its large water content.

[0224] A "polymer" is a large molecule made up of a series of repeating subunits. The basic repeating subunit is called a "monomer." As a whole, polymers are known for their tensile strength and elasticity.

[0225] 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 cause polymerization of the polymerizable solution. Examples of photoinitiators that can be used in the present method include, but are not limited to, lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), 1-[4-(2-hydroxyethoxy)phenyl]-2-methylpropan-1-one ( 2959), Eosin Y + triethanolamine or riboflavin.

[0226] Methacrylates are esters or salts derived from methacrylic acid. Methacrylates are common monomers in polymer plastics, forming acrylate polymers. Addition of methacrylate groups to collagen results in photocurable collagen methacrylate (rhCollagen-MA or MA-rhCollagen). Addition of methacrylate groups to hyaluronic acid (HA) results in photocurable hyaluronic acid-methacrylate (HAMA or MA-HA).

[0227] In some embodiments, the rhCollagen used in the dermal fillers described herein includes a combination of unmodified rhCollagen and MA-rhCollagen. In some embodiments, the ratio of unmodified rhCollagen to MA-rhCollagen 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-rhCollagen includes about 0-12 mg / ml. In some embodiments, the final concentration range of unmodified rhCollagen includes about 0-12 mg / ml. In some embodiments, the final concentration range of MA-rhCollagen includes about 0-12 mg / ml, and the final concentration range of unmodified rhCollagen includes about 0-12 mg / ml. In some embodiments, the final concentration range of MA-rhCollagen includes about 0 mg / ml-6 mg / ml. In some embodiments, the final concentration range of unmodified rhCollagen includes about 0 mg / ml-6 mg / ml. In some embodiments, the final concentration of MA-rhCollagen comprises about 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 mg / ml. In some embodiments, the final concentration of unmodified rhCollagen comprises about 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 mg / ml.

[0228] Thiols are organosulfur compounds containing carbon-bonded sulfhydryl (R-SH) groups (where R represents an alkyl group or other organic substituent). Thiolation of collagen can improve cohesive and mucoadhesive properties and affect swelling capacity.

[0229] Light is a form of electromagnetic radiation. "Visible light" is light with a wavelength in the range of 380-800nm, or at least 390-700nm. "Ultraviolet light" has a shorter wavelength, while "infrared" has a longer wavelength.

[0230] The irradiation device can be a light source suitable for activating the photoinitiator used, and it can activate the photoinitiator from outside the body. Although thermal initiators can be used (and therefore infrared sources can be used), and ultraviolet-activated initiators can be used (and therefore suitable ultraviolet sources can be used), the preferred light source is a white light source. Therefore, a suitable photoinitiator is used to adjust the maximum absorption of the initiator and the light source. As mentioned above, one such visible light source is a light emitting diode (LED). As long as gelation occurs in the body, at a site, below the surface of the skin, etc., other suitable light sources can be used, such as by applying electromagnetic radiation to the body, to a desired site, or from above the surface of the skin. Electromagnetic radiation is applied at a certain intensity, for a certain time, and for a certain duration to cause gelation. The light source can be located above the surface of the skin or directly on the surface of the skin, typically above the location of the molded or engraved polymerizable solution.

[0231] The monomer solution of some embodiments may contain any of a variety of other materials, such as inert materials known in the pharmaceutical field (such as preservatives, fillers, excipients or diluents), pharmacologically active molecules or agents (such as small molecules or organisms), cells, etc. Therefore, suitable inert agents or biologically active agents can be added to the monomer solution. In the latter case, the active agent can exert a pharmacological effect locally at or near the site of the polymer or network structure of interest, or can be released from the formed scaffold, matrix or network to move through adjacent tissue spaces, or can enter the circulatory system to reduce local effects.

[0232] As noted above, the polymerizable solution methods of interest may also be used in combination with other dermatological, plastic surgery, cosmetic, and other medical treatments.

[0233] In some embodiments, the polymerizable solution is mixed with a known filler to provide a composition that is moldable, contoured, has a long residence time, etc. Examples of fillers include, but are not limited to, hyaluronic acid (HA), polyvinyl alcohol (PVA), polyethylene glycol (PEG), oxidized cellulose (OC), or a modified derivative thereof, or a combination thereof. In some embodiments, a polymerizable solution in a semi-liquid phase is injected into the dermis independently, and a known filler that is also in a semi-liquid phase is injected as well, which together will provide a composition that is moldable, contoured, has a long residence time, etc. Examples of fillers that can be injected independently include, but are not limited to, hyaluronic acid (HA), polyvinyl alcohol (PVA), polyethylene glycol (PEG), oxidized cellulose (OC), or a modified derivative thereof, or a combination thereof. In some embodiments, a polymerizable solution in a semi-liquid phase is injected into the dermis as a mixture, which together will provide a composition that is moldable, contoured, has a long residence time, etc. Examples of fillers that can be mixed with rhCollagen for injection include, but are not limited to, hyaluronic acid (HA), polyvinyl alcohol (PVA), polyethylene glycol (PEG), oxidized cellulose (OC) or modified derivatives thereof, or combinations thereof.

[0234] In yet another aspect, disclosed herein is a method for inducing a cell growth-promoting scaffold in a subepidermal tissue space, the method comprising introducing a solution into the tissue space, the solution comprising: (a) plant-derived human collagen; and (b) at least one growth factor or a source thereof.

[0235] In one embodiment, the source of at least one growth factor comprises plasma or platelet-rich plasma.

[0236] In one embodiment, the cell growth promoting scaffold promotes healing or replacement due to degradation or damage of collagen-containing tissue. In one embodiment, the collagen-containing tissue is selected from the group consisting of tendon, ligament, skin, cornea, cartilage, blood vessel, intestine, intervertebral disc, muscle, bone or tooth. In a specific embodiment, the cell growth promoting scaffold promotes the healing of tendinitis.

[0237] In one embodiment, the plant-derived collagen comprises rhCollagen. In one embodiment, the plant-derived collagen is obtained from a genetically modified plant. In various embodiments, the genetically modified plant is a genetically modified plant 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.

[0238] In one 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.

[0239] In a specific embodiment, the plant-derived human collagen comprises: at least one modified human collagen α-1 chain as shown in SEQ ID NO:3 and expressed in a genetically modified plant; and at least one modified human collagen α-2 chain as shown in SEQ ID NO:6 and expressed in a genetically modified plant; and the genetically modified plant further expresses exogenous prolyl 4-hydroxylase (P4H).

[0240] In another specific embodiment, the method further comprises expressing an exogenous polypeptide selected from the group consisting of: lysyl hydroxylase (LH), proteinase N, and proteinase C.

[0241] In a specific embodiment, the human collagen α-1 chain is encoded by the sequence shown in SEQ ID NO: 1. In another specific embodiment, the human collagen α-2 chain is encoded by the sequence shown in SEQ ID NO:2.

[0242] In one embodiment, the exogenous P4H is mammalian P4H. In a specific embodiment, the exogenous P4H is human P4H.

[0243] In one embodiment, the method further comprises targeting human collagen alpha-1 to the vacuole of the plant or genetically modified plant and digesting with ficin. In one embodiment, the method further comprises targeting human collagen alpha-2 to the vacuole of the plant or genetically modified plant and digesting with ficin.

[0244] In a specific embodiment, the plant-derived human collagen is atelocollagen.

[0245] The skilled person will understand that in some embodiments, the term "dermal filler" encompasses solutions comprising plant-derived human collagen, such as type 1 recombinant human collagen (rhCollagen) or derivatives thereof. In some embodiments, the term "dermal filler" also encompasses solutions comprising plant-derived human collagen, such as type 1 recombinant human collagen (rhCollagen) or derivatives thereof, and fillers or derivatives thereof or cross-linked fillers or derivatives thereof, the fillers having the same meaning and qualities, wherein the dermal filler can be used to augment tissue structure, or can be used to reduce lines, folds, fine lines, wrinkles or scars, or any combination thereof.

[0246] The skilled artisan will appreciate that the dermal fillers described herein include different formulations such as, but not limited to:

[0247] ●rhCollagen or its MA or thiol derivatives;

[0248] ● IPN or semi-IPN or double cross-linked network, which includes rhCollagen or rhCollagen-MA or rhCollagen-thiol and filler or its derivative;

[0249] ● IPN or semi-IPN or double cross-linked network, which includes rhCollagen or rhCollagen-MA or rhCollagen-thiol and HA or MA-HA or thiol-HA;

[0250] ● IPN or semi-IPN or double cross-linked network, which includes rhCollagen or rhCollagen-MA or rhCollagen-thiol and PVA or MA-PVA or thiol-PVA;

[0251] ● IPN or semi-IPN or double cross-linked network, which includes rhCollagen or rhCollagen-MA or rhCollagen-thiol and PEG or MA-PEG or thiol-PEG;

[0252] ● IPN or semi-IPN or double cross-linked network, which includes rhCollagen or rhCollagen-MA or rhCollagen-thiol and OC or MA-OC or thiol-OC;

[0253] IPN or semi-IPN or double cross-linked network or cell growth promoting scaffold, which includes rhCollagen and autologous platelet-rich plasma (PRP) fraction containing high concentration of platelets in blood;

[0254] IPN or semi-IPN or double cross-linked network or cell growth promoting scaffold, each comprising rhCollagen and an autologous platelet-rich plasma (PRP) portion of blood containing a high concentration of platelets, wherein the platelets release various types of growth factors (GFs), including vascular endothelial growth factor (VEGF), transforming beta growth factor (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;

[0255] ● Dual cross-linked dermal fillers comprising rhCollagen or rhCollagen-MA or rhCollagen-thiol cross-linked with a cross-linked filler or derivative thereof;

[0256] ● Dual cross-linked dermal fillers comprising rhCollagen or rhCollagen-MA or rhCollagen-thiol cross-linked with cross-linked HA or cross-linked MA-HA or cross-linked thiol-HA;

[0257] ● Dual cross-linked dermal fillers comprising rhCollagen or rhCollagen-MA or rhCollagen-thiol cross-linked with cross-linked PVA or cross-linked MA-PVA or cross-linked thiol-PVA;

[0258] ● a dual cross-linked dermal filler comprising rhCollagen or rhCollagen-MA or rhCollagen-thiol cross-linked with cross-linked PEG or cross-linked MA-PEG or cross-linked thiol-PEG; or

[0259] - Double cross-linked dermal fillers comprising rhCollagen or rhCollagen-MA or rhCollagen-thiol cross-linked with cross-linked OC or cross-linked MA-OC or cross-linked thiol-OC.

[0260] One skilled in the art will appreciate that in some embodiments, the term "cell growth promoting scaffold" encompasses dermal fillers comprising collagen and an autologous platelet-rich plasma (PRP) portion of blood or its components. In some embodiments, PRP does not comprise "cells" but rather comprises (cell-derived) membrane vesicles containing growth factors and plasma components such as fibrinogen and prothrombin. In some embodiments, a "cell growth promoting scaffold" encompasses a dermal filler comprising collagen and an autologous platelet-rich plasma (PRP) portion of blood or its components, and at least an additional filler component.

[0261] In some embodiments, the cell growth promoting scaffold comprises a dermal filler, which may be an IPN network, a semi-IPN network or a double cross-linked dermal filler, wherein the double cross-linked dermal filler further comprises an autologous platelet-rich plasma (PRP) portion of the blood containing a high concentration of platelets, wherein the autologous PRP portion of the blood contains a high concentration of platelets, wherein the platelets release various types of growth factors (GFs), including 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) or hepatocyte growth factor (HGF) or a combination thereof. In some embodiments, the cell growth promoting scaffold comprises a dermal filler comprising an IPN network, a semi-IPN network, or a double cross-linked dermal filler, the double cross-linked dermal filler further comprising at least one growth factor comprising vascular endothelial growth factor (VEGF), transforming beta growth factor (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 growth promoting scaffold comprises a dermal filler comprising an IPN network, a semi-IPN network, or a double cross-linked dermal filler, the double cross-linked dermal filler further comprising a subset or portion of a PRP component.

[0262] In some embodiments, the dermal fillers described herein include a polymerizable solution. In some embodiments, the dermal fillers described herein include a non-polymerizable solution. In some embodiments, polymerization of the dermal filler solution occurs in vivo. In some embodiments, the components of the polymerizable dermal filler solution are injected together and then polymerized to form a solidified dermal filler. In some embodiments, the components of the polymerizable dermal filler solution are injected separately and then polymerized to form a solidified dermal filler. In some embodiments, examples of unique methods of independently injecting dermal filler components may include injecting a filler into the dermis of the skin, such as but not limited to HA or a derivative thereof, and injecting methacrylated or thiol-rhCollagen into the dermis of the skin in the vicinity of the first injection, respectively, wherein the components are in a semi-liquid phase and then cross-linked in situ. In some embodiments, the method allows for easier injection and in situ culturing before the dermal filler components are solidified together by photopolymerization.

[0263] In some embodiments, the dermal fillers provided herein are used in a method of soft tissue augmentation. In some embodiments, the dermal fillers provided herein enhance cell proliferation. In some embodiments, the dermal fillers provided and used in the method of soft tissue augmentation degrade over time. In some embodiments, the dermal fillers provided herein are used in a method of soft tissue augmentation, wherein the dermal filler fills the tissue space below the epidermis. In some embodiments, the dermal fillers provided herein are used in a method of soft tissue augmentation, wherein the use reduces lines, folds, fine lines, wrinkles or scars.

[0264] In one embodiment, a solution comprising plant-derived human collagen has a reduced viscosity at room temperature compared to a similar solution comprising tissue-extracted human or animal-derived collagen at the same concentration and formulation. In another embodiment, a solution comprising plant-derived human collagen has a reduced viscosity at 37° C. compared to a similar solution comprising tissue-extracted human or animal-derived collagen at the same concentration and formulation. In yet another embodiment, a solution comprising plant-derived human collagen is introduced into a tissue space at room temperature with reduced force compared to a similar solution comprising tissue-extracted human or animal-derived collagen at the same concentration and formulation. In still another embodiment, a solution comprising plant-derived human collagen is introduced into a tissue space at 37° C. with reduced force compared to a similar solution comprising tissue-extracted human or animal-derived collagen at the same concentration and formulation. In a specific embodiment, a solution comprising plant-derived human collagen has increased scaffold formation or promotes increased cell growth compared to a similar solution comprising tissue-extracted human or animal-derived collagen at the same concentration and formulation.

[0265] In yet another aspect, disclosed herein is a method of injecting a solution into a subepidermal tissue space to induce a cell growth promoting scaffold, the solution comprising plant-derived human collagen and at least one growth factor or a source thereof to promote healing or replacement due to degradation or damage of collagen-containing tissue. In a specific embodiment, the source of at least one growth factor comprises plasma or platelet-rich plasma.

[0266] In an embodiment, the collagen-containing tissue is selected from the group consisting of tendon, ligament, skin, cornea, cartilage, blood vessel, intestine, intervertebral disc, muscle, bone or tooth. In another embodiment, the cell growth promoting scaffold promotes the healing of tendinitis. In an embodiment, the collagen-containing tissue is skin.

[0267] In some embodiments, a genetically modified plant is provided that is capable of expressing at least one type of collagen alpha chain and accumulating it in a subcellular compartment that lacks endogenous P4H activity.

[0268] As used herein, the phrase "genetically modified plant" refers to any lower (e.g., moss) or higher (vascular) plant or tissue or isolated cells thereof (e.g., cell suspensions) that have been stably or transiently transformed with an exogenous polynucleotide sequence. Examples of plants include tobacco, corn, alfalfa, rice, potato, soybean, tomato, wheat, barley, canola, cotton, carrot, and lower plants (e.g., mosses).

[0269] As used herein, the phrase "collagen chain" refers to a collagen subunit, such as an α1 or 2 chain of a collagen fiber (preferably, type I fiber). As used herein, the phrase "collagen" refers to an assembled collagen trimer, which, in the case of type I collagen, comprises two α1 chains and one α2 chain. Collagen fibers are collagens lacking the terminal propeptides C and N.

[0270] As used herein, the phrase "subcellular compartment lacking endogenous P4H activity" refers to any compartment of a cell that does not contain a plant P4H or an enzyme having plant-like P4H activity. Examples of such subcellular compartments include vacuoles, apoplasts, and cytoplasm, as well as organelles (such as chloroplasts, mitochondria, etc.).

[0271] The genetically modified plants of the present invention may express any type of collagen chain. Examples include collagens that form fibrils (types I, II, III, V, and XI), collagens that form networks (types IV, VIII, and X), collagens that are associated with the surface of fibrils (types IX, XII, and XIV), collagens that appear as transmembrane proteins (types XIII and XVII), or collagens that form 11-nm periodic beaded filaments (type VI).

[0272] In one embodiment, the expressed collagen chains are α1 and / or 2 chains of type I collagen. The expressed collagen α chains can be encoded by any polynucleotide sequence derived from any mammal. In a specific embodiment, the sequence encoding the collagen α chain is human and is shown in SEQ ID NO: 1 and 4.

[0273] Typically, alpha collagen chains expressed in plants may or may not include their terminal propeptides (ie, propeptide C and propeptide N).

[0274] Processing of procollagen by plant proteolytic activity differs from normal processing in humans, and although the cleavage site is unknown, propeptide C may be removed by plant proteolytic activity. Cleavage of the C-propeptide may occur on the procollagen peptide prior to assembly of the trimer (association of three C-propeptides is essential to initiate assembly of the trimer).

[0275] Cleavage of the N-propeptide by plant proteolytic activity occurs in mature plants but not in young plants. This cleavage removes 2 amino acids (2 of 17) from the N-terminal peptide.

[0276] C-propeptides (and to a lesser extent, N-propeptides) keep procollagen soluble during its passage through animal cells (Bulleid et al., 2000) and are expected to have similar effects in plant cells. 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 the spontaneous self-assembly of collagen molecules into fibrils. Removal of propeptides by procollagen N-proteinases and C-proteinases reduces the solubility of procollagen by more than 10,000 times and is necessary and sufficient to initiate the self-assembly of collagen into fibers. Crucial to this assembly process is a short non-triple helical peptide called a telopeptide at the end of the triple helical domain, which ensures that the collagen molecules are properly aligned within the fibril structure and reduces the critical concentration for self-assembly. Pepsin can cleave propeptides during the production of collagen. However, pepsin destroys the telopeptide, so pepsin-extracted collagen cannot form an ordered fibril structure.

[0277] It has been shown that protein disulfide isomerase (PDI), which forms the beta subunit of human P4H, binds to the C-propeptide prior to trimer assembly, thereby also acting as a chaperone during chain assembly.

[0278] The use of human procollagen I N-proteinase and procollagen C-proteinase expressed in different plants can produce collagen that is more similar to native human collagen and can form an ordered fibril structure.

[0279] In the case where the expressed collagen chains contain N or C propeptides or both, the genetically modified plants of the present invention may also express the respective proteases (i.e., C or N or both). The polynucleotide sequences encoding such proteases are exemplified by SEQ ID NO: 18 (Protease C) and 20 (Protease N). Such proteases may be expressed so that they accumulate in subcellular compartments as the collagen chains.

[0280] Accumulation of expressed collagen chains in subcellular compartments lacking endogenous P4H activity can be achieved by any of several methods.

[0281] For example, the expressed collagen chain may include a signal sequence for targeting the expressed protein to a subcellular compartment such as the apoplast or an organelle (e.g., chloroplast). Examples of suitable signal sequences include the chloroplast transit peptide (contained in Swiss-Prot entry P07689, amino acids 1-57) and the mitochondrial transit peptide (contained in Swiss-Prot entry P46643, amino acids 1-28). The subsequent Examples section provides additional examples of suitable signal sequences, as well as guidance for employing such signal sequences in the expression of collagen chains in plant cells.

[0282] Alternatively, the sequence of the collagen chains can be modified in a way that alters the cellular localization of the collagen when expressed in plants.

[0283] As described above, the ER of plants contains P4H that cannot properly hydroxylate collagen chains. Collagen alpha chains naturally contain an ER targeting sequence that directs the expressed collagen into the ER where it is post-translationally modified (including incorrect hydroxylations). Therefore, removal of the ER targeting sequence will result in cytoplasmic accumulation of collagen chains that lack post-translational modifications (including any hydroxylations).

[0284] Example 1 of the Examples section that follows describes the generation of collagen sequences lacking ER sequences.

[0285] Still alternatively, the collagen chains may be expressed and accumulated in organelles containing DNA, such as chloroplasts or mitochondria. Further description of chloroplast expression is provided below.

[0286] As described above, the assembly of stable type I collagen requires hydroxylation of the α chain. Since the α chain expressed by the genetically modified plants of the present invention accumulates in compartments lacking endogenous P4H activity, it is necessary to isolate this chain from the plant, plant tissue or cell and perform hydroxylation in vitro. This hydroxylation can be achieved by the method described by Turpeenniemi-Hujanen and Myllyla ("Simultaneous hydroxylation of proline and lysine residues in collagen in vitro using purified enzymes" Biochim Biophys Acta. 1984 July, 16; 800 (1): 59-65).

