Dermal fillers and their uses

Abstract

To provide a polymerizable solution which can be used as improved injectable dermal fillers having adjustable rheological and mechanical properties.SOLUTION: Provided is a polymerizable solution that contains: (i) crosslinkable plant-derived human collagen; (ii) hyaluronic acid or modified derivatives thereof, poly(vinyl alcohol) or modified derivatives thereof, polyethylene glycol or modified derivatives thereof, cellulose oxide or modified derivatives thereof, polymethyl methacrylate microspheres or modified derivatives thereof, tricalcium phosphate or modified derivatives thereof, calcium hydroxyl apatite or modified derivative thereof, carboxymethyl cellulose or modified derivative thereof, crystalline nanocellulose or modified derivative thereof, or a combination thereof; and (iii) a photoinitiator.SELECTED DRAWING: Figure 27

Description

[Technical Field]

[0001] Disclosed herein are photoinitiated, dual-crosslinked dermal fillers comprising plant-derived human collagen and a cell growth-promoting scaffold, and in some cases, methods of using the dermal fillers for soft tissue augmentation. [Background technology]

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

[0003] One of the common aspects of aging is the appearance of lines, fine lines or wrinkles. Treatments involving the use of collagen extracted from tissue have been used to reduce or eliminate lines, fine lines or wrinkles. Similar treatments have been used to reduce scars.

[0004] Collagen is also a component of tendons. Tendinopathy, a common disorder usually associated with sports and physical activity, is associated with the degeneration and misalignment of tendon collagen fibers. Healing of injured tendons requires the coordinated activity of specific cells and the prolonged presence of associated growth factors (GFs) near the injury. Tendinopathy has become a major cause of consultation for musculoskeletal complaints in recent years (Kaux et al. (2011) J. Sport. Sci. Med. January: 238-253). Tendinopathy refers to a variety of painful conditions that develop in and around tendons and ligaments, potentially resulting from an imbalance between pathological changes caused by tendon overuse and the resulting regenerative response (Andres et al. (2008) Clin. Orthop. Relat. Res. 466: 1539-1554). Tendinopathy is associated with collagen degeneration and misalignment (Maffulli et al. (2003) Clin. Sport. Med. 22:675-692) and may be associated with microtears of fibers, increased vascularity, and mild inflammation (Khan et al. (1999) Sport. Med. 27(6):393-408). Clinically, tendon disorders are characterized by tendon stiffness, activity-related pain, decreased function, and sometimes localized swelling (Kaux 2011; Andres 2008). Collagen fibers no longer have the usual dense, parallel, bundled appearance, but instead exhibit uneven, irregular crimping, loosening, and increased waviness (Mafulli 2003). As people remain active into old age, the incidence of tendon injuries is expected to increase over the next few decades. Although a wide variety of treatments for tendinopathy are available, including physical therapy, pharmacological treatment, and combinations thereof, clinical outcomes are unsatisfactory and recurrence of symptoms is common (Kaux 2011).Injection of autologous platelet-rich plasma (PRP) for the treatment of tendon disorders has received widespread attention in the past few decades (Delong et al. (2016) Curr. Orthpaedic Pract. 22:514-523; Kaux et al. (2012) Wound Repair Regen. 20:748-756; Yuan et al. (2013) Muscles. Ligaments Tendons J. 3(3):139-49; Di Matteo et al. (2015) Musculoskelet. Surg. 99(1):1-9). PRP is a plasma fraction of blood that contains a high concentration of platelets. When injected into the injury site, platelets release various types of growth factors (GFs), which are thought to promote the healing process. Among the GFs associated with PRP, 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), and hepatocyte growth factor (HGF) have been reported (Delong 2016, Yuan 2013, Harrison et al. (2011) Am. J. Sports Med. 39(4):729-734). Numerous in vitro studies and in vivo models have shown that PRP treatment improves collagen expression and extracellular matrix production, stimulates angiogenesis, and increases cell migration, differentiation, and proliferation, thus aiding in the healing of tendon injuries (Yuan 2013, Kajikawa et al. (2008) J. Cell. Physiol. 215(3):837-845, Zhang et al. (2010) Am. J. Sports Med. 38(12):2477-2486). However, clear clinical evidence regarding the efficacy of PRP treatment is limited (Delong 2016, Yuan 2013, Moraes et al. (2014)).