[0287] Although such in vitro hydroxylation can result in correctly hydroxylated collagen chains, it can be difficult and expensive to achieve.

[0288] To overcome the limitations of in vitro hydroxylation, the genetically modified plants of the present invention preferably also co-express P4H, which is able to correctly hydroxylate one or more collagen α chains [i.e., only hydroxylates the proline (Y) position of the Gly-XY triplet]. P4H is an enzyme composed of two subunits (α and β). Both are required to form an active enzyme, and the β subunit also possesses a chaperone function.

[0289] The P4H expressed by the genetically modified plant of the present invention is preferably human P4H encoded by, for example, SEQ ID NOs: 12 and 14. In addition, P4H mutants or P4H homologs that exhibit enhanced substrate specificity may also be used.

[0290] A suitable P4H homologue is exemplified by the Arabidopsis oxidoreductase identified by NCBI accession number NP_179363. Pairwise alignment of this protein sequence with the human P4H alpha subunit performed by the present inventors revealed the highest homology between the functional domains of any known P4H homologues in plants.

[0291] Since P4H needs to be co-accumulated with the expressed collagen chains, its coding sequence is preferably modified accordingly (addition of signal sequences, deletions that may prevent ER targeting, etc.).

[0292] In mammalian cells, collagen is also modified by lysyl hydroxylases, galactosyltransferases, and glucosyltransferases. These enzymes sequentially modify lysyl residues in specific positions to hydroxylysyl, galactosylhydroxylysyl, and glucosylgalactosylhydroxylysyl residues. A single human enzyme, lysyl hydroxylase 3 (LH3), can catalyze all three sequential steps in the formation of hydroxylysine-linked carbohydrates.

[0293] Therefore, the genetically modified plants of the present invention preferably also express mammalian LH3. LH3 coding sequences such as the sequence shown in SEQ ID NO: 22 can be used for this purpose.

[0294] The above-mentioned one or more collagen chains and modifying enzymes can be expressed from a stably integrated or transiently expressed nucleic acid construct comprising a polynucleotide sequence encoding an alpha chain and / or modifying enzyme (e.g., P4H and LH3) under the transcriptional control of a plant functional promoter. Such nucleic acid constructs (also referred to herein as expression constructs) can be configured to be expressed in whole plants, defined plant tissues, or defined plant cells or at defined developmental stages of plants. Such constructs can also comprise selection markers (e.g., antibiotic resistance), enhancer elements, and origins of replication for bacterial replication.

[0295] It should be understood that a construct comprising two expressible inserts (e.g., two α chain types, or an α chain and P4H) preferably comprises a separate promoter for each insert, or alternatively, such a construct can express a single transcript chimera comprising both inserts from a single promoter. In this case, the chimeric transcript comprises an IRES sequence between the two inserts, from which the downstream insert can be translated.

[0296] The constructs of the present invention may utilize a wide variety of plant functional expression promoters and enhancers, which may be tissue-specific, developmentally-specific, constitutive or inducible, some examples of which are provided below.

[0297] As used herein in the specification and in the claims section that follows, the phrase "plant promoter" or "promoter" encompasses a promoter that can direct gene expression in plant cells (including organelles containing DNA). Such promoters can be derived from plants, bacteria, viruses, fungi, or animal sources. Such promoters can 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 a specific plant tissue), inducible (i.e., capable of directing gene expression under stimulation), or chimeric (i.e., formed from portions of at least two different promoters).

[0298] Thus, the plant promoter employed may be a constitutive promoter, a tissue-specific promoter, an inducible promoter or a chimeric promoter.

[0299] Examples of constitutive plant promoters include, but are not limited to, the CaMV35S and CaMV19S promoters, the FMV34S promoter, the sugarcane bacilliform adenovirus promoter, the CsVMV promoter, the Arabidopsis ACT2 / ACT8 actin promoter, the Arabidopsis ubiquitin UBQI promoter, the barley thionin BTH6 promoter, and the rice actin promoter.

[0300] Examples of tissue-specific promoters include, but are not limited to, bean phaseolin storage protein promoter, DLEC promoter, PHS promoter, zein storage protein promoter, mucin gamma promoter from soybean, AT2S1 gene promoter, ACT11 actin promoter from Arabidopsis thaliana, napA promoter from Brassica napus, and potato glycoprotein gene promoter.

[0301] Inducible promoters are promoters induced by specific stimuli (such as stress conditions, including, for example, light, temperature, chemicals, drought, high salinity, osmotic shock, oxidant conditions or pathogenic conditions), and include but are not limited to the light-inducible promoter derived from the pea rbcS gene, the promoter from the alfalfa rbcS gene, the promoters DRE, MYC and MYB active under drought; the promoters INT, INPS, prxEa, Ha hsp17.7G4 and RD21 active under high salinity and osmotic stress, and the promoters hsr203J and str246C active under pathogenic stress.

[0302] Preferably, the promoter used in the present invention is a strong constitutive promoter, thereby achieving overexpression of the construct insert after plant transformation.

[0303] It should be understood that any construct type used in the present invention can be co-transformed into the same plant using the same or different selection markers in each construct type. Alternatively, a first construct type can be introduced into a first plant, and a second construct type can be introduced into a second isogenic plant, and the resulting transgenic plants can be subsequently hybridized and offspring selected for double transformants. Further selfing of this offspring can be used to produce strains that are homozygous for the two constructs.

[0304] There are a variety of methods for introducing nucleic acid constructs into monocotyledonous and dicotyledonous plants (Potrykus, I., "Annu. Rev. Plant. Physiol., Plant. Mol. Biol." (1991) 42: 205-225; Shimamoto et al., "Nature" (1989) 338: 274-276). Such methods rely on the stable integration of nucleic acid constructs or parts thereof into the plant genome, or rely on transient expression of nucleic acid constructs, in which case these sequences are not inherited by the progeny of the plant.

[0305] Additionally, there are several methods by which nucleic acid constructs can be introduced directly into the DNA of DNA-containing organelles such as chloroplasts.

[0306] There are two principle methods for achieving stable genomic integration of exogenous sequences, such as those comprised in the nucleic acid constructs of the present invention, into the plant genome:

[0307] (i) Agrobacterium-mediated gene transfer: Klee et al. (1987) Annu. Rev. Plant Physiol. 38:467-486; Klee and Rogers, Cell Culture and Somatic Cell Genetics of Plants, Vol. 6, Molecular Biology of Plant Nuclear Genes, eds. Schell, J. and Vasil, L. K., Academic Publishers, San Diego, CA (1989) pp. 2-25; Gatenby, in Plant Biotechnology, eds. Kung, S. and Arntzen, C. J., Butterworth Publishers, Boston, MA (1989) pp. 93-112.

[0308] (ii) Direct DNA uptake: Paszkowski et al., Cell Culture and Somatic Cell Genetics of Plants, Vol. 6, Molecular Biology of Plant Nuclear Genes, eds. Schell, J. and Vasil, LK, Academic Press, San Diego, CA (1989) pp. 52-68; including methods for direct DNA uptake into protoplasts, Toriyama, K. et al. (1988) Bio / Technology 6: 1072-1074. DNA uptake induced by brief electric shock of plant cells: Zhang et al. Plant Cell Rep. (1988) 7: 379-384. Fromm et al. Nature (1986) 319: 791-793. DNA is introduced into plant cells or tissues by particle bombardment, Klein et al. Bio / Technology (1988) 6:559-563; McCabe et al. Bio / Technology (1988) 6:923-926; Sanford, Physiol. Plant. (1990) 79:206-209; by using a micropipette system: Neuhaus et al., Theor. Appl. Genet. (1987) 75:30-36; Neuhaus and Spangenberg, Plant Physiology (1990) 79:213-217; or by direct incubation of DNA with germinating pollen, DeWet et al. Experimental Manipulation of Ovule Tissue Tissue, eds. Chapman, GP and Mantell, SH and Daniels, W. Longman, London (1985) pp. 197-209; and Ohta, PNAS (1986) 83:715-719.

[0309] Agrobacterium system comprises the use of plasmid vector, and the plasmid vector contains the DNA segment of the limit that is integrated into the plant genomic DNA.The method of inoculating plant tissue varies according to plant species and Agrobacterium delivery system.A widely used method is leaf disc procedure, and the procedure can be carried out with any tissue explant, thereby providing a good source for starting the differentiation of whole plant.Horsch et al. Plant Molecular Biology Manual A5, Kluwer Academic Publishers, Dordrecht (1988) pp. 1-9.A kind of complementary method is to combine the Agrobacterium delivery system with vacuum infiltration.Agrobacterium system is particularly feasible in creating transgenic dicotyledonous plants.

[0310] There are a variety of methods for transferring DNA directly into plant cells. In electroporation, protoplasts are briefly exposed to a strong electric field. In microinjection, DNA is mechanically injected directly into cells using a very small micropipette. In microparticle bombardment, DNA is adsorbed onto microparticles (such as magnesium sulfate crystals, tungsten particles, or gold particles), and the microparticles are physically accelerated into cells or plant tissues.

[0311] Plant propagation is performed after transformation. The most common method of plant propagation is through seeds. However, the disadvantage of regeneration through seed propagation is that the crop lacks uniformity due to heterozygosity, because seeds are produced by plants according to genetic variation controlled by Mendel's rules. Basically, each seed is genetically different and each seed has its own special traits. Therefore, it is preferred to produce transformed plants so that the regenerated plants have the same traits and characteristics as the parent transgenic plant. Therefore, it is preferred to regenerate transformed plants by micropropagation, which can provide rapid and consistent reproduction of transformed plants.

[0312] Transient expression methods that can be used to transiently express the isolated nucleic acid contained in the nucleic acid construct of the present invention include, but are not limited to, microinjection and bombardment as described above, but are performed under conditions that are conducive to transient expression and viral-mediated expression, wherein a packaged or unpackaged recombinant viral vector containing the nucleic acid construct is used to infect plant tissues or cells, such that the reproductive recombinant virus established therein expresses a non-viral nucleic acid sequence.

[0313] Viruses that have been shown to be useful for transformation of plant hosts include CaMV, TMV, and BV. Plant transformation using plant viruses is described in the following literature: U.S. Pat. No. 4,855,237 (BGV), EP-A 67,553 (TMV), Japanese Published Application 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 expressing foreign DNA in many hosts (including plants) are described in WO 87 / 06261.

[0314] The above references and the following documents demonstrate the construction of plant RNA viruses for introducing and expressing non-viral exogenous nucleic acid sequences in plants: 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.

[0315] When the virus is a DNA virus, the virus itself can be constructed. Alternatively, the virus can be cloned into a bacterial plasmid first to easily construct a desired viral vector with exogenous DNA. The virus can then be excised from the plasmid. If the virus is a DNA virus, the bacterial origin of replication can be attached to the viral DNA and then replicated by the bacteria. The transcription and translation of the DNA will produce coat proteins that will encapsulate the viral DNA. If the virus is an RNA virus, the virus is usually cloned as cDNA and inserted into a plasmid. The plasmid is then used to construct all constructs. RNA viruses are then produced by transcribing the viral sequence of the plasmid and translating the viral gene to produce one or more coat proteins that encapsulate the viral RNA.

[0316] The above references as well as US Pat. No. 5,316,931 demonstrate the construction of plant RNA viruses for the introduction and expression of non-viral exogenous nucleic acid sequences, such as those contained in the constructs of the present invention, in plants.

[0317] In one embodiment, a plant viral nucleic acid is provided, wherein the natural coat protein coding sequence has been deleted from the viral nucleic acid, the non-natural plant viral coat protein coding sequence and the non-natural promoter, preferably the subgenomic promoter of the non-natural coat protein coding sequence has been inserted, and the subgenomic promoter can express, package the recombinant plant viral nucleic acid in the plant host and ensure that the host is infected by the recombinant plant viral nucleic acid. Alternatively, the coat protein gene can be inactivated by inserting the non-natural nucleic acid sequence in the coat protein gene, thereby producing protein. The recombinant plant viral nucleic acid can contain one or more additional non-natural subgenomic promoters. Each non-natural subgenomic promoter can transcribe or express adjacent genes or nucleic acid sequences in the plant host, and cannot recombine with each other or with the natural subgenomic promoter. If more than one nucleic acid sequence is included, a non-natural (exogenous) nucleic acid sequence can be inserted near the natural plant viral subgenomic promoter or the natural and non-natural plant viral subgenomic promoter. The non-natural nucleic acid sequence is transcribed or expressed in the host plant under the control of the subgenomic promoter to produce the desired product.

[0318] In a second embodiment, a recombinant plant viral nucleic acid is provided as in the first embodiment, except that the native coat protein coding sequence is placed adjacent to one of the non-native coat protein subgenomic promoters rather than adjacent to the non-native coat protein coding sequence.

[0319] In a third embodiment, a recombinant plant viral nucleic acid is provided in which a native coat protein gene is adjacent to its subgenomic promoter, and one or more non-native subgenomic promoters have been inserted into the viral nucleic acid. The inserted non-native subgenomic promoters are capable of transcribing or expressing adjacent genes in a plant host and are not capable of recombining with each other or with a native subgenomic promoter. A non-native nucleic acid sequence can be inserted near a non-native subgenomic plant viral promoter such that the sequence is transcribed or expressed in a host plant under the control of the subgenomic promoter to produce a desired product.

[0320] In a fourth embodiment, a recombinant plant viral nucleic acid is provided as in the third embodiment, except that the native coat protein coding sequence is replaced by a non-native coat protein coding sequence.

[0321] Viral vectors are encapsidated with coat proteins encoded by recombinant plant viral nucleic acids to produce recombinant plant viruses. Recombinant plant viral nucleic acids or recombinant plant viruses are used to infect appropriate host plants. Recombinant plant viral nucleic acids are capable of replicating in the host, spreading systemically in the host, and being able to transcribe or express one or more exogenous genes (isolated nucleic acids) in the host to produce desired proteins.

[0322] The technology of introducing exogenous nucleic acid sequences into chloroplast genomes is known. This technology involves the following procedures. First, plant cells are chemically treated to reduce the number of chloroplasts per cell to about one. Then, exogenous nucleic acids are introduced into cells by particle bombardment, with the purpose of introducing at least one exogenous nucleic acid molecule into chloroplasts. Exogenous nucleic acids are selected so that they can be integrated into the genome of chloroplasts by homologous recombination, which is easily affected by enzymes inherent to chloroplasts. For this reason, in addition to the gene of interest, the exogenous nucleic acid also includes at least one nucleic acid fragment derived from the chloroplast genome. In addition, the exogenous nucleic acid includes a selectable marker, which determines by a sequential selection procedure that all or substantially all copies of the chloroplast genome after this selection will include exogenous nucleic acids. More details related to this technology can be found in U.S. Patents Nos. 4,945,050 and 5,693,507, which are incorporated herein by reference. Therefore, polypeptides can be produced by the protein expression system of chloroplasts and integrated into the inner membrane of chloroplasts.

[0323] The above-described transformation methods can be used to produce collagen chains and / or modifying enzymes and assembled collagen (with or without propeptides) in any species of plant, or plant tissue or isolated plant cells derived therefrom.

[0324] Preferred plants are plants that can accumulate a large amount of collagen chains, collagen and / or processing enzymes as described herein. Such plants can also be selected according to the resistance of plants to stress conditions and the ease of extracting the collagen of the expression components or assembly. Examples of preferred plants include tobacco, corn, alfalfa, rice, potato, soybean, tomato, wheat, barley, canola and cotton.

[0325] Collagen fibers are widely used in the food and cosmetic industries. Therefore, although collagen fiber components (α chains) and plant-expressed modifying enzymes can be used for industrial synthesis of collagen, the production of collagen is preferably accomplished in plants due to its simplicity and cost-effectiveness.

[0326] Several methods can be used to produce type I collagen in plants. For example, collagen α1 chain can be isolated from plants expressing collagen α1 and P4H (and optionally LH3) and mixed with collagen α2 chain isolated from plants expressing collagen α2 and P4H (and optionally LH3 and proteinase C and / or N). Since collagen α1 chain self-assembles into a triple helix, it may be necessary to denature this homotrimer before mixing and renaturing with collagen α2 chain.

[0327] Preferably, a first plant expressing collagen α1 and P4H (and optionally LH3 and proteinase C and / or N) can be crossed with a second (and preferably, isogenic) plant expressing collagen α2, or alternatively, a first plant expressing both α chains can be crossed with a second plant expressing P4H and optionally LH3 and proteinase C and / or N.

[0328] It should be noted that although the above-described plant breeding methods utilize two individually transformed plants, methods using three or more individually transformed plants, each expressing one or both components, may also be utilized.

[0329] Various plant breeding techniques will be apparent to those of ordinary skill in the art, and thus further description of such techniques is not provided herein.

[0330] Although plant breeding methods are preferred, it should be noted that a single plant expressing collagen alpha 1 and 2, P4H and LH3 (and optionally proteinase C and / or N) can be produced by several transformation events, each of which is designed to introduce one or more expressible components into the cell. In this case, specific selection markers can be used to verify the stability of each transformation event.

[0331] In any case, the transformation and plant breeding methods can be used to produce any plant expressing any number of components. Currently preferred are plants expressing collagen alpha 1 and 2 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 exhibits stability at temperatures up to 42°C.

[0332] By verifying the presence of exogenous mRNA and / or polypeptides using nucleic acid or protein probes (e.g., antibodies), progeny produced by breeding or, alternatively, by multiple transformations of plants can be selected. The latter approach is preferred because it enables the localization of expressed polypeptide components (e.g., by probing fractionated plant extracts), thereby also verifying the potential for correct processing and assembly. Examples of suitable probes are provided in the Examples section below.

[0333] Once collagen-expressing progeny are identified, such plants are further cultured under conditions that maximize expression of the collagen chains as well as the modifying enzymes.

[0334] Since the accumulation of free proline can promote the overproduction of various proline-rich proteins including collagen chains expressed by the genetically modified plants of the present invention, preferred culture conditions are those that increase the accumulation of free proline in the cultivated plants.

[0335] Free proline accumulates in many plants in response to a wide range of environmental stresses, including water shortage, salinization, low temperature, high temperature, pathogen infection, heavy metal toxicity, anaerobic bacteria, nutrient deficiency, atmospheric pollution and UV irradiation (Hare and Cress, 1997).

[0336] 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.

[0337] Thus, progeny expressing collagen can be grown under different stress conditions (e.g., different NaCl concentrations ranging from 50 mM to 250 mM). To further enhance collagen production, the effects of various stress conditions on collagen expression will be studied and optimized with respect to plant viability, biomass, and collagen accumulation.

[0338] The plant tissue / cells are preferably harvested at maturity and the collagen fibers isolated using well known prior art extraction methods, one such method being described in detail below.

[0339] Leaves of transgenic plants were ground into powder under liquid nitrogen and the homogenate was extracted in 0.5M acetic acid containing 0.2M NaCl for 60 hours at 4°C. Insoluble material was removed by centrifugation. The supernatant containing recombinant collagen was salt graded in 0.4M and 0.7M NaCl. The 0.7M NaCl precipitate containing recombinant heterotrimeric collagen was dissolved in 0.1M acetic acid and dialyzed and then stored at -20°C (following Ruggiero et al., 2000).

[0340] In one embodiment, disclosed herein is a method of processing procollagen to produce homogenous, soluble, fibrillating atelocollagen.

[0341] In some embodiments, as shown in the analysis of protein hydrolysis results by SDS PAGE herein, certain plant-derived proteases (e.g., papain) are unable to cleave the propeptide portion from soluble procollagen without proteolytic cleavage within the helical region (even though they are able to remove the telopeptide from animal-derived terminal collagen), while other proteases (e.g., esperase, savinase) are unable to effectively cleave the propeptide region from soluble procollagen, thereby hindering fibril formation. Through careful experiments, the inventors have found that only specific plant-derived proteases (e.g., ficin) and bacterial-derived proteases (e.g., neutral enzymes and subtilisin) can be used to correctly cleave the propeptide portion (including the telopeptide) from soluble procollagen without digesting the helical region of non-animal procollagen to produce a homogenous preparation of soluble atelocollagen ( Fig.13 , 15, 17, 19 and 20). In addition, the present inventors have shown that recombinant trypsin can also correctly cleave ( Fig.26 The inventors have further shown that cleavage with ficin allows the resulting atelocollagen to retain its fibril-forming ability (Table 5 of the Examples section below).

[0342] Therefore, according to one aspect, a method for producing atelocollagen is provided. The method comprises contacting human recombinant telopeptide-containing collagen with a protease selected from the group consisting of: neutral enzyme, subtilisin, recombinant trypsin, recombinant pepsin and ficin, wherein the human recombinant telopeptide-containing collagen is expressed in non-animal cells, thereby producing atelocollagen.

[0343] As used herein, the phrase "telopeptide-containing collagen" refers to a soluble collagen molecule that includes a telopeptide that is longer than the telopeptide residue included in atelocollagen. Thus, the telopeptide-containing collagen can be a procollagen that includes a full-length propeptide. Alternatively, the telopeptide-containing collagen can be a procollagen molecule that includes a partially digested propeptide. Still alternatively, the telopeptide-containing collagen can be a telopeptide.

[0344] As used herein, the term "procollagen" refers to a collagen molecule (eg, human) that includes an N-terminal propeptide, a C-terminal propeptide, or both. Exemplary human procollagen amino acid sequences are shown in SEQ ID NOs: 30, 31, 36, and 37.

[0345] As used herein, the term "telopeptide" refers to a collagen molecule that lacks the N-terminal and C-terminal propeptides that are usually included in procollagen but still contains a telopeptide. As described in the background section above, the telopeptide of fibrillar collagen is the residue of the N-terminal propeptide and the C-terminal propeptide after digestion with a natural N / C protease.

[0346] Recombinant human atelocollagen can be produced in cells that have been transformed to express both exogenous human procollagen and the corresponding proteases (i.e., C or N or both). Polynucleotide sequences encoding such proteases are exemplified by SEQ ID NO: 39 (Protease C) and 40 (Protease N). Such proteases can be expressed so that they accumulate in subcellular compartments like collagen chains, as further described below.

[0347] As used herein, the term "atelocollagen" refers to a collagen molecule that lacks both the N-terminal and C-terminal propeptides typically included in procollagen and at least a portion of their telopeptides, but contains a sufficient portion of their telopeptides to enable the atelocollagen to form fibrils under appropriate conditions.

[0348] Any type of atelocollagen can be produced according to the methods disclosed herein. Examples include collagens that form fibrils (types I, II, III, V, and XI), collagens that form networks (types IV, VIII, and X), collagens associated with fibril surfaces (types IX, XII, and XIV), collagens that appear as transmembrane proteins (types XIII and XVII), or collagens that form 11-nm periodic beaded filaments (type VI). According to one embodiment, the atelocollagen includes α-1 and / or α-2 chains of type I collagen.

[0349] It is understood that in some embodiments, disclosed herein are genetically modified forms of collagen / atelocollagen - eg, collagen that is resistant to collagenase, etc.

[0350] Recombinant human procollagen or telomere can be expressed in any non-animal cell, including but not limited to plant cells and other eukaryotic cells such as yeast and fungi.

[0351] Plants that can produce (i.e., express) human procollagen or terminal collagen can be lower (e.g., mosses and algae) or higher (vascular) plant species, including tissues or their separated cells and extracts (e.g., cell suspensions). Preferred plants are plants that can accumulate large amounts of collagen chains, collagens, and / or processing enzymes as described below. Such plants can also be selected based on the resistance of the plant to stress conditions and the ease of extracting expressed components or assembled collagen. Examples of plants that can express human procollagen include, but are not limited to, tobacco, corn, alfalfa, rice, potato, soybean, tomato, wheat, barley, canola, carrots, lettuce, and cotton.

[0352] Production of recombinant human procollagen is typically achieved by stable or transient transformation with an exogenous polynucleotide sequence encoding human procollagen.

[0353] Exemplary polynucleotide sequences encoding human procollagen are shown in SEQ ID NOs: 32, 33, 41 and 42.

[0354] As described above, production of human atelocollagen is typically achieved by stable or transient transformation with an exogenous polynucleotide sequence encoding human procollagen and at least one exogenous polynucleotide sequence encoding an associated protease.

[0355] The stability of the triple helical structure of collagen requires that proline be hydroxylated by prolyl-4-hydroxylase (P4H) to form hydroxyproline residues within the collagen chains. Although plants are able to synthesize hydroxyproline-containing proteins, the prolyl hydroxylases responsible for hydroxyproline synthesis in plant cells exhibit relatively relaxed substrate sequence specificity compared to mammalian P4H. Therefore, the production of hydroxyproline-containing collagen only in the Y position of the Gly-XY triplet requires co-expression of collagen with human or mammalian P4H genes.

[0356] Thus, according to one embodiment, the procollagen or telomere is expressed in a subcellular compartment of a plant lacking endogenous P4H activity, thereby avoiding its erroneous hydroxylation. As used herein, the phrase "subcellular compartment lacking endogenous P4H activity" refers to any compartment of a cell that does not contain a plant P4H or an enzyme with plant-like P4H activity. According to one embodiment, the subcellular compartment is a vacuole.

[0357] Accumulation of expressed procollagen in subcellular compartments lacking endogenous P4H activity can be effected by any of several methods.

[0358] For example, the expressed procollagen / teloclagen may comprise a signal sequence for targeting the expressed protein to a subcellular compartment such as the apoplast or an organelle (e.g., chloroplast). Examples of suitable signal sequences include the chloroplast transit peptide (contained in Swiss-Prot entry P07689, amino acids 1-57) and the mitochondrial transit peptide (contained in Swiss-Prot entry P46643, amino acids 1-28).

[0359] Alternatively, the sequence of the procollagen may be modified in such a way that the cellular localization of the procollagen is altered when expressed in plants.

[0360] In some embodiments, disclosed herein are genetically modified cells that co-express both human procollagen and P4H, which are capable of correctly hydroxylating one or more procollagen α chains [i.e., hydroxylating only the proline (Y) position of the Gly-XY triplet]. P4H is an enzyme composed of two subunits, α and β, as described in Genbank Nos. P07237 and P13674. Both subunits are required for the formation of an active enzyme, and the β subunit also possesses a chaperone function.

[0361] The P4H expressed by the genetically modified cells of the present invention is preferably a human P4H encoded by, for example, SEQ ID NOs: 34 and 35. In addition, P4H mutants or P4H homologs exhibiting enhanced substrate specificity may also be used. Suitable P4H homologs are exemplified by the Arabidopsis thaliana oxidoreductase identified by NCBI Accession No.: NP__179363.

[0362] Since it is essential that P4H co-accumulates together with the expressed procollagen chains, its coding sequence is preferably modified accordingly (eg by adding or deleting a signal sequence).

[0363] In mammalian cells, collagen is also modified by lysyl hydroxylase, galactosyltransferase and glucosyltransferase. These enzymes sequentially modify the lysyl residues in the specific position to hydroxylysyl, galactosylhydroxylysyl and glucosylgalactosylhydroxylysyl residues at the specific position. Single human enzyme (lysyl hydroxylase 3 (LH3), as described in Genbank No. O60568) can catalyze all three continuous modification steps, as seen in the formation of carbohydrates connected by hydroxylysine.

[0364] Thus, the genetically modified cells disclosed herein may also express mammalian LH3. LH3 coding sequences such as the sequence shown in SEQ ID NO: 38 may be used for this purpose.

[0365] The above-mentioned one or more procollagens and modifying enzymes can be expressed from a stably integrated or transiently expressed nucleic acid construct comprising a polynucleotide sequence encoding a procollagen alpha chain and / or a modifying enzyme (e.g., P4H and LH3) under transcriptional control of a functional promoter. Such nucleic acid constructs (also referred to herein as expression constructs) can be configured to express in a whole organism (e.g., a plant, a defined tissue, or a defined cell) and / or to express at a defined developmental stage in a plant. Such constructs can also include a selection marker (e.g., antibiotic resistance), an enhancer element, and a replication origin for bacterial replication.

[0366] It should be understood that a construct comprising two expressible inserts (e.g., two alpha procollagen chain types, or procollagen alpha chain and P4H) preferably comprises a separate promoter for each insert, or alternatively, such a construct can express a single transcript chimera comprising two inserts under a single promoter. In this case, the chimeric transcript can comprise an internal ribosome entry region (IRES) sequence between the two inserts, from which the downstream insert can be translated.

[0367] The constructs of the invention may utilize a wide variety of functional expression promoters and enhancers, which may be tissue-specific, developmentally-specific, constitutive, or inducible, some examples of which are provided below.

[0368] Regardless of the transformation technique used, once progeny expressing procollagen are identified, such plants are further cultivated under conditions that maximize their expression. Progeny produced by transformed plants can be selected by verifying the presence of exogenous mRNA and / or polypeptides using nucleic acid or protein probes (e.g., antibodies). The latter method enables the location of the expressed polypeptide components (e.g., by probing fractionated plant extracts), thereby also verifying the potential of the plant to correctly process and assemble exogenous proteins.

[0369] After planting such plants, usually the telopeptide collagen is harvested. Preferably, plant tissue / cells are harvested when mature, and the procollagen molecules are separated using an extraction method. Preferably, harvesting is performed so that the procollagen remains in a state that can be cleaved by proteases. According to one embodiment, a crude extract is produced from transgenic plants of the present invention and then contacted with proteases. An exemplary method for producing a crude plant extract is described in the Examples section hereinafter.

[0370] It should be understood that the collagen containing the propeptide or telopeptide can be purified from the genetically engineered cells of the present invention before incubation with the protease, or alternatively, the collagen containing the propeptide or telopeptide can be purified after incubation with the protease. Still alternatively, the collagen containing the propeptide or telopeptide can be partially purified before the protease treatment and then fully purified after the protease treatment. Alternatively again, the collagen containing the propeptide or telopeptide can be treated with the protease together with other extraction / purification procedures.

[0371] Exemplary methods for purifying or semi-purifying the telopeptide-containing collagen of the present invention include, but are not limited to, salting out with ammonium sulfate or the like and / or removing small molecules by ultrafiltration.

[0372] As described above in the background, there are risks in using animal-derived materials for medical purposes. This risk is also relevant when selecting proteolytic enzymes for processing procollagen expressed in plants into atelocollagen. The use of animal-derived source enzymes (such as trypsin or pepsin) may itself contaminate the final preparation of the disease vector. Therefore, it is desirable to design a production system in which all components are free of animal origin.

[0373] It has been disclosed herein that only specific proteases can correctly cleave recombinant collagen containing propeptides or telopeptides. These proteases include certain plant-derived proteases, such as ficin (EC 3.4.22.3), and certain bacterial-derived proteases, such as subtilisin (EC 3.4.21.62), neutral enzymes. In certain embodiments, the use of recombinant enzymes (such as rhTrypsin and rhPepsin) is disclosed herein. These enzymes are commercially available, such as ficin from fig tree latex (Sigma, catalog number #F4125 and European Biochem), subtilisin from Bacillus licheniformis (Sigma, catalog number #P5459), neutral enzymes from Bacillus amyloliquefaciens (Novozymes, catalog number #PW201041) and TrypZean.TM., a recombinant human trypsin expressed in corn (Sigma catalog number #T3449).

[0374] Preferably, the procollagen or telopeptide is contacted with the protease under conditions that allow the protease to cleave the propeptide or telopeptide from the procollagen or telopeptide. Typically, the conditions are determined by the specific protease selected. Thus, for example, the procollagen may be incubated with the protease at a concentration of 1-25 mg / ml and at a temperature of about 10-20° C. for up to 15 hours.

[0375] After protease digestion, the resulting atelocollagen can 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 that has been processed from procollagen produced by plants or plant cells by a protease selected from the group consisting of: neutral enzyme, subtilisin, ficin, and recombinant human trypsin, and analyzed using methods known in the art (e.g., size analysis by Coomassie staining, protein analysis, etc.).

[0376] After purification, the atelocollagen can be re-dissolved by adding an acidic solution (e.g., 10 mM HCl). This acidic solution can be used to store the purified atelocollagen.

[0377] The inventors have shown that after digestion with ficin, atelocollagen retains its ability to form fibrils upon neutralization of the above acidic solution. According to one embodiment, at least 70% of the purified and re-dissolved atelocollagen produced according to the method of the present invention is capable of forming fibrils. According to one embodiment, at least 88% of the purified and re-dissolved atelocollagen produced according to the method of the present invention is capable of forming fibrils.

[0378] The ability to form fibrils suggests that the resulting atelocollagen may be useful for medical purposes including, but not limited to, cosmetic surgery, as a therapeutic adjunct for burn victims, reconstruction of bone, and various dental, orthopedic, and surgical purposes.

[0379] As described in the Background section, type I collagen is considered an ideal candidate for use as a primary component of building materials in 3D bioprinting. Despite the obvious advantages of this natural polymer, many factors still hinder its use in 3D bioprinting. Due to the sensitivity of tissue-derived collagen to temperature and ionic strength, its use for this purpose is limited, and the collagen drives spontaneous gel formation at temperatures above 20°C under physiological conditions [see, for example, PureCol, Advanced BioMatrix, Inc.]. During printing, the typical temperature-dependent gel formation of tissue-derived collagen significantly hinders precise fluidity. Keeping the printed medium at low temperature until use is a possible solution to this phenomenon, but this implies serious technical limitations. Another solution is to use gelatin, a denatured form of collagen that does not become gel-like under these conditions. However, gelatin lacks the true tissue and cell interactions of natural collagen and therefore loses key biological functions.

[0380] Recent advances in technology have led to the development of a system for purifying pure human type I collagen (rhCollagen) by introducing five human genes encoding heterotrimeric type I collagen into tobacco plants (COLLPLANT, Israel TM ; SIGMA- Also available). The protein is purified to homogeneity by a cost-effective industrial process that exploits the unique properties of collagen. See also WO 2006 / 035442, WO 2009 / 053985, and patents and patent applications derived therefrom, all of which are incorporated herein by reference as if fully set forth herein.

[0381] Thus, according to one aspect, disclosed herein is a genetically modified plant capable of expressing at least one type of collagen alpha chain and accumulating it in a subcellular compartment lacking endogenous P4H activity.

[0382] Type I collagen and rhCollagen are considered candidates for use as the main components of building materials in 3D bioprinting. Various types of scaffolds have been used in cosmetic and other reconstructive applications.

[0383] In addition, the use of dermal fillers for soft tissue augmentation (e.g., wrinkle reduction) has increased. One possible method for using dermal fillers involves injecting a polymerizable dermal filler material into a desired area and then contouring or molding the filler into a desired conformation. Polymerizing and cross-linking the material by one of a variety of methods can convert the monomers in the injected material into polymers and chains that can form a network, thereby retaining the desired molded conformation. There are many methods for forming polymers and cross-linking polymers. One method involves a photoreactive agent and a light-induced reaction that produces a reactive species in a monomer solution.

[0384] However, at least some of these methods continue to focus on tissue-derived collagen or non-collagen polymers (e.g., polyvinyl alcohol or hyaluronic acid). In addition, due to the sensitivity of tissue-extracted collagen to temperature and ionic strength, its use is limited, and the collagen drives spontaneous 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 hinders precise fluidity. Keeping collagen at low temperatures until use is a possible solution to this phenomenon, but this means serious technical limitations. Another solution is to use gelatin, which is a denatured form of collagen that does not become gelatinous under these conditions. However, gelatin lacks the true tissue and cell interactions of natural collagen, and therefore loses key biological functions. Moreover, the viscosity of gelatin makes it more difficult to inject it into the subdermis using a fine-gauge needle, and also makes it more difficult to spread and mold into a smaller cavity.

[0385] Examples of dermal fillers and uses thereof disclosed herein include, but are not limited to:

[0386] 1. A method for filling a subepidermal tissue space, the method comprising:

[0387] a. introducing a polymerizable solution into the tissue space, the polymerizable solution comprising:

[0388] i. cross-linkable plant-derived human collagen; and

[0389] ii. a photoinitiator; and

[0390] b. applying light to the surface of the spatial superficial epidermis to induce polymerization.

[0391] 2. A method for filling a subepidermal tissue space, the method further comprising:

[0392] (a) The step of molding or sculpting the polymerizable solution into a desired configuration in the tissue space, wherein the step is performed simultaneously with or after the step of applying light.

[0393] 3. A method of filling subepidermal tissue space, wherein the molding or sculpting step reduces lines, folds, fine lines, wrinkles or scars.

[0394] 4. A method for filling subepidermal tissue spaces, wherein the cross-linkable plant-derived human collagen is methacrylated or thiolated.

[0395] 5. A method for filling subepidermal tissue space, wherein the polymer solution further comprises hyaluronic acid (HA) or a modified derivative thereof, polyvinyl alcohol (PVA) or a modified derivative thereof, polyethylene glycol (PEG) or a modified derivative thereof, oxidized cellulose (OC) or a modified derivative thereof, polymethyl methacrylate (PMMA) microspheres or a modified derivative thereof, tricalcium phosphate (TCP) or a modified derivative thereof, calcium hydroxyapatite (CaHA) or a modified derivative thereof, carboxymethyl cellulose or a modified derivative thereof, crystalline nanocellulose (CNC) or a modified derivative thereof, or a combination thereof.

[0396] 6. A method for filling subepidermal tissue space, wherein the modified derivatives of hyaluronic acid (HA), polyvinyl alcohol (PVA), polyethylene glycol (PEG), oxidized cellulose (OC), polymethyl methacrylate (PMMA) microspheres, tricalcium phosphate (TCP), calcium hydroxyapatite (CaHA), carboxymethyl cellulose or crystalline nanocellulose (CNC) include photopolymerizable modified derivatives.

[0397] 7. The method of claim 5, wherein the modified derivatives of hyaluronic acid (HA), polyvinyl alcohol (PVA), polyethylene glycol (PEG), oxidized cellulose (OC), polymethyl methacrylate (PMMA) microspheres, tricalcium phosphate (TCP), calcium hydroxyapatite (CaHA), carboxymethyl cellulose or crystalline nanocellulose (CNC) include methacrylated or thiolated derivatives.

[0398] 8. A method for filling subepidermal tissue spaces, wherein the plant-derived collagen comprises rhCollagen.

[0399] 9. A method for filling subepidermal tissue spaces, wherein the plant-derived collagen is obtained from a genetically modified plant.

[0400] 10. A method of filling subepidermal tissue space, wherein the genetically modified plant is a genetically modified plant selected from the group consisting of tobacco, corn, alfalfa, rice, potato, soybean, tomato, wheat, barley, canola, carrot and cotton.

[0401] 11. A method of filling subepidermal tissue spaces, wherein the genetically modified plant is a tobacco plant.

[0402] 12. A method for filling subepidermal tissue space, wherein the genetically modified plant comprises an expressible sequence of at least one gene sequence of human deoxyribonucleic acid (DNA), wherein the human deoxyribonucleic acid is selected from the group consisting of: COL1, COL2, P4H-α, P4H-β and LH3.

[0403] 13. A method for filling subepidermal tissue spaces, wherein the plant-derived human collagen comprises: at least one modified human collagen α-1 chain, as shown in SEQ ID NO:3 and expressed in a genetically modified plant; and at least one modified human collagen α-2 chain, as shown in SEQ ID NO:6 and expressed in a genetically modified plant; and wherein the genetically modified plant further expresses exogenous prolyl 4-hydroxylase (P4H).

[0404] 14. A method for filling a subepidermal tissue space, the method further comprising expressing an exogenous polypeptide selected from the group consisting of: lysyl hydroxylase (LH), proteinase N, and proteinase C.

[0405] 15. A method for filling subepidermal tissue spaces, wherein the human collagen α-1 chain is encoded by the sequence shown in SEQ ID NO: 1.

[0406] 16. A method for filling subepidermal tissue spaces, wherein the human collagen α-2 chain is encoded by the sequence shown in SEQ ID NO:2.

[0407] 17. A method for filling subepidermal tissue spaces, wherein the exogenous P4H is mammalian P4H.

[0408] 18. A method for filling subepidermal tissue space, wherein the exogenous P4H is human P4H.

[0409] 19. A method for filling subepidermal tissue spaces, the method further comprising targeting the human collagen alpha-1 to the vacuole of the plant or the genetically modified plant and digesting with ficin.

[0410] 20. A method of filling subepidermal tissue spaces, the method further comprising targeting the human collagen alpha-2 to the vacuole of the plant or the genetically modified plant and digesting with ficin.

[0411] 21. A method for filling subepidermal tissue spaces, wherein the plant-derived human collagen is atelocollagen.

[0412] 22. A method of filling a subepidermal tissue space, wherein the light source comprises a light emitting diode (LED), a laser, or a xenon lamp.

[0413] 23. A method of filling a subepidermal tissue space, wherein the photoinitiator induces polymerization of the polymerizable solution in response to visible light.

[0414] 24. A method for filling subepidermal tissue space, wherein the visible light has a wavelength of 390-700 nm.

[0415] 25. A method of filling subepidermal tissue space, wherein the photoinitiator is selected from the group consisting of: Eosin Y + triethanolamine or riboflavin.

[0416] 26. A method of filling a subepidermal tissue space, wherein the photoinitiator induces polymerization of the polymerizable solution in response to ultraviolet (uv) light.

[0417] 27. A method for filling a subepidermal tissue space, wherein the photoinitiator is selected from the group consisting of lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) or 1-[4-(2-hydroxyethoxy)phenyl]-2-methylpropan-1-one ( 2959).