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

[0006] The morphology and most properties of native collagen are determined by its triple-helical domain, which comprises more than 95% of the molecule. This domain is composed of three alpha chains, each containing approximately 1,000 amino acids, wrapped in a rope-like manner to form a tight triple-helical structure. This triple helix is ​​wound so that the peptide bonds connecting adjacent amino acids are buried within the molecule, making the collagen molecule resistant to attack by proteases such as pepsin.

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

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

[0009] A key feature of fibril-forming collagens is that they are synthesized as precursor procollagens containing globular N- and C-terminally extended propeptides. Procollagen biosynthesis is a complex process involving several different post-translational modifications, including proline and lysine hydroxylation, N- and O-linked glycosylation, and the formation of both intra- and interchain disulfide bonds. The enzymes that carry out these modifications act in a coordinated manner to ensure the folding and organization of a correctly aligned and thermally stable triple-helical molecule.

[0010] The tripartite polypeptide chain assembles within the rough endoplasmic reticulum (ER) to form procollagen. As the polypeptide chain translocates cotranslationally across the ER membrane, prolyl-4-hydroxylase (P4H)-dependent hydroxylation of proline and lysine residues occurs within the Gly-XY repeat region. The stability of the final triple helix structure of collagen is highly dependent on P4H-mediated hydroxylation of the collagen chain. 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 sequentially modify lysyl residues at specific positions to hydroxylysyl, galactosyl-hydroxylysyl, and glucosylgalactosyl-hydroxylysyl residues. These structures are unique to collagens and essential for their functional activity (Wang et al. (2002) Matrix Biology, 21(7):559-566). A single human enzyme, lysyl hydroxylase 3 (LH3), can catalyze all three successive steps in the formation of hydroxylysine-linked carbohydrates (Wang et al. (2002) Matrix Biology, 21(7):559-566). Once the polypeptide chain is fully translocated into the lumen of the endoplasmic reticulum, three pro-α chains join via the C-propeptide to form a trimeric molecule, and a Gly-XY repeat region forms a nucleation point at its C-terminus, ensuring the correct alignment of the chains. The Gly-XY region then folds from the C-direction to the N-direction, forming a triple helix (Khoshnoodi et al. (2006) J. Biol. Chem. 281:38117-38121).

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

[0012] Native collagen generally exists in connective tissues as collagen molecules containing telopeptides packed side by side in the form of fibrils. Each longitudinal track is composed of molecules aligned in an end-to-end configuration, with a slight longitudinal staggered spacing relative to the next successive laterally adjacent longitudinal track. In this way, gaps are created between the opposing end regions of consecutive molecules in a given longitudinal track, which are joined by the staggered sides of molecules in laterally adjacent, parallel longitudinal tracks.

[0013] Dispersion and solubilization of native animal collagen can be achieved using a variety of proteolytic enzymes that disrupt intermolecular bonds and remove immunogenic non-helical telopeptides without affecting the fundamental rigid triple-helical structure that confers collagen's desirable properties (see, e.g., U.S. Patent 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 atelocollagen can then be purified by repeated precipitation at low pH and high ionic strength, followed by washing at low pH and resolubilization. Nevertheless, soluble preparations are typically contaminated with cross-linked collagen chains, which reduces the homogeneity of the protein preparation.

[0014] Collagen, due to its unique characteristics and diverse profile in the human body, is a biocompatible material of choice for use in tissue repair, supporting structural integrity, guiding cell infiltration, and promoting tissue regeneration. Among the five major collagen types, type I collagen is the most abundant form of collagen in the human body.