[0418] 28. A method of filling a subepidermal tissue space, wherein the photoinitiator induces polymerization of the polymerizable solution in response to infrared light.

[0419] 29. A method of filling a subepidermal tissue space wherein the polymerizable solution is introduced into the tissue space through a hollow needle or cannula in the range of 27 gauge to 33 gauge.

[0420] 30. A method of filling a subepidermal tissue space, wherein the polymerizable solution in the tissue space is molded or sculpted into the desired configuration by manual massage.

[0421] 31. A method of filling a subepidermal tissue space, wherein the polymerizable solution in the tissue space is molded or sculpted into the desired configuration using a molding or sculpting tool.

[0422] 32. A method of filling a subepidermal tissue space, wherein the polymerizable solution in the tissue space is substantially non-gellable at room temperature.

[0423] 33. A method of filling a subepidermal tissue space, wherein the polymerizable solution in the tissue space is substantially non-gellable at 37°C.

[0424] 34. A method of filling subepidermal tissue spaces, wherein said polymerizable solution comprising said plant-derived human collagen has a reduced viscosity at room temperature compared to a similar polymerizable solution comprising tissue-extracted human or bovine collagen at the same concentration and formulation.

[0425] 35. A method of filling subepidermal tissue spaces, wherein said polymerizable solution comprising said plant-derived human collagen has a reduced viscosity at 37°C compared to a similar polymerizable solution comprising tissue-extracted human or bovine collagen at the same concentration and formulation.

[0426] 36. A method for filling a subepidermal tissue space, wherein the polymerizable solution comprising the plant-derived human collagen is introduced into the tissue space at room temperature with reduced force compared to a similar polymerizable solution comprising tissue-extracted human or bovine collagen at the same concentration and formulation.

[0427] 37. A method for filling a subepidermal tissue space, wherein the polymerizable solution comprising the plant-derived human collagen is introduced into the tissue space at 37°C with reduced force compared to a similar polymerizable solution comprising tissue-extracted human or bovine collagen at the same concentration and formulation.

[0428] 38. A method of filling subepidermal tissue spaces, wherein said polymerizable solution comprising said plant-derived human collagen has increased tissue augmentation compared to a similar polymerizable solution comprising tissue-extracted human or bovine collagen at the same concentration and formulation.

[0429] 39. A method of injecting a polymerizable solution into a subepidermal tissue space to reduce lines, folds, fine lines, wrinkles or scars, wherein the polymerizable solution comprises cross-linkable plant-derived human collagen and a photoinitiator that induces polymerization prior to or simultaneously with the application of visible light, and molding or sculpting the polymerizable solution into a desired configuration to reduce lines, folds, fine lines, wrinkles or scars.

[0430] 40. A use of injecting a polymerizable solution into a subepidermal tissue space to reduce lines, folds, fine lines, wrinkles or scars, wherein the cross-linkable plant-derived human collagen is methacrylated or thiolated.

[0431] 41. A method of injecting a polymerizable solution into a subepidermal tissue space to reduce lines, folds, fine lines, wrinkles or scars, wherein the polymer solution further comprises hyaluronic acid (HA) or a modified derivative thereof, polyvinyl 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 hydroxyapatite (CaHA) or a modified derivative thereof, carboxymethyl cellulose or a modified derivative thereof, crystalline nanocellulose (CNC) or a modified derivative thereof, or a combination thereof.

[0432] 42. A method of injecting a polymerizable solution into a subepidermal tissue space to reduce lines, folds, fine lines, wrinkles or scars, wherein the modified derivatives of hyaluronic acid (HA), polyvinyl alcohol (PVA), polyethylene glycol (PEG), oxidized cellulose (OC), polymethyl methacrylate (PMMA) microspheres, tricalcium phosphate (TCP), calcium hydroxyapatite (CaHA), carboxymethyl cellulose or crystalline nanocellulose (CNC) include photopolymerizable modified derivatives.

[0433] 43. A method of injecting a polymerizable solution into a subepidermal tissue space to reduce lines, folds, fine lines, wrinkles or scars, wherein the modified derivatives of hyaluronic acid (HA), polyvinyl alcohol (PVA), polyethylene glycol (PEG), oxidized cellulose (OC), polymethyl methacrylate (PMMA) microspheres, tricalcium phosphate (TCP), calcium hydroxyapatite (CaHA), carboxymethyl cellulose or crystalline nanocellulose (CNC) include methacrylated or thiolated derivatives.

[0434] 44. A method of filling a subepidermal tissue space, the method comprising:

[0435] A polymerizable solution is introduced into the tissue space, the polymerizable solution comprising cross-linkable plant-derived human collagen.

[0436] 45. A method of filling a subepidermal tissue space, the method further comprising:

[0437] (a) The step of molding or sculpting the polymerizable solution into a desired configuration in the tissue space.

[0438] 46. ​​A method of filling a subepidermal tissue space, wherein the molding or sculpting step reduces lines, folds, fine lines, wrinkles or scars.

[0439] 47. A method for filling subepidermal tissue space, wherein the polymerizable solution further comprises hyaluronic acid (HA) or a modified derivative thereof, polyvinyl alcohol (PVA) or a modified derivative thereof, polyethylene glycol (PEG) or a modified derivative thereof, oxidized cellulose (OC) or a modified derivative thereof, polymethyl methacrylate (PMMA) microspheres or a modified derivative thereof, tricalcium phosphate (TCP) or a modified derivative thereof, calcium hydroxyapatite (CaHA) or a modified derivative thereof, carboxymethyl cellulose or a modified derivative thereof, crystalline nanocellulose (CNC) or a modified derivative thereof, or a combination thereof.

[0440] 48. A method for filling subepidermal tissue spaces, wherein the plant-derived collagen comprises rhCollagen.

[0441] 49. A method of filling subepidermal tissue spaces, wherein the plant-derived collagen is obtained from a genetically modified plant.

[0442] 50. A method of filling subepidermal tissue space, wherein the genetically modified plant is a genetically modified plant selected from the group consisting of tobacco, corn, alfalfa, rice, potato, soybean, tomato, wheat, barley, canola, carrot and cotton.

[0443] 51. A method of filling subepidermal tissue spaces, wherein the genetically modified plant is a tobacco plant.

[0444] 52. A method for filling subepidermal tissue spaces, wherein the genetically modified plant comprises an expressible sequence of at least one gene sequence of human deoxyribonucleic acid (DNA), wherein the human deoxyribonucleic acid is selected from the group consisting of: COL1, COL2, P4H-α, P4H-β and LH3.

[0445] 53. A method for filling subepidermal tissue spaces, wherein the plant-derived human collagen comprises: at least one modified human collagen α-1 chain, as shown in SEQ ID NO:3 and expressed in a genetically modified plant; and at least one modified human collagen α-2 chain, as shown in SEQ ID NO:6 and expressed in a genetically modified plant; and wherein the genetically modified plant further expresses exogenous prolyl 4-hydroxylase (P4H).

[0446] 54. A method of filling a subepidermal tissue space, the method further comprising expressing an exogenous polypeptide selected from the group consisting of: lysyl hydroxylase (LH), proteinase N, and proteinase C.

[0447] 55. A method for filling a subepidermal tissue space, wherein the human collagen alpha-1 chain is encoded by the sequence shown in SEQ ID NO: 1.

[0448] 56. A method for filling a subepidermal tissue space, wherein the human collagen alpha-2 chain is encoded by the sequence shown in SEQ ID NO:2.

[0449] 57. A method of filling a subepidermal tissue space, wherein the exogenous P4H is mammalian P4H.

[0450] 58. A method of filling a subepidermal tissue space, wherein the exogenous P4H is human P4H.

[0451] 59. A method for filling subepidermal tissue spaces, the method further comprising targeting the human collagen alpha-1 to the vacuole of the plant or the genetically modified plant and digesting with ficin.

[0452] 60. A method of filling subepidermal tissue spaces, the method further comprising targeting the human collagen alpha-2 to the vacuole of the plant or the genetically modified plant and digesting with ficin.

[0453] 61. A method for filling subepidermal tissue spaces, wherein the plant-derived human collagen is atelocollagen.

[0454] 62. A method of filling a subepidermal tissue space wherein the polymerizable solution is introduced into the tissue space through a hollow needle or cannula in the range of 27 gauge to 33 gauge.

[0455] 63. A method of filling a subepidermal tissue space, wherein the polymerizable solution in the tissue space is molded or sculpted into the desired configuration by manual massage.

[0456] 64. A method of filling a subepidermal tissue space, wherein the polymerizable solution in the tissue space is molded or sculpted into the desired configuration using a molding or sculpting tool.

[0457] 65. A method of filling a subepidermal tissue space, wherein the polymerizable solution in the tissue space is substantially non-gellable at room temperature.

[0458] 66. A method of filling a subepidermal tissue space, wherein the polymerizable solution in the tissue space is substantially non-gellable at 37°C.

[0459] 67. A method of filling subepidermal tissue spaces, wherein said polymerizable solution comprising said plant-derived human collagen has a reduced viscosity at room temperature compared to a similar polymerizable solution comprising tissue-extracted human or bovine collagen at the same concentration and formulation.

[0460] 68. A method of filling subepidermal tissue spaces, wherein said polymerizable solution comprising said plant-derived human collagen has a reduced viscosity at 37°C compared to a similar polymerizable solution comprising tissue-extracted human or bovine collagen at the same concentration and formulation.

[0461] 69. A method for filling a subepidermal tissue space, wherein the polymerizable solution comprising the plant-derived human collagen is introduced into the tissue space at room temperature with reduced force compared to a similar polymerizable solution comprising tissue-extracted human or bovine collagen at the same concentration and formulation.

[0462] 70. A method for filling a subepidermal tissue space, wherein the polymerizable solution comprising the plant-derived human collagen is introduced into the tissue space with reduced force at 37°C compared to a similar polymerizable solution comprising tissue-extracted human or bovine collagen at the same concentration and formulation.

[0463] 71. A method of filling subepidermal tissue spaces, wherein said polymerizable solution comprising said plant-derived human collagen has increased tissue augmentation compared to a similar polymerizable solution comprising tissue-extracted human or bovine collagen at the same concentration and formulation.

[0464] 72. A method of injecting a polymerizable solution into a subepidermal tissue space to reduce lines, folds, fine lines, wrinkles or scars, wherein the polymerizable solution comprises cross-linkable plant-derived human collagen, and molding or sculpting the polymerizable solution into a desired configuration to reduce lines, folds, fine lines, wrinkles or scars.

[0465] 73. A method for inducing a cell growth scaffold in a subepidermal tissue space, the method comprising introducing a solution into the tissue space, the solution comprising:

[0466] (a) human collagen of plant origin; and

[0467] (b) at least one growth factor or a source thereof.

[0468] 74. A method of inducing a cell growth scaffold in a subepidermal tissue space, wherein the source of at least one growth factor comprises plasma or platelet-rich plasma.

[0469] 75. A method of inducing a cell growth scaffold in a subepidermal tissue space, wherein the cell growth scaffold promotes healing or replacement due to degradation or injury of collagen-containing tissue.

[0470] 76. A method of inducing a cell growth scaffold in a subepidermal tissue space, wherein the collagen-containing tissue is selected from the group consisting of tendon, ligament, skin, cornea, cartilage, blood vessel, intestine, intervertebral disc, muscle, bone or tooth.

[0471] 77. A method of inducing a cell growth scaffold in a subepidermal tissue space, wherein the cell growth scaffold promotes healing of tendinitis.

[0472] 78. A method for inducing cell growth scaffolding in a subepidermal tissue space, wherein the plant-derived collagen comprises rhCollagen.

[0473] 79. A method for inducing a cell growth scaffold in a subepidermal tissue space, wherein the plant-derived collagen is obtained from a genetically modified plant.

[0474] 80. A method for inducing a cell growth scaffold in a subepidermal tissue space, wherein the genetically modified plant is a genetically modified plant selected from the group consisting of tobacco, corn, alfalfa, rice, potato, soybean, tomato, wheat, barley, canola, carrot and cotton.

[0475] 81. A method of inducing a cell growth scaffold in a subepidermal tissue space, wherein the genetically modified plant is a tobacco plant.

[0476] 82. A method for inducing a cell growth scaffold in a tissue space beneath the epidermis, wherein the genetically modified plant comprises an expressible sequence of at least one gene sequence of human deoxyribonucleic acid (DNA), the human deoxyribonucleic acid being selected from the group consisting of: COL1, COL2, P4H-α, P4H-β and LH3.

[0477] 83. A method for inducing a cell growth scaffold in a subepidermal tissue space, wherein the plant-derived human collagen comprises: at least one modified human collagen α-1 chain, as shown in SEQ ID NO:3 and expressed in a genetically modified plant; and at least one modified human collagen α-2 chain, as shown in SEQ ID NO:6 and expressed in a genetically modified plant; and wherein the genetically modified plant further expresses exogenous prolyl 4-hydroxylase (P4H).

[0478] 84. A method of inducing a cell growth scaffold in a subepidermal tissue space, the method further comprising expressing an exogenous polypeptide selected from the group consisting of: lysyl hydroxylase (LH), proteinase N, and proteinase C.

[0479] 85. A method for inducing cell growth scaffold in a subepidermal tissue space, wherein the human collagen alpha-1 chain is encoded by the sequence shown in SEQ ID NO: 1.

[0480] 86. A method for inducing a cell growth scaffold in a subepidermal tissue space, wherein the human collagen α-2 chain is encoded by the sequence shown in SEQ ID NO:2.

[0481] 87. A method of inducing a cell growth scaffold in a subepidermal tissue space, wherein the exogenous P4H is a mammalian P4H.

[0482] 88. A method of inducing a cell growth scaffold in a subepidermal tissue space, wherein the exogenous P4H is human P4H.

[0483] 89. A method for inducing a cell growth scaffold in a subepidermal tissue space, the method further comprising targeting the human collagen alpha-1 to the vacuole of the plant or the genetically modified plant and digesting with ficin.

[0484] 90. A method of inducing a cell growth scaffold in a subepidermal tissue space, the method further comprising targeting the human collagen alpha-2 to the vacuole of the plant or the genetically modified plant and digesting with ficin.

[0485] 91. A method of inducing a cell growth scaffold in a subepidermal tissue space, wherein the plant-derived human collagen is atelocollagen.

[0486] 92. A method of inducing a cellular growth scaffold in a subepidermal tissue space, wherein said solution comprising said plant-derived human collagen has a reduced viscosity at room temperature compared to similar solutions comprising tissue-extracted human or bovine collagen at the same concentration and formulation.

[0487] 93. A method of inducing a cell growth scaffold in a subepidermal tissue space, wherein said solution comprising said plant-derived human collagen has a reduced viscosity at 37°C compared to similar solutions comprising tissue-extracted human or bovine collagen at the same concentration and formulation.

[0488] 94. A method for inducing a cell growth scaffold in a subepidermal tissue space, wherein the solution comprising said plant-derived human collagen is introduced into said tissue space at room temperature with reduced force compared to a similar solution comprising tissue-extracted human or bovine collagen at the same concentration and formulation.

[0489] 95. A method for inducing a cell growth scaffold in a subepidermal tissue space, wherein the solution comprising the plant-derived human collagen is introduced into the tissue space at 37°C with reduced force compared to a similar solution comprising tissue-extracted human or bovine collagen at the same concentration and formulation.

[0490] 96. A method for inducing a cell growth scaffold in a subepidermal tissue space, wherein the solution comprising said plant-derived human collagen has increased scaffold formation or promotes increased cell growth compared to a similar solution comprising tissue-extracted human or bovine collagen at the same concentration and formulation.

[0491] 97. A use of injecting a solution into a subepidermal tissue space to induce a cell growth scaffold, the solution comprising plant-derived human collagen and at least one growth factor or a source thereof to promote healing or replacement due to degradation or damage of collagen-containing tissue.

[0492] 98. A use of injecting a solution into a subepidermal tissue space to induce a cell growth scaffold, wherein the source of the at least one growth factor comprises plasma or platelet-rich plasma.

[0493] definition

[0494] As used herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "molecule" also includes a plurality of molecules.

[0495] As used herein, the term "about" refers to ±10% or ±5%.

[0496] The terms "comprises / comprising", "includes / including", "having" and their cognates mean "including, but not limited to".

[0497] The term "consisting of" means "including and limited to."

[0498] The term "consisting essentially of" means that the composition, method or structure may include additional ingredients, steps and / or parts, but only if these additional ingredients, steps and / or parts do not materially change the basic and novel characteristics of the claimed composition, method or structure.

[0499] As used herein, the term "method" refers to ways, means, techniques and procedures for accomplishing a given task, including but not limited to those ways, means, techniques and procedures known to practitioners in the fields of chemistry, pharmacology, biology, biochemistry and medicine or readily developed by known ways, means, techniques and procedures.

[0500] As used herein, the phrase "genetically modified plant" encompasses any lower (e.g., moss) or higher (vascular) plant or tissue or isolated cells thereof (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, corn, alfalfa, rice, potato, soybean, tomato, wheat, barley, canola, cotton, carrot, and lower plants (e.g., mosses).

[0501] As used herein, the phrase "collagen chain" encompasses collagen subunits, such as α1 or 2 chains of collagen fibers (preferably, type I fibers). As used herein, the phrase "collagen" refers to an assembled collagen trimer, which, in the case of type I collagen, comprises two α1 chains and one α2 chain. Collagen fibers are collagens lacking the terminal propeptides C and N.

[0502] As used herein, the phrase "telopeptide-containing collagen" encompasses soluble collagen molecules that include telopeptides that are longer than the telopeptide residues included in atelocollagen. Thus, the telopeptide-containing collagen can be a procollagen that includes full-length propeptides. Alternatively, the telopeptide-containing collagen can be a procollagen molecule that includes partially digested propeptides. Still alternatively, the telopeptide-containing collagen can be telopeptide collagen.

[0503] As used herein, the term "procollagen" encompasses collagen molecules (eg, human) that include an N-terminal propeptide, a C-terminal propeptide, or both. Exemplary human procollagen amino acid sequences are shown in SEQ ID NOs: 1, 2, 7, and 8.

[0504] As used herein, the term "telopeptide" encompasses collagen molecules that lack the N-terminal and C-terminal propeptides that are typically included in procollagen but still contain telopeptides. The telopeptide of fibrillar collagen is the residue of the N-terminal propeptide and the C-terminal propeptide after digestion with natural N / C proteases. Recombinant human telopeptides can be produced in cells that have been converted to express both exogenous human procollagen and the corresponding proteases (i.e., C or N or both). The polynucleotide sequences encoding such proteases are exemplified by SEQ ID No: 10 (Protease C) and 11 (Protease N). Such proteases can be expressed so that they accumulate in subcellular compartments like collagen chains, as further described below.

[0505] As used herein, the term "atelocollagen" encompasses collagen molecules that lack both the N-terminal and C-terminal propeptides typically included in procollagen and at least a portion of their telopeptides, but contain a sufficient portion of their telopeptides to enable the atelocollagen to form fibrils under suitable conditions. Any type of atelocollagen can be produced according to the methods of the present invention. Examples include fibril-forming collagens (types I, II, III, V, and XI), network-forming collagens (types IV, VIII, and X), collagens associated with fibril surfaces (types IX, XII, and XIV), collagens that appear as transmembrane proteins (types XIII and XVII), or collagens that form 11-nm periodic beaded filaments (type VI). According to one embodiment, the atelocollagen includes α1 and / or 2 chains of type I collagen.

[0506] It should be understood that, in some embodiments, the dermal fillers disclosed herein may include genetically modified forms of collagen / atelocollagen - such as collagenase-resistant collagen and the like.

[0507] As used herein, the phrase "plant promoter" or "promoter" encompasses promoters that can direct gene expression in cells (including organelles containing DNA) of plants, fungi, and yeast. Such promoters can be derived from plants, bacteria, viruses, fungi, or animal sources. Such promoters can 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), inducible (i.e., capable of directing gene expression under stimulation), or chimeric (i.e., formed from portions of at least two different promoters).

[0508] As used herein, the phrase "subcellular compartment lacking endogenous P4H activity" refers to any compartment of a cell that does not contain a plant P4H or an enzyme having plant-like P4H activity. Examples of such subcellular compartments include vacuoles, apoplasts, and cytoplasm, as well as organelles (such as chloroplasts, mitochondria, etc.).

[0509] Throughout this document, the phrase "building material" encompasses the phrases "uncured building material" or "uncured building material formulations", and together describe the materials used to sequentially form the layers, as described herein. The phrase encompasses the uncured material that forms the final object, i.e., one or more uncured molding material formulations, and optionally, the uncured material used to form the support, i.e., the uncured support material formulation. The uncured building material may include one or more molding formulations, and may be distributed so that different parts of the object are made when different molding formulations are cured, so that the uncured building material is made of different cured molding materials or different mixtures of the cured molding materials.

[0510] As used herein, "bioprinting" refers to the practice of additive manufacturing processes while utilizing one or more bio-ink formulations that include biological components by methods compatible with an automated or semi-automated computer-assisted additive manufacturing system (e.g., a bioprinter or bioprinting system) described herein.

[0511] Throughout this document, in the context of bioprinting, the term "object" describes an additively manufactured final product that includes a biological component in at least a portion thereof. The term refers to the product obtained by the bioprinting methods described herein after removal of the support material (if it has been used as part of the uncured building material). In some embodiments, the biological component comprises recombinant human collagen, as described, for example, in WO 2006 / 035442, WO 2009 / 053985, and patents and patent applications derived therefrom, all of which are incorporated herein by reference as if fully set forth herein.

[0512] As used throughout this document, the term "object" refers to an entire object or a portion thereof.

[0513] Throughout this article, a "curable material" is a compound (monomeric or oligomeric or polymeric) that cures or hardens to form a cured molding material as defined herein when exposed to curing conditions as described herein. A curable material is typically a polymerizable material that polymerizes and / or crosslinks when exposed to a suitable energy source. Alternatively, a curable material is a thermoresponsive material that cures or hardens when exposed to a temperature change (e.g., heating or cooling). Optionally, a curable material is a biomaterial that reacts to form a hardened or solid material during a biological reaction (e.g., an enzyme-catalyzed reaction).

[0514] "Curing conditions" encompass curing energy (eg, temperature, radiation) and / or materials or agents that promote curing.

[0515] In some embodiments of any of the embodiments described herein, the curable material is a photopolymerizable material, as described herein, which polymerizes or cross-links when exposed to radiation, and in some embodiments, the curable material is a UV-curable material or a visible light-curable material, as described herein, which polymerizes or cross-links when exposed to UV-vis radiation.

[0516] In some embodiments of any of the embodiments described herein, the curable material can be a monomer, an oligomer, or a short-chain polymer, each of which can be polymerized as described herein.

[0517] In this context, the term "curable" encompasses the terms "polymerizable" and "cross-linkable".

[0518] As used herein, "aeroponics" is the process of growing plants in an air or mist environment without the use of soil or aggregate media (known as "agronomy").

[0519] As used herein, "hydroponics" is the process of growing plants without soil using mineral nutrient solutions in a water solvent ("aquaponics").

[0520] As used herein, "endosphere" includes all endophytes of a plant.

[0521] As used herein, "exudate" is fluid that is discharged from an organism through a hole or wound. "Exudation" is the process of discharging "exudate".

[0522] As used herein, "hydroponics" is the process of growing plants without soil using mineral nutrient solutions in a water solvent ("aquaponics").

[0523] As used herein, "invasion" or "invasive hybridization" is the movement of genes from the gene pool of one species to the gene pool of another species (i.e., "gene flow") through repeated backcrossing of an interspecific hybrid to one of its parental species, which is distinct from simple hybridization and results in a complex admixture of the parental genes.

[0524] As used herein, "metabolome" is a full set of small molecule chemicals (including but not limited to cells, organelles, organs, tissues, tissue extracts, biological fluids or organisms) found in a "biological sample". The small molecule 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 heterogeneous organisms. "Endogenous metabolome" is composed of endogenous metabolites, and "exogenous metabolome" is composed of "exogenous chemicals". "Endogenous metabolome" is composed of "primary metabolites" and "secondary metabolites", especially in plants, fungi and prokaryotes. The "primary metabolome" consists of "primary metabolites" (i.e., metabolites that are directly involved in the normal growth, development, and reproduction of an organism), while the "secondary metabolome" consists of "secondary metabolites" (i.e., metabolites that are not directly involved in the normal growth, development, or reproduction of an organism). Secondary metabolites often have important ecological functions.

[0525] As used herein, "metabolites" are generally small molecules with a molecular weight of less than 1500 Da. "Metabolites" may include, but are not limited to, glycolipids, polysaccharides, short peptides, small oligonucleotides, organic acids, taxol, alkaloids, and abietolactones, while very large macromolecules (e.g., proteins, mRNA, rRNA, and DNA) are generally not metabolites and are not part of the metabolome.

[0526] As used herein, the "SILVA database" is the SILVA ribosomal RNA database.

[0527] All samples obtained from an organism (including samples that have undergone further processing of any kind) are considered to be obtained from the organism.

[0528] Methods for DNA separation, sequencing, amplification and / or cloning are known to those skilled in the art. The most commonly used DNA amplification method is PCR (polymerase chain reaction; for example, see "PCR Basics: from background to Bench", Springer Verlag, 2000; Eckert et al., 1991. "PCR Methods and Applications" 1: 17). Other suitable amplification methods include ligase chain reaction (LCR), transcription amplification and automatic maintenance sequence replication, and nucleic acid-based sequence amplification (NASBA). Similarly, methods for separation, characterization, etc. of RNA and proteins and methods for protein expression are known to those skilled in the art.

[0529] The following examples are presented to more fully illustrate some embodiments of the dermal fillers and their uses disclosed herein. However, the examples should in no way be construed as limiting the broad scope of the dermal fillers or their uses disclosed herein. Many variations and modifications of the principles disclosed herein can be readily devised by those skilled in the art without departing from the scope of the invention.

[0530] Examples

[0531] Example 1. Construction and transformation protocol.

[0532] Figure 1a-1d The construction of expression cassettes and vectors used in this work is shown in (see also U.S. Patent 8,455,717). All coding sequences in this work were optimized for expression in tobacco and chemically synthesized with the desired flanking regions (SEQ ID NO: 1, 4, 7, 12, 14, 16, 18, 20, 22). Figure 1a : Synthetic genes encoding Col1 and Col2 (SEQ ID NOs: 1, 4) fused to either vacuolar or apoplast signals (encoded by SEQ ID NO: 7) or without signals were cloned into 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 into the multiple cloning site 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) fused to a vacuolar or apoplast signal (encoded by SEQ ID NO: 7) or without a signal were cloned into an expression cassette consisting of the CaMV 35S promoter and TMVω sequence and the Agrobacterium nopaline synthase (NOS) terminator carried by the vector pJD330 (Galili et al., 1987, Nucleic Acids Res 15: 3257-3273). The complete expression cassette was cloned into the multiple cloning site of the pBINPLUS vector carrying the Col1 or Col2 expression cassette. Figure 1c : Synthetic genes encoding proteinase C and proteinase N (SEQ ID NO: 18, 20) fused to vacuolar or apoplast signals (encoded by SEQ ID NO: 7) were cloned into 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 into the multiple cloning site of the pBINPLUS plant transformation vector. Figure 1d A synthetic gene encoding LH3 (SEQ ID NO: 22) fused to either a vacuolar or apoplast signal (encoded by SEQ ID NO: 7) or no signal with a flanking Strawberry Vein Banding Virus (SVBV) promoter (NCBI accession number AF331666 region: 623.950 version AF331666.1 GI: 13345788) and terminated with the Agrobacterium tumefaciens octopine synthase (OCS) terminator (NCBI accession number Z37515 region: 1344.1538 version Z37515.1 GI: 886843) was cloned into the multiple cloning site of the pBINPLUS vector carrying the Col1 and P4Hβ expression cassettes.

[0533] Figure 2 The use of Figure 1a-1d 1. Co-transformation scheme for the expression cassettes described in 1 into the host plant. Each expression cassette insert is represented by the abbreviation of the coding sequence. The coding sequences and the associated SEQ ID NOs are described in Table 1. Each co-transformation was performed by two pBINPLUS binary vectors. Each rectangle represents a single pBINPLUS vector carrying one, two or three expression cassettes. Figure 1a-1d The promoter and terminator are specified in .

[0534] Example 2. Expression of plant collagen.

[0535] Synthetic polynucleotide sequences encoding the proteins listed in Table 1 below were designed and optimized for expression in tobacco plants.

[0536] Table 1

[0537]

[0538] Signal peptide

[0539] 1. The vacuolar signal sequence of the barley gene for the thiol protease aleurain precursor (NCBI accession number P05167 GI: 113603) MAHARVLLLALAVLATAAVAVASSSSFADSNPIRPVTDRAASTLA (SEQ ID NO: 24).

[0540] 2. Apoplast signal of endogenous 1,4-β-glucanase from Arabidopsis thaliana (cell, NCBI accession number CAA67156.1GI:2440033); SEQ ID NO.9, encoded by SEQ ID NO.7.

[0541] Plasmid construction

[0542] Plant expression vectors were constructed according to the method in Example 1, and the composition of each constructed expression vector was confirmed by restriction enzyme analysis and sequencing.

[0543] An expression vector containing the following expression cassette was constructed:

[0544] 1. Collagen α1

[0545] 2. Collagen α1 + human P4Hβ subunit

[0546] 3. Collagen α1 + human P4Hβ subunit + human LH3

[0547] 4. Collagen α2

[0548] 5. Collagen α2 + human P4Hα subunit

[0549] 6. Collagen α2+ Arabidopsis P4H

[0550] 7. Human P4Hβ subunit + human LH3

[0551] 8. Human P4Hα subunit

[0552] Each of the above coding sequences is translationally fused to a vacuolar transit peptide or to an apoplast transit peptide, or does not contain any transit peptide sequence, in which case cytoplasmic accumulation is expected.

[0553] Plant transformation and PCR screening

[0554] according to Figure 2 The above expression vectors were used to transform tobacco plants (Nicotiana tabacum, Samsun NN) according to the transformation protocol taught in .

[0555] The resulting transgenic plants were screened by multiplex PCR using four primers designed to amplify a 324 bp fragment of collagen α1 and a 537 bp fragment of collagen α2 (Table 2). Figure 3 Shown are the results of a multiplex PCR screen.

[0556] Table 2

[0557]

[0558] Example 3. Detection of human collagen in transgenic tobacco plants.

[0559] By grinding 500mg leaf blade in 0.5ml 50mM Tris-HCl pH=7.5, and adding "Complete" protease inhibitor mixture (product #1836145 from Roche Diagnostics GmbH, 1 tablet per 50ml buffer), total soluble protein was extracted from tobacco transformants 2, 3 and 4. The crude extract was mixed with 250μl 4X. The sample application buffer containing 10% β-mercaptoethanol and 8% SDS was boiled for 7 minutes and centrifuged for 8 minutes at 13000rpm. 20μl supernatant was added to 10% polyacrylamide gel and tested with anti-collagen I (denatured) antibody (#AB745 from Chemical Reagent International) with standard Western blotting procedure ( Figure 4 ). WT is wild type tobacco. Positive collagen bands are seen in plants that are PCR positive for type I collagen alpha 1 or alpha 2 or both. The positive control band of 500 ng type I collagen from human placenta (#CC050 from Chemical Reagents International) represents approximately 0.3% of the total soluble protein (approximately 150 μg) in the transgenic plant samples.

[0560] When collagen was targeted to the vacuole, plants expressing collagen at the expected molecular weight were detected at up to about 1% by weight of total soluble protein ( Figure 4 ). The subcellular targeting of full-length collagen to the apoplast was successfully achieved ( Figure 5 Plants expressing collagen in the cytoplasm (i.e. without the targeting peptide) did not accumulate collagen to detectable levels, suggesting that subcellular targeting of collagen in plants is critical for success.

[0561] In addition, in contrast to the studies of Ruggiero et al. (2000) and Merle et al. (2002), which showed that collagen lacking the N-propeptide underwent significant proteolysis, using the methods of the present invention, full-length collagen with C-propeptide and N-propeptide accumulated at high levels in subcellular compartments.

[0562] The present data also clearly demonstrate that crossing two plants that each express a different collagen chain type is advantageous as it enables selection of plants expressing optimal levels of each chain type and subsequent crossing of the plants to achieve the desired collagen producing plants.

[0563] The collagen produced by the plants of the present invention contains native propeptides and is therefore expected to form larger proteins than human controls purified by proteolysis. The calculated molecular weights of the collagen α1 and α2 chains without hydroxylation or glycosylation are as follows: Col1 with propeptide - 136 kDa, Col1 without propeptide - 95 kDa, Col2 with propeptide - 127 kDa, Col2 without propeptide - 92 kDa.

[0564] like Figure 4 As shown, the Col1 bands in transformants 3-5 and 3-49 appeared larger than the Col1 bands in other plants. This suggests that proline is hydroxylated in the collagen chains by the human proline-4-hydroxylase holoenzyme composed of α and β subunits, which are co-expressed in these plants and targeted to the same subcellular compartments (e.g., vacuoles) as the human collagen chains.

[0565] Example 4. Collagen triple helix assembly and thermal stability in transgenic plants.

[0566] The assembly of collagen triple helices and the thermal stability of the helices in transgenic plants were tested by heat denaturation followed by trypsin or pepsin digestion of total crude protein extracts of transgenic plants ( Figure 6a-6b ).

[0567] In the first experiment, by grinding 500mg leaf blade in 0.5ml 50mM Tris-HCl pH=7.5, centrifuging at 13000rpm for 10 minutes and collecting supernatant, total soluble protein from tobacco 2-9 (expressing colα1 only but not P4H) and 3-5 (expressing both colα1+2 and P4H) was extracted. 0 μl supernatant was heat treated (at 33 ℃ or 43 ℃ for 15 minutes), then immediately placed on ice. By adding 6.mu.l of 1mg / ml trypsin in 50mM Tris-HCl pH=7.5 to each sample, trypsin digestion was started. Samples were incubated at room temperature (about 22 ℃) for 20 minutes. By adding 20 μl 4X sample application buffer containing 10% β-mercaptoethanol and 8% SDS, digestion was terminated, the sample was boiled for 7 minutes, and centrifuged at 13000rpm for 7 minutes. 50 μl of supernatant was loaded onto a 10% polyacrylamide gel and tested using standard Western blotting procedures with anti-collagen I antibody (#AB745 from Chemical Reagents International). The positive control was a 500 ng human collagen I sample (#CC050 from Chemical Reagents International, extracted from human placenta by pepsin digestion) added to 50 μl of total soluble protein extracted from wild-type tobacco.

[0568] like Figure 6a As shown, the collagen triple helix formed in plant #3-5 as well as the control human collagen resisted denaturation at 33° C. In contrast, the collagen formed by plant #2-9 denatured at 33° C. This difference in thermal stability suggests that triple helical assembly and post-translational proline hydroxylation were successfully completed in transformant #3-5, which expresses both collagen α1 and collagen α2 and P4H β and α subunits.

[0569] The two bands in transformant #2-9 may represent dimers or trimers, which are stable after boiling with SDS and mercaptoethanol for 7 minutes. Similar bands are seen in human collagen (above) and transformant #3-5. A possible explanation is the formation of a covalent bond between two peptides in different triple helices (crosslinking) after oxidative deamination of two lysines by lysine oxidase.

[0570] In the second experiment, total soluble protein from transgenic tobacco 13-6 (expressing collagen Iα1 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 100 mM Tris-HCl pH=7.5 and 300 mM NaCl, centrifuging at 10000 rpm for 7 minutes and collecting the supernatant. 50 μl of supernatant was heat treated (20 minutes at 33° C., 38° C. or 42° C.) and then immediately placed on ice. Pepsin digestion was initiated by adding 4.5 μl of 0.1 M HCl and 4 μl of 2.5 mg / ml pepsin (in 10 mM acetic acid) to each sample. The samples were incubated at room temperature (about 22° C.) for 30 minutes. Digestion was terminated by adding 5 μl of unbuffered 1 M Tris. Each sample is mixed with 22 μ l 4X sample application buffer containing 10% beta-mercaptoethanol and 8% SDS, boiled for 7 minutes, and centrifuged for 7 minutes at 13000rpm. 40 μ l supernatant is added in 10% polyacrylamide gel, and tested with standard western blot procedure with anti-collagen I antibody (from the #AB745 of Chemical Reagent International). Positive control is about 50ng human collagen I sample (from the #CC050 of Chemical Reagent International, extracted from human placenta by pepsin digestion), which is added in the total soluble protein from wild-type tobacco.

[0571] like Figure 6b As shown, the collagen triple helix formed in plant #13-6 resists denaturation at 42°C. Cleavage by propionic acid is first seen at 33°C, with increasing efficiency as the temperature is raised to 38°C and again to 42°C. The cleaved collagen triple helical domains show a migration on the gel similar to that of human collagen treated with pepsin. The human collagen used in this experiment was extracted from human placenta by pepsin hydrolysis and therefore lacks propeptides and some telopeptides.

[0572] Example 5. Expression of plant P4H.

[0573] Induction of natural plant P4H

[0574] The tobacco P4H cDNA was cloned and used as a probe to determine conditions and treatments that would induce endogenous P4H expression. Northern blot analysis ( Figure 7 ) clearly showed that P4H was expressed at relatively high levels in the shoot apex and at low levels in the leaves. After 4 hours of abrasion treatment, P4H levels were significantly induced in the leaves ("damaged" in the figure below). Similar results were obtained using other stress conditions (not shown).

[0575] Detection of human P4H α and β subunits and collagen α1 and α2 chains in transgenic tobacco plants

[0576] Human P4H α and β subunits and type I collagen α1 and α2 chains were detected in transgenic tobacco plants using anti-human P4H α subunit antibody (#63-163 from ICN Biochemicals), anti-human P4H β subunit antibody (#NMAB2701 from Chemical Reagents International), and anti-collagen I antibody (#AB745 from Chemical Reagents International). The results of Western blots probed with these antibodies are shown in Figure 8 middle.

[0577] The expression of P4Hα, P4Hβ and collagen 1α1 and α2 bands was confirmed in plant 13-6 (also transformed with human LH3). The calculated molecular weights of P4Hα and β, including the vacuolar signal peptide, were 65.5 kDa and 53.4 kDa, respectively. The calculated molecular weights of collagen α1 and α2 chains with propeptides without hydroxylation or glycosylation were 136 kDa and 127 kDa, respectively.

[0578] Example 6. Stable expression of vacuole-targeted collagen in plants grown in the dark.

[0579] Plants that express collagen:

[0580] 20-279 parental tobacco plant lines were generated by co-transformation with an expression vector expressing P4Hβ+LH3 and another expression vector expressing P4Hα. Each gene was preceded by the vacuolar targeting determinant aleurain, a plant vacuolar thiol protease,

[0581] 2-300 parental tobacco plant lines were generated by co-transformation with an expression vector expressing col1 and another expressing col2. Each gene was preceded by the vacuolar targeting determinant aleurain, a plant vacuolar thiol protease.

[0582] 13-652 plants were generated by co-transforming tobacco plants with expression vectors encoding Col1, P4Hβ and LH3 and a second expression vector encoding Col2 and P4Hα. Each gene was preceded by the vacuolar targeting determinant aleurain, a plant vacuolar thiol protease, and the cassette sequence contained in the vector is described in Example 1 above.

[0583] Light and dark experiments

[0584] Six 13-6 / 52 homozygous plants were analyzed. Samples were taken from leaves #4+5 / 6 at the same time every day (12:30) for 8 days from 3 plants grown under conventional conditions (16 hours in light, 8 hours in the dark) and 3 plants grown only in the dark.

[0585] Total protein extraction and western blot analysis

[0586] At 4 ℃, 90mg of tobacco leaves were homogenized in extraction buffer (100mM Tris HCl pH=7.5, protease inhibitor cocktail obtained from Roche (Roche) catalog number, 04-693-116-001) by MM301 type mixer mill (Retsch). After centrifugation for 30 minutes (at 4 ℃, 20,000Xg), the supernatant was collected. Protein samples were graded on 8% SDS-PAGE (Laemmli 1970) and purified by BIO-RAD TM Protein TRANS-BLOT TM The device was transferred to a nitrocellulose membrane. The membrane was blocked with 3% (g / v) skim milk (Difco) at room temperature for 30 minutes and then reacted with any commercial rabbit anti-human type I collagen polyclonal antibody (Chemicon) at room temperature overnight (on). The membrane was rinsed with water 3-5 times and then washed in TBS for 30 minutes. After incubation with the secondary antibody [goat anti-rabbit IgG antibody (Chemicon) coupled to alkaline phosphatase (AP)] for 2 hours at room temperature, the membrane was rinsed with water 3-5 times and washed in TBS for 30 minutes. Immunodetection was performed at room temperature with tetrazolium blue chloride (NBT, Sigma) and 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt (BCIP, Sigma) for 2 hours to overnight.

[0587] result

[0588] like Fig. 9 As shown, tobacco plants transgenic for vacuolar-targeted collagen expressed Pro-α-1 and Pro-α-2 (lane 1). Collagen from vacuolar-targeted plants grown in the dark showed similar stability (lane 2), thus confirming the exceptional stability of collagen produced according to the teachings of the present invention.

[0589] Example 7-13.