[0015] Type I collagen can self-assemble into fibrous hydrogels that can support tissue cells through bioactive adhesive sites. Loading collagen with methacrylate groups produces collagen methacrylate (CMA), making it more resistant to degradation (Gaudet et al., Biointerphases (2012) 7:25-33). Thiolation of collagen can improve cohesion and mucoadhesion and affect swelling capacity (Duggan et al., Eur. J. Pharm. Biopharm. (April 2015) 91:75-81).

[0016] The unique properties of collagen contribute to its use in tissue engineering products. Collagen provides a biomaterial with the necessary characteristics for a myriad of applications, including pharmaceuticals (hemostatic compresses, sponges, healing bandages), medical (prosthetics such as heart valves, tendons and ligaments, skin substitutes, and fillers), dentistry (gum grafts / periodontal disease), and cosmetics (microcontainers for additives, anti-wrinkle agents, and fragrance materials). Collagen-based products manufactured in all of the aforementioned markets require large quantities of collagen raw material for their manufacture.

[0017] Human and animal-derived collagen, such as cadaveric or animal-derived (bovine, porcine, or equine), and collagen-based products have been used for application, injection, implantation, and oral ingestion. Uses include premolding into desired shapes for repair or partial replacement of damaged bone or cartilage structures, injection into damaged joints, and as a dermal filler.

[0018] The use of animal-derived collagen (including human-derived collagen) is problematic due to the potential risk of contamination with nontraditional infectious agents. While the risks posed by bacterial and viral contamination can be completely controlled, prions are difficult to contain and pose a significant health risk. These infectious agents, which appear to have protein-like properties, are involved in the development of degenerative animal encephalopathies (sheep trembling disease, bovine spongiform encephalopathy) and human encephalopathies (Creutzfeldt-Jakob disease, Gerstmann-Straussler syndrome, and kuru). Other diseases (e.g., acquired immunodeficiency syndrome [AIDS], hepatitis, rabies, and some cancers) can also be transmitted to recipients. The long time it takes for some of these encephalopathies and other diseases to develop makes formal management 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.)

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

[0020] Plants that express collagen chains are known in the art (see, e.g., WO2005 / 035442, U.S. Patent No. 6,617,431, U.S. Publication No. 2002 / 0098578, U.S. Publication No. 2002 / 0142391, Merle et al. (2002) FEBS Letters 515:114-118, Ruggiero et al. (March 3, 2000) FEBS Lett. 469(1):132-6). Such plants can be used to produce collagen chains, not just collagen, but these chains are not properly hydroxylated, and therefore their self-assembly, whether in planta or outside, results in inherently unstable collagen. For example, although plants can synthesize proteins containing hydroxyproline, the prolyl hydroxylases involved in the synthesis of hydroxyproline in plant cells exhibit relatively loose substrate sequence specificity compared to mammalian P4H. Therefore, plant coexpression of collagen and P4H genes is required to produce collagen containing hydroxyproline only at the Y position of the Gly-XY triplet (Olsen et al. (2003) Adv. Drug Deliv. Rev., 55(12):1547-1567).

[0021] The processing of "insoluble collagen" of animal origin using plant-derived proteases 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).

[0022] Attempts to produce human collagen by relying on the naturally occurring hydroxylation machinery in plants have resulted in collagens with poor proline hydroxylation (Merle et al. (2002) FEBS Letters 515:114-118). Such collagens melt or lose their triple helical structure at temperatures below 30°C. Coexpression of collagen with prolyl hydroxylase results in stable hydroxylated collagens that are biologically relevant for body temperature applications (Merle et al. (2002) FEBS Letters 515:114-118).

[0023] Hydroxylysine in human collagen expressed in tobacco forms less than 2% of the hydroxylysine found in bovine collagen (0.04% of residues / 1.88% of residues), suggesting that endogenous lysyl hydroxylases in plants are unable to sufficiently hydroxylate lysines in collagen.