[0590] General Materials and Methods

[0591] Extraction and enzymatic reaction of collagen: 300 g of tobacco leaves were mixed in a blender in a cooled extraction buffer (600 ml of 100 mM Tris-HCl pH 7.5, containing 360 mg potassium metabisulfite, 530 mg L-cysteine ​​and 1 g EDTA) supplemented with 5 g PVPP and 2 g activated carbon (see also U.S. Patent 8,759,487). Mixing was performed 5 times at 1 minute intervals to keep the temperature below 15°C. The crude extract was filtered through a gauze pad and centrifuged at 25000 g, 5°C for 30 minutes. The supernatant was collected; CaCl was added 2 To a final concentration of 10 mM. The supernatant was divided into 10 ml samples. The desired enzyme was added to each 10 ml sample according to the conditions listed in Table 3 below.

[0592] Table 3.

[0593]

[0594] Description of enzymes: Ficin from fig tree latex (Sigma, catalog #F4125); Subtilisin from Bacillus licheniformis (Sigma, catalog #P5459-5gr); Bromelain from pineapple stem (Sigma, catalog #B4882-10gr); Papain from papaya (Fluka, catalog #76220-25gr); Savinase 6.0t type W from the alkaliphilic bacterium Bacillus (Novozymes, catalog #PX92500501); Neutrase 1.5MG from Bacillus amyloliquefaciens (Novozymes, catalog #PW201041); Complex protease, a commercial Bacillus protease complex (Novozymes, catalog #PW2A1021); Alcalase 3.0T, alkaline protease from Bacillus subtilis (Novozymes, catalog #PJ90000901); Esperase 6.0T, alkaline-loving bacteria Bacillus (Novozymes, catalog #PE90110401); Alcalase 2.4L FG, alkaline protease from Bacillus subtilis (Novozymes, catalog #PLN05330); Esperase 8.0L, alkaline-loving bacteria Bacillus (Novozymes, catalog #PE00077), all donated by Novozymes. Trypsin, salt-free S-type pancreatic trypsin 6.0, from animal pancreas (Novozymes, catalog #P245-D20). TRYPZEAN TM , a recombinant trypsin expressed in maize, was purchased from Sigma Chemical Co. (Catalog #: T3449).

[0595] Determination of atelocollagen concentration: The concentration of atelocollagen produced according to Examples 9-10 was determined by the following two methods:

[0596] SIRCOL TM Assay: SIRCOL TM Collagen assay kit was purchased from Biocolor Ltd. (Cat. No. 85000). This assay is based on the interaction of Sirius red dye with collagen triple helix. The analysis was performed according to the supplier's instruction manual (4th edition, 2002). Calibration curves were prepared using bovine collagen standards (0 to 50 μg collagen). Three samples of 10-50 μl collagen solution in 10 mM HCl were placed in 1.5 ml Eppendorf tubes and the volume was adjusted to 100 μl with 0.5 M acetic acid. 1 ml SIRCOL was added to each test tube. TM Dye reagent and shake the test tube at room temperature for 30 minutes. Centrifuge the test tube at 12,000 rpm for 10 minutes at room temperature, aspirate the supernatant, and invert the test tube on absorbent paper to remove residual supernatant. Use a cotton swab to remove any contact drops on the tube wall. Add 1 ml of alkaline reagent to each tube, mix well and incubate at room temperature for 10 minutes. Measure the absorption at 540 nm using a spectrophotometer and calculate the concentration of collagen based on the calibration curve using 10 mM HCl as a blank sample.

[0597] SDS-PAGE Instant Blue Assay: The samples were boiled in SAB buffer for 5 minutes (reducing conditions) and centrifuged at 12,000 rpm for 5 minutes before loading on SDS PAGE (8% acrylamide). The gel was run in a Mini Protean 3 apparatus (BioRad #165-3301, 165-3302). Instant Blue reagent (Novexin #ISB01L) was applied to the gel until the proteins appeared as blue bands on the gel. The gel was rinsed with water and dried. The concentration of the collagen band was calculated by densitometry against a human standard loaded on the same gel.

[0598] Coomassie analysis: Collagen samples (in 10 mM HCl) were titrated to pH 7.5 using 1 M Tris. Sample application buffer containing 10% β-mercaptoethanol and 8% SDS was added by diluting it four-fold in 30 μl of the pH-titrated sample. The samples were boiled for 7 minutes. 30 μl of the supernatant was loaded onto a 10% polyacrylamide gel and separated at 100 volts for 2 hours. The gel was transferred to a Coomassie-based solution with shaking for 1 hour. The Coomassie dye was removed using a standard decolorization solution.

[0599] SDS-PAGE and Western Blot Analysis of α-1 and α-2 Collagen Chains: The samples were boiled in reducing sample application buffer (2.5% β-mercaptoethanol and 2% SDS) for 7 minutes 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 standard Western blotting methods. After transfer, the membranes were incubated with anti-collagen I antibodies (Chemical Reagents International, Inc., catalog # AB745) for immunodetection of α-1 and α-2 collagen chains. Molecular weight markers were purchased from Fermentas Inc. (catalog # SM0671).

[0600] Controls: A positive control of type I human skin collagen purchased from Calbiochem (#234138) was used as a marker for western blot analysis. The ground control sample reflects the precipitate immediately extracted from tobacco leaves prior to resuspension in extraction buffer. The "D" control sample reflects the same precipitate after resuspension in extraction buffer. The "K" control sample contains procollagen digested with ficin in 10 mM HCl. In order to monitor protease activity independent of background ficin, a ficin-free cleavage sample was always prepared in parallel with all ficin digestion tests.

[0601] Purification of collagen from transgenic plants: Digestion of propeptides in collagen-containing extracts was initiated by adding 30 mg / L trypsin or 5 mg / L (50 μl / L) subtilisin (Sigma #P5459) or 5 mg / L ficin (Sigma #F4125). Proteolysis was performed at 15°C for 4 hours. Insoluble contaminants were eliminated by centrifugation at 22,000g for 30 minutes at 15°C. The supernatant was recovered and the collagen was precipitated by slowly adding crystalline NaCl to a final concentration of 3.13 M with continuous stirring for 20 minutes at room temperature. The solution was incubated overnight in a cold room without stirring. Collection of collagen was achieved by centrifugation at 25,000g for 2 hours at 5°C.

[0602] Carefully pour the supernatant into four layers of gauze pads. Resuspend the pellet in 200 ml of 250 mM acetic acid and 2 M NaCl using a magnetic stirrer for 5 minutes. Centrifuge the suspension at 25,000 g for 40 minutes at 5 ° C. Remove a trace of supernatant from the glass bottle. Redissolve the pellet in 200 ml of 0.5 M acetic acid at room temperature for 1 hour. Eliminate insoluble material by centrifugation at 16,000 g for 30 minutes at 15 ° C. Pour the supernatant into 12 layers of gauze pads. Precipitate collagen by slowly adding NaCl to a final concentration of 3 M with constant stirring at room temperature for 20 minutes. Incubate the solution at 4 ° C for 8 hours to overnight. Collect collagen by centrifugation at 25,000 g for 2 hours at 5 ° C. After aspirating the supernatant, redissolve the pellet in 200 ml of 0.5 M acetic acid at room temperature using a magnetic stirrer for 1 hour. Eliminate insoluble material by centrifugation at 16,000g for 30 minutes at 15°C. Pour the supernatant into a 12-layer gauze pad. Precipitate collagen by slowly adding NaCl to a final concentration of 3M and stirring continuously at room temperature for 20 minutes. Incubate the solution at 4°C for 8 hours. Collect collagen by centrifugation at 2,000g for 2 hours at 5°C. Suck off the supernatant. Redissolve the precipitate in 40ml of 10mM HCl by pipetting and vortex at room temperature for 5 minutes. Transfer the solution to a dialysis bag (MWCO 14,000Da) and dialyze for 4 hours at 4°C in 4L of 10mM HCl. Repeat this dialysis.

[0603] The collagen was sterilized by first filtering the solution through a 0.45 μm filter and then filtering 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, #VS2041, MWCO 100'000). Centrifugation was performed at 5000 g for 45 minutes at 5°C until the volume was reduced to 0.75 ml.

[0604] Optimization of digestion kinetics and conditions for procollagen cleavage by food grade ficin: The precipitate (collected as described in Example 10 until saturation in 25% ammonium sulfate (AMS)) was resuspended in buffer (buffer A: 4.5 mM potassium metabisulfite, 12.5 mM L-cysteine, 7.5 mM EDTA dissolved in 0.1 M sodium phosphate buffer, titrated to pH 7.5 with 10 M NaOH or 6 N HCl) at a ratio of 4.36 g precipitate: 200 mL ice-cold buffer. The sample was then stirred at 15°C for 20 minutes. Aliquots of 10 mL in each 15 mL test tube were then prepared and increasing concentrations (5-15 mg / L) of ficin (fig tree latex, Biochem Europe food grade ficin) were then applied. Samples were incubated at 15°C for 1-3 hours and separated by SDS-PAGE and analyzed by Western blotting for the presence of collagen that migrates at a lower molecular weight than procollagen.

[0605] In the presence of 0-3M NaCl, the precipitate from tobacco leaves resuspended in phosphate buffer A (27.2 g: 800 mL buffer) of different pH values ​​(5.5, 7.5 or 8.5) was treated with 10 mg / L ficin at 15°C for 1 hour. The reaction was terminated by centrifuging 1 mL sample from each reaction mixture (10 minutes, 15000 g, 4°C). The precipitate was resuspended in 1 mL buffer A (pH 7.5), separated by SDS-PAGE, and analyzed by Western blotting.

[0606] Digestion kinetics and optimization of conditions for procollagen cleavage by pharmaceutical grade ficin: Tobacco leaf pellets were resuspended in extraction buffer (10 mg / L) containing pharmaceutical grade (Biochem-Europe pharmaceutical grade) ficin at different pH values ​​(7.5, 8.5, 9.5) and increasing NaCl concentrations (0-3M) for 5-45 minutes. Further experiments investigated the necessity, optimal conditions and concentrations of EDTA and L-cysteine ​​as extraction buffer additives. Samples were incubated in the digestion mixture at 15°C, pH 7.5 and without NaCl for 1-3 hours in the presence of 0-100 mM EDTA and 0-80 mM L-cysteine.

[0607] Fibril formation: Fibril formation is considered a test of collagen functionality. Therefore, the ability of the purified collagen digested by ficin to form fibrils is an essential property of the obtained product. Test method: The pH of the collagen-containing solution (duplicate samples) is neutralized to pH 6.7 with sodium phosphate (pH 11.2) and then incubated for 6 hours at 27+ / -2μC. The samples are centrifuged to precipitate the formed hydrogel. The protein concentration of the samples before and after neutralization (supernatant) is determined by the Lowry method. PURECOL TM (purchased from NUTACON, catalog number 5409) was used as a positive control, while gelatin was used as a negative control.

[0608] Example 7. Extraction and purification of collagen from transgenic plants in the presence of trypsin and pepsin.

[0609] The production of human collagen in plants was initiated in order to avoid the use of collagen from mammalian sources, since the use of mammalian proteins in human cosmetic or medical applications may be risky for human health due to the relatively close evolutionary proximity. One example is the known Creutzfeldt-Jakob disease (CJD), which is caused by humans consuming infected mammalian proteins.

[0610] Initially, collagen was purified from transgenic plants using bovine pancreatic trypsin and the digestive protease pepsin, both enzymes that catalyze the hydrolysis of proteins in the animal digestive system. The following example illustrates the identification of non-animal derived proteases suitable for use in the collagen purification process.

[0611] result

[0612] First, the propeptide digestion during collagen purification was performed by the pancreatic enzyme trypsin. Trypsin digested collagen propeptides at 300 mg / L, but at the end of the purification process, the collagen yield was very low ( Fig.10 When the trypsin concentration was reduced to 20 mg / L or 30 mg / L, the yield was higher, but procollagen digestion was only partial and inconsistent between the same samples ( Fig.11 ).

[0613] To overcome this problem, different incubation temperatures and times were tried; however, the results did not result in a change in yield (data not shown). Adding pepsin later in the purification process can solve the partial digestion problem ( Fig.12 ), and produced alpha-1 and alpha-2 collagen that migrated together with a control sample of porcine-derived collagen.

[0614] Example 8. Extraction of collagen and its enzymatic digestion.

[0615] However, a trypsin-pepsin solution is not optimal because it requires two different enzymes, which prolongs the purification process. In addition, both enzymes are from animals. To overcome these problems, a screen of different proteases from non-animal sources was conducted. By screening different enzymes, different digestion patterns can be obtained. Collagen and Savinase ( Fig.15 ) and Esperase ( Fig.17 ) little or no observable digestion of the propeptide was observed. Fig.14 )、Bromelain( Fig.13 ), Alcalase 2.4L and Esperase 8.0L( Fig.18 ) together with the protease resulted in over-digestion or under-digestion of the propeptide. Fig.16 ) resulted in the expected digestion pattern and level (25 mg / L, 6 hours), with the migration of the α1 and α2 chains similar to the porcine derived collagen samples. However, not all molecules were completely digested, so longer incubation times may be required. The best results were obtained when the procollagen was incubated with ficin (5 mg / L and 25 mg / L) ( Fig.15 ), where the bands of α1 and α2 chains showed no significant digestion impairment compared to the control sample of porcine collagen. Fig.13 ) and neutral enzyme 25mg / L for 6 hours ( Fig.17 ) demonstrated similar results.

[0616] Example 9. Extraction and purification of collagen from transgenic plants after digestion with subtilisin or ficin.

[0617] Collagen was purified from 450 gr of transgenic plant leaves (13-361 or 13-6-52) and then treated with ficin ( Fig.19 ) or subtilisin ( Fig. 20 ) for procollagen digestion. Collagen samples at various stages of the purification process were analyzed by protein analysis. Digestion of the propeptides with ficin and subtilisin resulted in the desired degree of processing of collagen 1 and collagen 2. Lower molecular weight bands were observed on Western blots throughout the purification process, but these bands were present in the plant extract prior to incubation with the enzymes (lanes 3-4) and in the control sample of porcine-derived collagen (positive control) ( Fig.19 ).

[0618] Example 10. Scale-up extraction and purification of collagen from transgenic plants after digestion with ficin.

[0619] In a 4L reactor (ESCO EL-3 type), 1kg of transgenic tobacco leaves were ground together with precooled 2L extraction buffer (100mM sodium phosphate buffer pH 7.5, 4.5mM potassium pyrosulfite, 12.23mM L-cysteine ​​and 7.5mM EDTA) for 20 minutes (5°C, scraper speed is 50% and homogenizer blade speed is 100%). 6.68g of charcoal and 16.67g of PVPP were added to the extract and stirred continuously for 20 minutes (5°C, scraper speed is 50%). The extract was centrifuged (11000rpm, 5°C, 0.5H), and the supernatant was saturated with 15% ammonium sulfate (stirred for 1 hour, 5°C). At 6880rpm, 5°C, after 30 minutes, the supernatant was saturated to 25% ammonium sulfate and stirred for 1 hour (5°C). After re-centrifugation, the precipitate (6880rpm, 5°C, 30 minutes) was resuspended (in extraction buffer) to 15% of the volume collected after the first centrifugation. The propeptide can be removed by digestion with 5mg / L ficin (Biochem Europe) at 15°C for 3 hours. The sample was centrifuged (11,000rpm, 15°C, 30 minutes) and the mature collagen was precipitated using 3M NaCl (NaCl was added slowly under stirring and left overnight at 4°C). After precipitation (13,000rpm, 5°C, 2 hours), the supernatant was discarded and the precipitate was resuspended in 0.5M acetic acid. After another round of 3M salting out (overnight) and centrifugation, the precipitate was resuspended in 40ml of 10mM HCl. The sample was transferred to a dialysis bag (12-14kDa) and dialyzed in 4L 10mM HCl at 4°C for 4 hours. The dialysis was repeated with fresh 4L 10mM HCl overnight. The dialyzed solution was filtered through a 0.45 micron filter (previously washed with 10 mM HCl) and then through a 0.25 micron filter. The sample was finally concentrated in a Vivaspin (Vivascience) filter tube (100 kDa).

[0620] Example 11. Solubility of atelocollagen produced as recombinant human procollagen in transgenic tobacco plants.

[0621] As described in the "Methods" section, the concentration of atelocollagen produced according to Examples 9-10 was determined by the following two methods. Table 4 lists the resulting concentrations obtained for several typical preparations digested with ficin:

[0622] Table 4.

[0623]

[0624] Example 12. Ficin-dependent proteolysis of tobacco leaf-derived procollagen.

[0625] Digestion kinetics of procollagen by food-grade ficin: To calibrate the appropriate ficin concentration and incubation time to achieve the highest collagen yield, tobacco leaf pellets expressing procollagen were incubated with increasing concentrations of food-grade ficin (5-15 mg / L) at 15°C for 1-3 hours. The samples were then analyzed by immunodetection of α-1 and α-2 collagen chains on Western blots. After a 1-hour incubation period, increasing ficin concentrations provided an increase in collagen chain yield ( Fig. 22 , lanes 5 vs. 6). However, increasing ficin concentrations with increasing reaction time resulted in overdigestion of collagen ( Fig. 22 , lanes 11 vs. 12-14 and lanes 17 vs. 18-20). Therefore, the optimal conditions for the digestion of procollagen to collagen were set as: adding 10 mg / L food grade ficin at 15°C for 1 hour.

[0626] Digestion Kinetics of Procollagen by Pharmaceutical Grade Ficin: Similar experiments were performed on tobacco leaf pellets expressing procollagen to determine the appropriate conditions for procollagen digestion by pharmaceutical grade ficin. The pellets were resuspended and incubated with increasing concentrations of pharmaceutical grade ficin (2.5-10 mg / L) at 15°C for 0.5-3 hours. The efficiency of digestion was determined by immunodetection of collagen chains on a Western blot. Figure 23A-23C As shown, increasing ficin concentrations resulted in increased collagen yields and decreased procollagen levels. Procollagen was most efficiently digested with 10 mg / L pharmaceutical grade ficin after 1 hour of reaction time.

[0627] Optimization of pH and salt concentration for ficin-dependent procollagen cleavage: The contribution of digestion buffer pH and salt concentration was then evaluated. Tobacco leaves following similar AMS precipitation were resuspended in extraction buffer titrated to pH 5.5, 7.5, 8.5, or 9.5 with salt content ranging from 0.5-3 M NaCl. Samples were then incubated with 10 mg / L pharmaceutical grade ficin at 15 °C for 1 h prior to immunoassay on a Western blot. Acidic assay conditions (pH 5.5) resulted in insufficient collagen yield ( Fig.24A , lanes 2-6), while an increase in pH demonstrated a ficin-dependent increase in collagen content, with a peak observed at pH 8.5 in the presence of 2 M NaCl ( Fig. 24B, channel 10). These results further confirm the results of the scaled-up extraction and purification experiments, which were performed on two 15kg precipitates that were pooled for ficin-induced procollagen digestion. In addition to the increase in collagen chain yield observed by immunoblotting, the samples digested in the buffer of pH 8.5 in the presence of 2M NaCl were as effective as the samples digested in buffer A (pH 7.5, 0mM NaCl) (see Table 5 below - YC1 batches and YC2 batches). Therefore, after ficin-induced procollagen digestion, higher pH and salt concentrations all provided improved collagen yields.

[0628] Determination of the activity of EDTA and L-cysteine ​​in the digestion reaction mixture: Both EDTA and L-cysteine ​​are additives present in the extraction buffer at the early stages of the collagen purification process. Here, the necessity of these two components for efficient ficin-dependent collagen cleavage was determined. Procollagen following AMS precipitation was 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 and pH 7.5 for 1 hour. In the presence of 10 mM L-cysteine, a significant enhancement of the digestion efficiency was observed ( Fig.25 , channels 7-10), whereas EDTA had no significant contribution to ficin-dependent collagen export ( Fig.25 , channels 7 vs. 8-10).

[0629] 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 another 1.5 hours. Western blot and fibril formation assays did not reveal any improvement in collagen yield or sample purity associated with increased reaction temperature.

[0630] Fibril formation of collagen extracted from ficin-induced procollagen cleavage: After ficin-induced digestion, a fibril formation assay was performed to determine the ability of the resulting collagen to form fibrils, which is the ultimate method to determine collagen function. Table 5 below summarizes the fibril formation results determined using two variant protocols after ficin cleavage of procollagen. Protocols A and B differed in reaction buffer pH and salt content, and both produced large amounts of collagen fibrils. Therefore, the proteolytic reaction parameters developed and optimized herein resulted in a high yield of functional collagen.

[0631] Table 5.

[0632]

[0633] Example 13. Determination of the effect of TRYPZEAN in procollagen cleavage TM The efficacy of proteases.

[0634] The procollagen-expressing tobacco leaf pellet was resuspended in an extraction buffer (pH 7.5) enriched with EDTA (7.5 mM) and L-cysteine ​​(12.5 mM) and incubated with TRYPZEAN TM (30-100 mg / L) were incubated at 15°C for 1-3 hours. Within 1 hour, 60 and 100 mg / L of TRYPZEAN TM Procollagen was efficiently cleaved to generate two distinct alpha collagen chains with no detectable overdigestion ( Fig.26 ). Therefore, use TRYPZEAN pH 7.5 TM Processing of procollagen results in efficient digestion of collagen chains alpha-1 and alpha-2.

[0635] discuss

[0636] Examples 7-13 above describe the identification of non-mammalian proteases suitable for use in the process of purifying collagen from plant sources. Proteases from bacterial and plant sources were examined and three enzymes were found to be suitable for digestion of collagen propeptides, namely, neutral enzyme, subtilisin, TRYPZEAN TM and ficin.

[0637] Both neutral enzyme and subtilisin are secreted by the bacterium Bacillus. Subtilisin is mainly (>90%) used in detergents and household cleaning products. About 10% of subtilisin is used in technical applications, such as protein hydrolysis, leather processing and textile and cosmetic industries. The standard use of subtilisin in collagen purification processes is problematic due to overdigestion of collagen. Neutral enzyme is mainly used in the beverage alcohol industry and in cheese ripening. In Examples 7-13 described above, neutral enzyme is only effective when digesting propeptides at high concentrations and requires at least 6 hours to obtain the desired digestion results.

[0638] Under the experimental conditions described so far, it was found that among the four proteases, recombinant trypsin and ficin were the most suitable, since there was no overdigestion of collagen after high enzyme concentrations or extended incubation times. In addition, as determined by SDS PAGE analysis, these enzymes apparently do not digest the helical regions of collagen. Ficin is a natural enzyme of the fig plant (Ficus carica) that is commercially available from a variety of sources in a variety of grades (including pharmaceutical grades) at low cost. Ficin is used in the food industry: alcohol and beer industries, protein hydrolysis, meat processing, baking industry, and in the preparation of pet foods and health foods. Ficin can also be used in the pharmaceutical industry: contact lens cleaners, cancer treatments, anti-arthritis treatments and digestive aids, as well as in the cosmetics and textile industries.

[0639] Example 14. Further analysis of rhCollagen properties.

[0640] Materials and methods

[0641] Material

[0642] Type I human recombinant collagen (rhCollagen) expressed and isolated from transgenic tobacco plants was produced and provided by CollPlantLtd (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-methylacetophenone (Irgacure 2959), anhydrous sodium dihydrogen phosphate, HCl 1N (HCl ≥ 37%), sodium bicarbonate and NaOH were purchased from Sigma Aldrich Ltd, Israel. Phosphate buffered saline (PBS), x10PBS, fetal bovine serum (FBS), DMEM high glucose and penicillin / streptomycin were purchased from iologicalIndustries Ltd, Israel. Anhydrous disodium hydrogen phosphate was purchased from Canton, India. Anhydrous ethanol and acetone were purchased from Bio-Lab Ltd, Israel. Hyaluronic acid was purchased from Lifecore, USA.

[0643] Preparation of buffer and photoinitiator stock solutions

[0644] Fibril formation buffer (FB): Disodium hydrogen phosphate was dissolved in double distilled water (DDW) to a final concentration of 162 mM. The solution was titrated to pH 11.2 with 10 N NaOH.

[0645] Preparation of culture medium: Under sterile conditions, add 50 ml of fetal bovine serum and 5 ml of penicillin / streptomycin (10,000 units / mL and 10 mg / mL, respectively) to 500 ml of DMEM high glucose medium. Mix the medium gently and store in the refrigerator.

[0646] Preparation of phosphate buffered saline: Mix 39 ml of 0.1 M sodium dihydrogen phosphate solution with 61 ml of 0.1 M sodium dihydrogen phosphate solution and adjust the final volume to 200 ml with DDW. Adjust the final pH to 7 with concentrated NaOH or HCl as needed. Add NaCl to a final concentration of 150 mM.

[0647] Wash Buffer: Add HCl to the fibrillation buffer to reach a final concentration of 16.2 mM sodium phosphate dibasic and 10 mM HCl. Adjust the pH to 7.2-7.4 with 10 N NaOH.

[0648] Photoinitiator 10% (v / v) stock solution: Dissolve Irgacure 2959 in a 1:1 solution of absolute ethanol / PBS to a final concentration of 100 mg / mL.

[0649] Methacrylation of rhCollagen

[0650] Fibrous rhCollagen-methacrylamide and monomeric rhCollagen-methacrylamide were prepared by reacting lysine and hydroxylysine collagen residues with methacrylic anhydride in aqueous medium as described below and stored in the light at 4°C until further use.

[0651] Fibrous rhCollagen-Methacrylamide

[0652] Synthesize 3 to 10 mg / mL of fibrillar rhCollagen-methacrylamide in wash buffer, fibril formation buffer or DDW at room temperature (RT) or 12°C. For example, briefly, fibrillar collagen-MA was synthesized in DDW as follows: monomeric rhCollagen (3-4 mg / mL solution in 10 mM HCl) (COLLAGE TM) was mixed with fibril formation buffer at a ratio of 9:1 v / v, and stirred at room temperature for 1 hour to accept fibrils. The solution was centrifuged at 7500 rpm for 30 minutes at 4 ° C, thereby discarding the supernatant. The precipitate was resuspended in an equal volume of wash buffer and centrifuged under the same conditions. Afterwards, the precipitate fibrils were resuspended in DDW to 10 mg / mL. Concentration was confirmed by solid volume percentage. Methacrylic anhydride (MA) was added dropwise at room temperature under nitrogen flow with a molar ratio of 10 to 20 relative to collagen lysine, and the pH of the reaction solution was monitored over time and adjusted to pH 7 with 10N NaOH. After 24 hours of reaction, the mixture was dialyzed in wash buffer (pH 7) at 4 ° C using a 10kDa cut-off dialysis tube (Spectrum Laboratories Inc, California, the United States) for 3 days, and at least 6 dialysate changes (wash buffer in this case) were performed to remove reaction byproducts, and finally lyophilized for 3-4 days.

[0653] rhCollagen-methacrylamide monomer

[0654] Use 200 mM MOPS, phosphate, or Tris buffer supplemented with 150 mM NaCl. For example, add 200 mM MOPS and 150 mM NaCl to 3-4 mg / mL COLLAGE TM , and stirred at room temperature until a clear solution was obtained. Thereafter, 10 to 20 times excess methacrylic anhydride was added dropwise at 12°C under a nitrogen stream, and the pH was adjusted to pH 7 over time with 10N NaOH. After 24 hours of reaction, the mixture was dialyzed in 10mM HCl and 20mM NaCl (pH 2) at 4°C for 3 days using a 10kDa cut-off dialysis tube, and at least 6 dialysate changes were performed, followed by lyophilization for 3-4 days.

[0655] Methacrylate esterification of hyaluronic acid (HA)

[0656] 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 added to 50 ml of a 10 mg / mL HA solution in DDW and mixed thoroughly before adding the next component. The reaction was mixed overnight at room temperature, and then the HAMA was precipitated in 20 volumes of acetone and redissolved in DDW. The precipitation process was repeated twice to eliminate all reaction residues. The material was finally freeze-dried.

[0657] Solution preparation for viscosity measurements

[0658] PureCol and Collage in PBS TM : 8 ml of monomeric collagen solution (3 mg / mL, in 10 mM HCl), rhCollagen (COLLAGE TM ) or bovine collagen (PureColl). The solution was then brought to pH 7-7.5 by titration with 0.1 N NaOH. Double distilled water was finally added to a final volume of 10 ml. The samples were incubated at 37°C for at least 90 minutes before measurement (37°C or 4°C).

[0659] COLLAGE in fibrillation buffer TM : Neutralize 9 ml of monomeric rhCollagen (COLLAGE TM ) solution (3.79 mg / mL in 10 mM HCl). Samples were incubated at 37°C for at least 90 minutes before measurements were taken (37°C or 4°C).

[0660] Fibrous rhCollagen-methacrylamide in PBS: Lyophilized fibrillar rhCollagen-MA prepared in DDW and dialyzed against wash buffer (with 10-fold excess MA as described above) was dissolved in PBS to a concentration of 10 mg / mL. Samples were incubated at 37°C for at least 90 minutes before measurements were made (37°C or 4°C).

[0661] Fibrillar rhCollagen-methacrylamide in DMEM: Lyophilized fibrillar rhCollagen-methacrylamide (15-fold excess of MA, prepared as described above and dialyzed in wash buffer) was dissolved in DMEM medium to a final concentration of 20 and 26 mg / mL.

[0662] rhCollagen-Methacrylamide / Hyaluronic Acid in DMEM: Hyaluronic acid was added to the solution of fibrillar rhCollagen-MA to obtain a final concentration of 10 mg / mL HA and 20 mg / mL rhCollagen-MA in DMEM medium.

[0663] rhCollagen-Methacrylamide / Hyaluronic Acid Methacrylate (HA-MA) in DMEM: Hyaluronic acid methacrylate (see above) was added to the solution of fibrillar rhCollagen-MA to obtain a final concentration of 10 mg / mL HA-MA and 20 mg / mL rhCollagen-MA in DMEM medium.

[0664] Photocrosslinking of rhCollagen-MA for loss modulus and storage modulus measurements

[0665] The cross-linked rhCollagen-MA scaffolds were formed in two different preparations, which were intended to be examined in two separate experiments. In the first preparation, 1-2wt% of fibrous rhCollagen-MA synthesized with a 10-fold excess of methacrylic acid reagent was dissolved in 0.1M PBS at room temperature, then 0.1% of Irgacure 2959 was added and a final volume of 1mL of the solution was injected into a disc-shaped mold. Subsequently, a curing process was performed using a mercury light source at a distance of 1.5m at an average intensity of 670mW / cm2 for 7 seconds and 10 seconds to finally form the cross-linked scaffold. The second preparation contained 2 different batches of fibrous rhCollagen-MA, which were synthesized with a 15-fold and 20-fold excess of methacrylic acid reagent. 1-2wt% was dissolved in 0.1M PBS, then 0.1% of Irgacure 2959 was added to reach a final volume of 1.5mL. To obtain highly cross-linked scaffolds, a mercury light source was used at a distance of 2m at an average intensity of 420mW / cm 2 The average strength of the curing process lasts 60 seconds.

[0666] TNBS assay

[0667] The assay protocol is 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 4% sodium bicarbonate solution containing 0.1-2 mg / mL fibrous rhCollagen-MA. After reacting for 2 hours at 40°C, 0.2 mL of 1N HCl and 0.4 mL of 10% (v / v) SDS were added. The absorbance was measured at 335 nm in 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 rhCollagen-MA solution. The absorbance of 1-2 mg / mL native fibrous rhCollagen prepared under the same conditions was recorded for calibration.

[0668] Rheological characterization

[0669] Viscosity: Viscosity measurements were performed on a HAAKE RHEOSTRESS600TM rheometer (Thermo Electron Corporation) with a temperature controlled cell chamber using a C60 / 1° Ti cone and plate apparatus. The viscosity of 1 mL samples was measured in a rotating ramp mode at 4°C, 25°C and 37°C with a shear rate range of 0.0001 to 1000 sec-1.

[0670] Storage and loss modulus of scaffolds: The rheological behavior of rhCollagen cross-linked disks was studied using a parallel plate system with a PP20 serrated spindle and a 20 mm serrated plate. For the characterization of uncross-linked rhCollagen-MA, a C60 / 1° Ti cone-plate element was used. To evaluate the rheological behavior of rhCollagen-MA, two sets of experiments were performed. First, an oscillating force was applied to a 1 mL sample in controlled stress mode and the storage modulus G' and loss modulus G" values ​​were recorded while applying a shear stress of 5 Pa at a frequency of 1 Hz and 37 °C for 300 s. The gap was adjusted to 90% of the original sample height in the range of 150-300 s and the G' and G" values ​​were averaged. In the second experiment, a 1.5 mL cross-linked disk was tested in a frequency swept oscillation at 37 °C, where G' was recorded at a shear stress of 1 Pa in the frequency range of 0.01-100 Hz. To start the measurement, the spindle was lowered to touch the hydrogel surface and then lowered further until the axial force of the instrument was equal to 0.4 N. Prior to all measurements, the sample was placed on a plate covered with a wet cover for 1 min to allow temperature equilibrium to be achieved.

[0671] result

[0672] TNBS assay

[0673] The modification degree of rhCollagen was quantified using TNBS colorimetric assay. The assay quantified the molar content of free, unreacted ε-amino groups derived from lysine and hydroxylysine, and the subsequent functionalization degree. The functionalization degree of 10, 15 and 20 times different batches of fibrous rhCollagen was determined by TNBS assay, as shown in Table 6.

[0674] Table 6: Functionalization degree of fibrous rhCollagen from different formulations determined by TNBS assay.

[0675]

[0676] The results demonstrate the high modification capacity of fibrillar rhCollagen and suggest that the addition of methacrylic acid reagent at a 10 molar ratio may be preferred for maximum functionalization of receptive fibrillar collagen.

[0677] Rheology

[0678] 1. Viscosity

[0679] Temperature dependence of rhCollagen / bovine collagen viscosity

[0680] Fig. 27 rhCollagen (COLLAGE TM) 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 the zero shear rate viscosity (η0), i.e., a viscosity plateau at low shear rate values, with η0 values ​​at 37 ° C (red) being an order of magnitude higher than those at 4 ° C (blue). In contrast, rhCollagen (dashed line) shows no significant difference between the η0 values ​​at 4 ° C and 37 ° C. rhCollagen neutralized in FB (see "Methods") shows a very similar behavior ( Fig.28 ), that is, the viscosity at 4°C and 37°C is almost the same. Fig.29 The viscosity of fibrous rhCollagen-MA at 4°C (blue line) and 37°C (red line) is shown. Although the curves are not identical, the zero shear rate values ​​at both temperatures are approximately 1000 cP.

[0681] Viscosity of rhCollagen-Methacrylamide

[0682] Fig.30 The viscosity of rhCollagen-MA dissolved in DMEM at 25°C is shown. rhCollagen-MA also maintained its viscosity at 25°C with and without the addition of HA / HA-MA. Fig. 27 and 28 Typical shear-thinning behavior of rhCollagen as seen in Figure 2. Increasing the concentration of rhCollagen from 20 to 26 mg / mL (green and red lines, respectively) increases the zero-shear viscosity and results in a final polymer concentration of 30 mg / mL due to the later addition of 10 mg / mL HA or HAMA.

[0683] The skilled artisan will recognize that rhCollagen-MA is not cross-linked and that in order to achieve cross-linking, the addition of a photoinitiator and light is required.

[0684] 2. Loss modulus and storage modulus of the stent

[0685] In the first experiment, rheological analysis of 1 mL disks at 37 °C over time showed Fig.31The upper graph reports the loss and storage moduli as well as tan(δ) before UV curing, while the lower graph reports the values ​​after UV curing (upon addition of photoinitiator). The data show that the storage modulus of rhCollagen-MA increases 2-fold upon irradiation in the presence of photoinitiator. Moreover, the results point to the ability to control the properties of the scaffolds by varying the rhCollagen-MA concentration. The high difference between G' and G" values ​​and the near-zero tan(δ) values ​​of the cross-linked disks indicate that they have an elastic-like behavior. (G' - storage modulus; G" - loss modulus; G', "storage / elastic modulus", represents the G stored by the gel during deformation and used to subsequently recover its original shape. * G' measures the elastic behavior of a gel or how much of its shape it can recover after shear deformation. For example, vulcanized rubber is a purely elastic material that deforms immediately when stressed and fully recovers its shape after the stress is removed (i.e., G*≈G'). G", "loss / elastic modulus", represents the G lost during shear deformation due to internal friction * The energy fraction of “G” is not directly related to viscosity because HA fillers are not purely viscous. Rather, this term reflects the inability of the gel to fully recover its shape after the shear stress is removed.

[0686] In the second experiment, the 1.5 mL disk irradiated for 60 seconds showed higher G' values, e.g. Fig.32 The data show that G' increases with increasing rhCollagen-MA concentration and degree of methacrylateization, indicating the ability to control the properties of the scaffold.

[0687] Example 15. Procedure for obtaining and processing rhCollagen from tobacco plants.

[0688] Genetically modified tobacco plants were grown as described above, and leaves were harvested and prepared for initial upstream extraction and purification ( Figure 33A-Figure 33C ).like Fig.33A As shown, the leaves are mechanically crushed (step A) and the pulp is removed from the slurry, while the procollagen-containing fraction is retained and enzymatically digested to convert the procollagen into collagen (step BC). The pulp is discarded again and the collagen-containing fraction is retained from the slurry (step C). After the acidification step, the sample is subjected to a first centrifugation for a first clearing step and the precipitate is then discarded (step DF). After AMS precipitation and a second centrifugation, the protein is precipitated (H precipitate) and the supernatant is discarded (step GH). The H precipitate can be stored frozen at -20°C.

[0689] like Fig.33BAs shown, the H precipitate is resuspended to produce a protein suspension, followed by a third centrifugation, after which the precipitate is discarded (step IJ). The suspension is cleared using a depth filter, and the suspension is salted out with NaCl to precipitate collagen (step KL). The fourth centrifugation produces a collagen precipitate, and the supernatant is discarded (step M).

[0690] like Fig.33C As shown, the collagen precipitate is resuspended in HCl to produce solubilized collagen (step N). After 0.2-0.8 micron filtration, ultrafiltration (UF) (concentration and diafiltration) produces a large amount of concentrated collagen (step OP). After 0.2 micron filtration and filling, the purified collagen is stored in its final container (step Z).

[0691] Example 16. Viscosity and polymerization of rhCollagen methacrylate with additives.

[0692] Rich in different additives (polyvinyl alcohol methacrylate (PVAMA) ( Fig.34 and 37 )、hyaluronic acid methacrylate (HAMA) ( Fig.35 and 37 ) and oxidized cellulose (OC) ( Fig.36 and 37 The viscosity of 5 mg / ml rhCollagen methacrylate at collagenMA:additive ratios of 5:1, 2:1 and 1:2 is shown in Figure 31-37 The viscosity of 5 mg / ml rhCollagen methacrylate is reported in each figure for comparison (black curve). All samples were prepared in 0.1 M phosphate buffer pH 7.4 + 11.3 mM NaCl (physiological osmotic pressure) and measured at T = 22 °C. The data are compared and reported in Fig.37 Summarize in.

[0693] For a typical scaffold of 5 mg / ml collagenMA + different additives, the polymerization of rhCollagen methacrylate enriched with different additives is also shown at a collMA:additive ratio of 2:1 ( Fig.38 ). ColMA alone was compared with ColMA in combination with polyvinyl methacrylate (PVMA), hyaluronic acid methacrylate (HAMA) or oxidized cellulose (OC). The solutions were mixed with the photoinitiator 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylacetophenone (0.1%) and irradiated with ultraviolet (uv) light (365 nm) for 20 seconds.

[0694] Example 17. Injectable rhCollagen / Platelet-Rich Plasma Scaffold.

[0695] Injectable rhCollagen / platelet-rich plasma (PRP) scaffolds have been investigated as scaffolds and healing tools for tendinopathy. A slowly degrading rhCollagen matrix combined with a growth factor (GF) source such as platelet-rich plasma (PRP) is injected adjacent to the injured tendon to provide the desired support and thereby enhance healing of the injured tendon. The therapeutically used matrix is ​​composed of plant-derived recombinant human type I collagen (rhCollagen) mixed with PRP to support the prolonged release of growth factors at the site of injury and promote healing. In a rat model of collagenase-induced Achilles tendinopathy, the rhCollagen-PRP matrix was compared to PRP in vitro and in vivo in supporting fibroblast proliferation, clot degradation, GF release, and tendon healing. Both in vitro and in vivo, rhCollagen-PRP demonstrated superior performance compared to PRP alone. These results are encouraging in clinical trials using rhCollagen matrix in combination with PRP for tendinopathy indications.

[0696] Materials and methods

[0697] rhCollagen Matrix

[0698] By pH neutralization in phosphate solution, the monomer solution (CollPlant, Nice Ziona, Israel) of rhCollagen in 10mM HCl is fibrillated and cross-linked in 18mM 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (Sigma Aldrich, Israel).Then the cross-linked collagen is washed by repeated centrifugation in double distilled water, and calcium chloride (CaCl2) (Merck (Merck), Israel) is added, and the final concentration is calculated to be 20mM. The syringe filled with rhCollagen slurry is lyophilized and terminally sterilized with ethylene oxide.

[0699] Preparation of platelet-rich plasma (PRP)

[0700] Granulocyte-free PRP was prepared using the Tropocell PRP Kit (ESTAR, Israel) according to the manufacturer's instructions. For in vitro cell proliferation assays, human blood was collected from healthy human volunteers (Helsinky Permit No. 2012068). For in vivo animal studies, blood was drawn from Hsd: Sprague Dawley SD rats (Harlan).