[0024] Recent technological developments have led to the development of a system for purifying native human type I collagen (rh collagen) by introducing five human genes encoding heterotrimeric type I collagen into tobacco plants (COLLPLANT™, Israel, also available from SIGMA-ALDRICH®, St. Louis, MO, USA) [see, e.g., Stein H. (2009) Biomacromolecules 10:2640-5; Yaari et al. (2013) Tiss. Eng. Part A 19(13 / 14):1502-1506; Willard et al. (2013) Tiss. Eng. Part A 19(13 / 14):1507-1518; Shilo et al. (2013) Tiss. Eng. Part A 19(13 / 14):1519-1526; Shoseyov et al. (2013) Tissue Eng. Part A 19:1527-1533, and Shoseyov et al. (January / February 2014) Bioengineered 5:1, 1-4. The protein is purified to homogeneity through a cost-effective industrial process that exploits the unique properties of collagen. See also WO2006 / 035442, WO2009 / 053985, and patents and patent applications derived therefrom, all of which are incorporated by reference as if fully set forth herein.

[0025] Compared with tissue-extracted collagen, which may be partially denatured and lose its cell-binding domains, plant-derived human type I collagen has a more consistent structure and more cell-binding domains (Shoseyov et al. (January / February 2014) Bioengineered 5:1, 1-4; Majumdar et al. (2015) J. Biomed. Mater. Res. Part B: Appl. Biomater. 104B:300-307). For applications in additive manufacturing (AM), a process in which 3D objects are fabricated in a layer-by-layer manner using a computer model of the object via 3D bioprinting, rh collagen can form functional three-dimensional (3D) matrices and scaffolds. Furthermore, rh collagen generally lacks the immunogenicity and disease migration issues of tissue-extracted collagen.

[0026] Methods for producing collagen by expressing at least one type of collagen α-chain and allowing its accumulation in intracellular compartments lacking endogenous P4H activity (U.S. Pat. No. 8,455,717) are available, as are methods for producing atelocollagen from collagen containing human telopeptides derived from non-animal cells by treatment with proteases (U.S. Pat. No. 8,759,487).

[0027] Type I collagen and rh collagen are considered candidates for use as the main components of the build material for 3D bioprinting. Various types of scaffolds have been used in cosmetic and other reconstructive applications.

[0028] In addition, the use of dermal fillers for soft tissue augmentation, such as wrinkle reduction, has been increasing. One possible method for using dermal fillers involves injecting a polymerizable dermal filler material into the desired area, followed by contouring or molding the filler into the desired shape. Polymerization and crosslinking of the material by one of various methods can convert the monomers in the injected material to form polymers and chains, which form a network structure and can retain the desired molded shape. Several methods exist for forming polymers and crosslinking polymers. One method involves photoinduced reaction with a photoreactive reagent that generates reactive species in the monomer solution. See, for example, U.S. Pat. Nos. 9,795,711, 8,945,624, 6,352,710, and U.S. Publication No. 2009 / 0324722, and Elisseeff et al. (March 1999) Proc. Natl. Acad. Sci. USA 96:3104-3107.

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

[0030] Thus, there is a need for, and it would be highly desirable and advantageous to have, improved injectable dermal fillers with tunable rheological and mechanical properties, as well as methods and uses thereof. Summary of the Invention [Means for solving the problem]

[0031] In one aspect, there is provided a dual crosslinked dermal filler, comprising: (a) plant-derived human collagen; (b) cross-linked hyaluronic acid, Disclosed herein is a dual-crosslinked dermal filler in which plant-derived human collagen is crosslinked to crosslinked hyaluronic acid.

[0032] In a related aspect, the plant-derived human collagen is (a) type 1 recombinant human collagen (rh collagen), or (b) the cross-linked hyaluronic acid includes cross-linked hyaluronic acid and non-cross-linked hyaluronic acid, or (c) includes any combination thereof.