[0701] Preparation of RhCollagen matrix / PRP and control

[0702] RhCollagen Matrix / PRP: Syringes containing lyophilized cross-linked rhCollagen were hydrated with PRP or saline to obtain a final concentration of 20 mg / ml rhCollagen.

[0703] Thrombin-activated PRP (control): Human PRP was mixed with purified thrombin (Sigma Aldrich, Israel) to obtain a final concentration of 100 IU / ml.

[0704] CaCl2-activated PRP (control): Rat PRP was mixed with CaCl2 (Merck, Israel) to obtain a final concentration of 20 mM.

[0705] In vitro cell proliferation assay

[0706] In this study, the effect of GF on the viability and proliferation of normal human dermal fibroblasts (nHDF) was evaluated. The viability and proliferation of cells were compared when GF diffused from a matrix composed of cross-linked rhCollagen matrix combined with PRP or a clot composed of thrombin-activated PRP. The rhCollagen matrix combined with PRP or thrombin-activated PRP (200 μl each) was injected into a transwell (Thincerts Biosystems, Israel) placed on the top of a 24-well plate (Thermo science, Israel). TM 24-well 8.0 μm, Greiner bio-one, Israel) and incubated at 37°C for 20 minutes to form a clot. Normal human dermal fibroblasts (nHDF) (5,000 cells per 0.5 ml) were seeded in serum-deficient medium (Dule's modified Eagle medium, DMEM, containing 1% fetal bovine serum, FBS, Biological Industries, Israel) at the bottom of each well. A transwell containing matrix (rhCollagen matrix combined with PRP or thrombin-activated PRP) was placed on top of the inoculated wells and an additional 0.2 ml of medium was added on top of the sample. nHDF in 0.5 ml DMEM, 1% FBS was inoculated as a control. On days 7 and 10 after inoculation, samples were tested in triplicate using the cell proliferation kit WST-1 (Roche, Israel) according to the manufacturer's instructions.

[0707] In vivo studies

[0708] animal

[0709] Hsd: Sprague Dawley SD rats weighing 230g ± 20% were selected for animal experiments. Animals were assigned unique animal identification ear numbers and randomly assigned to specific groups. Animals were housed in individually ventilated (IVC) cages in a dedicated heating, ventilation, and air conditioning (HVAC) animal facility. Temperature and humidity were continuously monitored. Commercial rodent diets (Harlan Teklad TRM Ra / mouse diet) were provided to animals free of charge, and they were allowed free access to autoclaved water. The facility was not exposed to external light and maintained an automatic alternating cycle of 12 hours of light and 12 hours of darkness. All animals were treated in accordance with the 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 found in any animal throughout the study. No statistically significant differences were found in the average group weight values ​​and weight gain. At the time of termination, all weight gains were within the range of normal expected values.

[0710] In vivo clot degradation and growth factor release

[0711] The rhCollagen matrix combined with PRP, rhCollagen matrix alone, or CaCl were compared in a subcutaneous (SC) rat model (Science in Action Ltd., Ness Ziona, Israel). 2 Degradation time of activated PRP and GF content over time

[0712] The dorsal injection sites of 34 female Sprague Dawley rats (Harlan Laboratories, Nice Ziona, Israel) were shaved and marked. Each rat was injected with 0.5 ml of the same formulation on the dorsal plane at four separate locations, two sites in the front of the rat's back and two sites in the back of the rat's back. Animals were killed at the time points of the 1st, 7th, 14th, 21st, 30th and 45th days after treatment (10 or 12 animals per group, 2 animals at each time point). At each time point, the injection site was exposed and macroscopically evaluated. The skin and muscle of the injection site were gently separated with scissors, and the site was washed with 0.25 ml DMEM, 1% FBS (Bio-Industry, Israel), and then the clot was extracted and weighed. The wash medium was transferred to an Eppendorf tube (1.5-2 ml), and the extracted clot was transferred to a 6-well or 12-well plate at the same time. After weighing, the clots were combined with the respective washing media, cut with scissors and minced with a pestle to promote the release of GF from the clot to the surrounding medium. The Eppendorf tubes were then centrifuged for at least 5 minutes to separate between the precipitate of the clot and the culture medium. The supernatant was collected and stored at -80°C until assayed. A control (T0) containing ~0.5 ml of the corresponding formulation was formed in vitro according to the same steps as above without injecting it into the animal. At the end of the study, the content of PDGF and VEGF in the preserved supernatant was assessed by ELISA (QuantikineELISA mouse / rat PDGF and QuantikineELISA rat VEGF, R&D Systems, Israel).

[0713] In vivo tendinopathy induced in rats

[0714] The healing properties of rhCollagen matrix combined with PRP and PRP alone were compared in a collagenase-induced tendinopathy model in 36 male Sprague Dawley rats (18 rats per group, 6 animals per time point). The experiments were performed at Harlan Laboratories Israel Ltd. (Ness Ziona, Israel).

[0715] Skin incision was made on the common Achilles tendon above the proximal part of the right hind leg of the rat. Under appropriate magnification, the middle branch of the tendon was identified and separated, and tendinopathy was induced by injecting 0.3mg collagenase (10mg / ml, Sigma) under the common Achilles tendon sheath using a 0.5ml insulin syringe. Finally, the skin was closed by interrupted subcutaneous sutures using 4 / 0 Vicryl. One week after tendinopathy was induced, a stab incision was formed in the tendon sheath using an ophthalmic cornea / sclera knife. A cannula was then used to create a channel under the tendon sheath, and 50 μl of rhCollagen combined with PRP or PRP alone was injected into the pre-created tube. Animals were killed on the 3rd, 7th and 14th days after treatment. The treated tendons were excised and preserved for histopathological evaluation.

[0716] Histology

[0717] The tissues were embedded in paraffin and serially sectioned into 4-5 micron thick samples. The slides were stained with hematoxylin and eosin (H&E) for histopathological examination and evaluated blindly by a pathologist.

[0718] result

[0719] In vitro cell proliferation assay

[0720] In this study, the viability and proliferation of cells seeded near a clot consisting of a matrix of rhCollagen combined with PRP or PRP activated by thrombin were compared. Cells seeded in untreated wells were used as controls. The matrix (consisting of rhCollagen combined with PRP or PRP activated by thrombin) was placed in a transwell on top of the seeded wells to allow GFs to diffuse from the matrix to the wells without direct contact with the cell layer. The number of viable cells ( ) at day 7 and day 10 was reported. Fig.39 The above data are the average of two different experiments (the experiment was repeated 3 times), in which PRP was extracted from two different blood donors. Fig.39 As shown, in the presence of GFs released from the rhCollagen matrix combined with PRP, cell viability (at day 7 and day 10) was significantly higher than that in thrombin-activated PRP clots or controls. In addition, in the presence of the rhCollagen matrix combined with PRP, cell numbers increased from day 7 to day 10, whereas in the presence of thrombin-activated PRP and in the control group, numbers decreased, indicating that cell viability and proliferation were much superior in the presence of the rhCollagen matrix. The data were confirmed by microscopic analysis ( Fig.39 Cells cultured in the presence of rhCollagen matrix combined with PRP ( Fig.39Lower panel, A) showed an elongated shape and reached full confluency already 7 days after seeding, whereas cells cultured in the presence of thrombin-activated PRP showed little survival, which may indicate a toxic effect of thrombin in this experimental setting. Fig.39 Below, Panel B). Cells cultured in the presence of medium alone showed very limited viability ( Fig.39 below, panel C).

[0721] In vivo matrix degradation profile and growth factor release

[0722] Matrix degradation curve

[0723] The degradation profile of the injected formulations was determined by weighing the matrix at different time points after subcutaneous injection into rats.

[0724] When activated PRP is injected, the material disappears by day 1 ( Fig.40 ), indicating that the fibrin clot was completely degraded within the first 24 hours. On the other hand, the rhCollagen matrix alone or in combination with PRP had a biphasic degradation curve ( Fig.40 ), where there is a sharp drop in weight on the first day, followed by a relatively slow degradation rate, leading to complete elimination after 30–45 days (final weight < 0.5% of initial weight).

[0725] Growth factor content

[0726] The changes of GF content in the injection site over time were evaluated by ELISA for PDGF and VEGF ( Figure 41A-41B ). PDGF levels at time 0 were similar in the rhCollagen matrix combined with PRP and activated PRP treatments ( Fig.41A ), indicating that the PDGF content on day 0 was the only contribution of GF-rich platelets from PRP. However, when PRP was injected alone, PDGF levels at the injection site fell below the detection limit 1 day after injection and remained undetectable throughout the study, consistent with rapid clot degradation ( Fig.40 When PRP was incorporated into the rhCollagen matrix, a different picture was shown ( Fig.41A ). From day 1 to day 14, the PDGF content gradually increased until day 45 when it decreased again until the scaffold was completely degraded ( Fig.40 Interestingly, PDGF levels in the rhCollagen matrix group alone increased from day 7 and followed the pattern shown when the matrix was combined with the PRP group. On day 0, VEGF levels were below the detection limit for all formulations and remained at baseline levels in the activated PRP group ( Figure 41A-41Band 42). The VEGF curve of the rhCollagen matrix combined with PRP showed that the VEGF content increased rapidly on day 7, then dropped sharply by day 14 until it reached a stable level on day 30, and VEGF finally decreased on day 45 as the scaffold degraded ( Fig.41B ).

[0727] The increase in GFs seen from day 1 to day 14 in the PDGF assay and from day 0 to day 7 in the VEGF assay demonstrates the ability of the rhCollagen scaffold to accumulate GFs, likely reflecting cells migrating and proliferating within the scaffold. For each formulation, the nominal levels of PDGF and VEGF were integrated across the study in Fig.42 It is clear that the GF content at the injection site was much higher when the rhCollagen matrix was injected alone or in combination with PRP compared to activated PRP alone.

[0728] In vivo tendinopathy induced in rats

[0729] The healing properties of rhcollagen matrix combined with PRP compared to PRP alone were evaluated in a rat model for tendinopathy and assessed by histopathological analysis at different time points. Tendon healing and inflammation were quantified by scoring the level of mature fibrosis, the presence of mononuclear inflammatory cells, and the presence of immature granulation tissue (scored 0-5 as described in Table 7).

[0730] Table 7. Histopathological scores

[0731]

[0732] The cumulative values ​​of histopathological scores associated with each treatment are depicted in Figure 43A-43C The group treated with rhCollagen matrix combined with PRP showed more mature fibrosis compared to the PRP treated group, especially at days 3 and 14. This is consistent with and correlates with the lower level of immature granulation formation shown by the group treated with rhCollagen matrix combined with PRP at day 14 ( Fig.43C ). In addition, Fig.43BIn the present study, the group treated with rhCollagen matrix combined with PRP showed a reduction in inflammation as indicated by the low presence of mononuclear inflammatory cells at all time points, especially at days 3 and 14. Overall, the data suggest that treatment of injured tendons with rhCollagen matrix combined with PRP promotes faster healing as shown by higher levels of mature fibrosis, lower levels of immature granulation tissue, and a significant reduction in inflammatory monocytes compared to standard PRP injection treatment.

[0733] discuss

[0734] Damage to soft tissues, including tendons and ligaments, is extremely common and causes a significant clinical burden. Although several treatments are available, their clinical benefit remains limited. This has encouraged the search for new alternatives with the goal of improving treatment outcomes and reducing recovery time. An injectable matrix composed of human recombinant type I collagen was developed that, once mixed with PRP, forms a collagen-fibrin-PRP complex that slowly degrades, attracts cells to migrate and diffuse into the collagen scaffold, and allows for a prolonged release of GFs at the site of injury, thereby better supporting the healing process. In vitro experiments ( Fig.39 ) showed significantly superior nHDF viability and proliferation around rhCollagen matrices combined with PRP compared to thrombin-activated PRP. The results suggest that sustained growth factor release from the collagen matrix promotes and enhances cell proliferation. Combining type I rhCollagen with PRP provides a supportive environment that promotes and enhances cell proliferation even when not in direct contact with the cell layer. SC injections in rats demonstrated for the first time that the GF-containing fibrin clot formed in situ at the time of PRP injection had degraded within twenty-four hours, such that the GF content at the injection site was below the detection limit of ELISA ( Figure 41A-41B Once platelets are complexed with the rhCollagen matrix, GF is released over a 45-day period, which coincides with scaffold degradation ( Fig.40 and 41A - Fig.41B Interestingly, the GF content curve was not as monotonic as expected from a standard release curve. PDGF curves in rhCollagen matrix and PRP treatment ( Figure 41A-41B) shows that the first day drops sharply, which is very similar to the case of using PRP alone, but then gradually increases until the 21st day, and finally drops to an undetectable level, which is consistent with the degradation of the scaffold. Interestingly, rhCollagen alone shows a similar pattern of gradually increasing PDGF content in the first few weeks, and declines with the complete degradation of the scaffold. Therefore, the scaffold of rhCollagen and PRP combines the benefits provided by the captured PRP (early GF release) with the benefits provided by the rhCollagen scaffold itself (can highly promote and enhance cell recruitment and proliferation). As for the content curve of VEGF, although the level of VEGF remains very low when PRP is injected throughout the study, the injection of rhCollagen matrix combined with PRP causes VEGF levels to increase and eventually decline in the first week, which is consistent with the degradation of the scaffold. Interestingly, compared with PDGF, the VEGF level of the group treated with the rhCollagen matrix alone is still higher than that of the group using PRP alone, but its curve is different from that of the rhCollagen matrix with PRP, especially on the 7th day. This observation emphasizes the contribution of PRP to GF levels once combined with the collagen scaffold. Finally, the healing properties of rhColla...

Claims

1. A polymerizable solution comprising: Cross-linkable plant-derived recombinant human collagen type 1 (rhcollagen); Hyaluronic acid (HA) or a modified derivative thereof, polyvinyl alcohol (PVA) or a modified derivative thereof, polyethylene glycol (PEG) or a modified derivative thereof, oxidized cellulose (OC) or a modified derivative thereof, polymethyl methacrylate (PMMA) microspheres or a modified derivative thereof, tricalcium phosphate (TCP) or a modified derivative thereof, calcium hydroxyapatite (CaHA) or a modified derivative thereof, carboxymethyl cellulose or a modified derivative thereof, crystalline nanocellulose (CNC) or a modified derivative thereof, or a combination thereof; and Photoinitiator.

2. The polymerizable solution according to claim 1, wherein the cross-linkable plant-derived human collagen is methacrylated or thiolated plant-derived type 1 recombinant human collagen (rhcollagen).

3. The polymerizable solution of claim 1, wherein the modified derivatives of hyaluronic acid (HA), polyvinyl alcohol (PVA), polyethylene glycol (PEG), oxidized cellulose (OC), polymethyl methacrylate (PMMA) microspheres, tricalcium phosphate (TCP), calcium hydroxyapatite (CaHA), carboxymethyl cellulose or crystalline nanocellulose (CNC) comprise methacrylated or thiolated derivatives.

4. The polymerizable solution of claim 1, wherein the hyaluronic acid (HA), polyvinyl alcohol (PVA), polyethylene glycol (PEG), oxidized cellulose (OC), polymethyl methacrylate (PMMA) microspheres, tricalcium phosphate (TCP), calcium hydroxyapatite (CaHA), carboxymethyl cellulose or crystalline nanocellulose (CNC) comprises cross-linked hyaluronic acid (HA), cross-linked polyvinyl alcohol (PVA), cross-linked polyethylene glycol (PEG), cross-linked oxidized cellulose (OC), cross-linked polymethyl methacrylate (PMMA) microspheres, cross-linked tricalcium phosphate (TCP), cross-linked calcium hydroxyapatite (CaHA), cross-linked carboxymethyl cellulose or cross-linked crystalline nanocellulose (CNC).

5. The polymerizable solution according to claim 1, comprising: Methacrylated or thiolated plant-derived recombinant human collagen type 1 (rhcollagen); Cross-linked hyaluronic acid (HA), cross-linked polyvinyl alcohol (PVA), cross-linked polyethylene glycol (PEG), cross-linked oxidized cellulose (OC), cross-linked polymethyl methacrylate (PMMA) microspheres, cross-linked tricalcium phosphate (TCP), cross-linked calcium hydroxyapatite (CaHA), cross-linked carboxymethyl cellulose or cross-linked crystalline nanocellulose (CNC); and The photoinitiator.

6. The polymerizable solution according to claim 1, comprising: Methacrylated plant-derived recombinant human collagen type 1 (rhcollagen); Hyaluronic acid (HA), cross-linked hyaluronic acid (HA), methacrylated or thiolated hyaluronic acid (HA), or any combination of said hyaluronic acid (HA), said cross-linked hyaluronic acid (HA), and said methacrylated or thiolated hyaluronic acid (HA); and Photoinitiator.

7. The polymerizable solution of claim 6, wherein the ratio of the hyaluronic acid (HA), the cross-linked hyaluronic acid (HA), the methacrylated or thiolated hyaluronic acid (HA), or the combination thereof to the methacrylated plant-derived type 1 recombinant human collagen (rhcollagen) is in the range of from 6:1 to 1:

6.

8. The polymerizable solution of claim 7, wherein the ratio is in the range of from 4:1 to 1:4 or from 3:1 to 1:3 or from 3:1 to 2:

1.

9. A kit comprising: (i) a solution comprising cross-linkable plant-derived recombinant human collagen type 1 (rhcollagen) and a photoinitiator; and (ii) hyaluronic acid (HA) or a modified derivative thereof, polyvinyl alcohol (PVA) or a modified derivative thereof, polyethylene glycol (PEG) or a modified derivative thereof, oxidized cellulose (OC) or a modified derivative thereof, polymethyl methacrylate (PMMA) microspheres or a modified derivative thereof, tricalcium phosphate (TCP) or a modified derivative thereof, calcium hydroxyapatite (CaHA) or a modified derivative thereof, carboxymethyl cellulose or a modified derivative thereof, crystalline nanocellulose (CNC) or a modified derivative thereof, or a combination thereof.

10. The polymerizable solution of claim 1, further comprising unmodified plant-derived recombinant human collagen type 1.

11. The polymerizable solution of claim 1, wherein the concentration of the cross-linkable plant-derived recombinant human collagen type 1 is in the range of from 1 mg / ml to 12 mg / ml.

12. The polymerizable solution of claim 1 which is an injectable solution at the temperature of use and / or has a texture and viscosity that allows flow through a suitable delivery device.

13. The polymerizable solution according to claim 1, which is a photocurable solution.

14. The polymerizable solution of claim 1, wherein the photoinitiator induces polymerization of the polymerizable solution in response to visible light having a wavelength of 390-800 nm.

15. The polymerizable solution of claim 1 which is a dermal filler.

16. The polymerizable solution of claim 1, capable of augmenting tissue of a subject.

17. The polymerizable solution of claim 16, wherein the tissue is skin tissue.

18. The polymerizable solution of claim 1, capable of filling subepidermal tissue spaces.

19. A double cross-linked dermal filler comprising: (a) Plant-derived recombinant human collagen type 1; as well as (b) cross-linked hyaluronic acid; wherein the plant-derived human collagen is cross-linked with the cross-linked hyaluronic acid.

20. The dual cross-linked dermal filler of claim 19, further comprising non-cross-linked hyaluronic acid.

21. The dual cross-linked dermal filler of claim 19, wherein the cross-linking agent attaching the cross-linked hyaluronic acid is different from the cross-linking agent attaching the plant-derived recombinant human collagen type 1 to the cross-linked hyaluronic acid.

22. The dual cross-linked dermal filler of claim 19, wherein the ratio of the cross-linked hyaluronic acid to the plant-derived recombinant human collagen type 1 is in the range of from 6:1 to 1:

6.

23. The dual cross-linked dermal filler of claim 19, wherein the concentration of the cross-linked hyaluronic acid ranges from 5 mg / ml to 50 mg / ml.

24. The dual cross-linked dermal filler of claim 22, wherein the concentration of the cross-linked hyaluronic acid ranges from 5 mg / ml to 50 mg / ml.

25. The dual cross-linked dermal filler according to claim 19, wherein the cross-linking agent that cross-links the hyaluronic acid and the cross-linking agent that cross-links the plant-derived recombinant human collagen type 1 are each independently selected from 1,4-butanediol diglycidyl ether (BDDE), 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide methyl iodide (EDC), N,N'-dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide (DIC), divinyl sulfone (DVS) and glutaraldehyde.

26. A method of preparing a dual cross-linked dermal filler comprising plant-derived recombinant human collagen type 1 cross-linked with cross-linked hyaluronic acid, the method comprising: cross-linking hyaluronic acid; Neutralizes cross-linked hyaluronic acid; Neutralizes plant-derived human collagen; Mixing neutralized cross-linked hyaluronic acid with neutralized plant-derived human collagen; Addition of lower molecular weight hyaluronic acid (MW HA); cross-linking a mixture of cross-linked hyaluronic acid and plant-derived human collagen; as well as Dialyzed double cross-linked hyaluronic acid-plant-derived human collagen dermal filler.

Images