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

[0034] In one aspect, there is provided a method for preparing a dual crosslinked dermal filler comprising plant-derived human collagen crosslinked to crosslinked hyaluronic acid, comprising: (a) cross-linking hyaluronic acid; (b) neutralizing the cross-linked hyaluronic acid; (c) neutralizing the plant-derived human collagen; (d) mixing the neutralized cross-linked hyaluronic acid with the neutralized plant-derived human collagen; (e) adding low molecular weight hyaluronic acid (MW HA); (f) cross-linking the mixture of cross-linked hyaluronic acid and plant-derived human collagen; (g) dialyzing the doubly crosslinked crosslinked hyaluronic acid-plant-derived human collagen dermal filler.

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

[0036] Additionally, in one aspect, there is provided a method of filling a tissue space below the epidermis, comprising: (a) introducing a polymerizable solution into the tissue space, the polymerizable solution comprising: (i) cross-linkable plant-derived human collagen; (ii) hyaluronic acid (HA) or a modified derivative thereof, poly(vinyl alcohol) (PVA) or a modified derivative thereof, polyethylene glycol (PEG) or a modified derivative thereof, oxidized cellulose (OC) or a modified derivative thereof, polymethyl methacrylate (PMMA) microspheres or a modified derivative thereof, tricalcium phosphate (TCP) or a modified derivative thereof, calcium hydroxylapatite (CaHA) or a modified derivative thereof, carboxymethylcellulose or a modified derivative thereof, crystalline nanocellulose (CNC) or a modified derivative thereof, or a combination thereof; (iii) the introducing step, which includes a photoinitiator; (b) applying light to a surface of the epidermis at the surface of the space to induce polymerization.

[0037] In a related embodiment, the components of the polymerizable solution are introduced into the tissue space independently, at approximately the same location, and approximately simultaneously, and the crosslinkable plant-derived human collagen and photoinitiator are introduced together and independently from the hyaluronic acid (HA) or a modified derivative thereof, the poly(vinyl alcohol) (PVA) or a modified derivative thereof, the polyethylene glycol (PEG) or a modified derivative thereof, the oxidized cellulose (OC) or a modified derivative thereof, the polymethylmethacrylate (PMMA) microspheres or a modified derivative thereof, the tricalcium phosphate (TCP) or a modified derivative thereof, the calcium hydroxylapatite (CaHA) or a modified derivative thereof, the carboxymethylcellulose or a modified derivative thereof, the crystalline nanocellulose (CNC) or a modified derivative thereof, or any combination thereof, and are introduced into the tissue space independently and approximately simultaneously. In another related embodiment, the method further includes a step of molding or shaping the polymerizable solution or components of the polymerizable solution into a desired configuration within the tissue space, which step occurs simultaneously with or follows the step of applying light.

[0038] In another related aspect, the components of the polymerizable solution are introduced into the tissue space together as a mixture, wherein the crosslinkable plant-derived human collagen and photoinitiator are introduced together with the hyaluronic acid (HA) or a modified derivative thereof, or the poly(vinyl 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 hydroxylapatite (CaHA) or a modified derivative thereof, or the carboxymethylcellulose or a modified derivative thereof, or the crystalline nanocellulose (CNC) or a modified derivative thereof, or combinations thereof.

[0039] In another related aspect, the components of the polymerizable solution are introduced into the tissue space independently of each other, with the crosslinkable plant-derived human collagen and the photoinitiator being introduced together and independently from the hyaluronic acid (HA) or a modified derivative thereof, or the poly(vinyl 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 hydroxylapatite (CaHA) or a modified derivative thereof, or the carboxymethylcellulose or a modified derivative thereof, or the crystalline nanocellulose (CNC) or a modified derivative thereof, or any combination thereof.

[0040] In another related aspect, after introduction into the tissue space, the method further includes a step of molding or shaping the polymerizable solution or components of the polymerizable solution into a desired configuration within the tissue space, which step occurs simultaneously with or subsequent to the step of applying light.

[0041] In another related aspect, the method is non-therapeutic and the shaping or shaping step reduces lines, folds, fine lines, wrinkles, or scars, or a combination thereof.

[0042] In another related aspect, (a) the crosslinkable plant-derived human collagen is methacrylated or thiolated human recombinant collagen type 1 (rh collagen), or (b) Modified derivatives of hyaluronic acid (HA), poly(vinyl alcohol) (PVA), polyethylene glycol (PEG), oxidized cellulose (OC), polymethyl methacrylate (PMMA) microspheres, tricalcium phosphate (TCP), calcium hydroxylapatite (CaHA), carboxymethylcellulose, or crystalline nanocellulose (CNC), including methacrylated or thiolated derivatives; (c) hyaluronic acid (HA), poly(vinyl alcohol) (PVA), polyethylene glycol (PEG), oxidized cellulose (OC), polymethyl methacrylate (PMMA) microspheres, tricalcium phosphate (TCP), calcium hydroxylapatite (CaHA), carboxymethyl cellulose, or crystalline nanocellulose (CNC), or cross-linked hyaluronic acid (HA), cross-linked poly(vinyl 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 hydroxylapatite (CaHA), cross-linked carboxymethyl cellulose, or cross-linked crystalline nanocellulose (CNC); (d) A combination of (a) and (b) or (a) and (c).

[0043] In further related embodiments, when MA-rh collagen is selected, and when hyaluronic acid or a derivative thereof, or cross-linked hyaluronic acid is selected, the ratio of HA to MA-rh collagen is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 1:2, 1:3, 1:4, 1:5, 1:6, or 0:1.

[0044] In one aspect, a method of filling a tissue space below the epidermis comprises introducing into the tissue space a dual crosslinked dermal filler, the dual crosslinked dermal filler comprising: (a) plant-derived human collagen; (b) cross-linked hyaluronic acid (HA) or a modified cross-linked derivative thereof, cross-linked poly(vinyl 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 hydroxylapatite (CaHA) or a modified cross-linked derivative thereof, cross-linked carboxymethylcellulose or a modified cross-linked derivative thereof, cross-linked crystalline nanocellulose (CNC) or a modified cross-linked derivative thereof, or a combination thereof; Disclosed herein are methods in which plant-derived human collagen is crosslinked to crosslinked hyaluronic acid (HA) or its modified crosslinked derivatives, crosslinked poly(vinyl alcohol) (PVA) or its modified crosslinked derivatives, crosslinked polyethylene glycol (PEG) or its modified crosslinked derivatives, crosslinked oxidized cellulose (OC) or its modified crosslinked derivatives, crosslinked polymethyl methacrylate (PMMA) microspheres or its modified crosslinked derivatives, crosslinked tricalcium phosphate (TCP) or its modified crosslinked derivatives, crosslinked calcium hydroxylapatite (CaHA) or its modified crosslinked derivatives, crosslinked carboxymethylcellulose or its modified crosslinked derivatives, crosslinked crystalline nanocellulose (CNC) or its modified crosslinked derivatives.

[0045] In related aspects, the plant-derived human collagen is type 1 human recombinant collagen (rhcollagen), or MA or a thiolated derivative thereof, or modified derivatives of hyaluronic acid (HA), poly(vinyl alcohol) (PVA), polyethylene glycol (PEG), oxidized cellulose (OC), polymethylmethacrylate (PMMA) microspheres, tricalcium phosphate (TCP), calcium hydroxylapatite (CaHA), carboxymethylcellulose, or crystalline nanocellulose (CNC), including methacrylated or thiolated derivatives, or combinations thereof.

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

[0047] In a related aspect, the method is non-therapeutic and reduces the appearance of lines, folds, fine lines, wrinkles, or scars, or a combination thereof.

[0048] In one aspect, a polymerizable or non-polymerizable solution for use in tissue augmentation, comprising: (a) the polymerizable solution comprises crosslinkable plant-derived human collagen and a photoinitiator for inducing polymerization prior to and simultaneously with the application of visible light; or (b) the non-polymerizable solution comprises a dual-crosslinked dermal filler comprising plant-derived human collagen and crosslinked hyaluronic acid or crosslinked PVA or crosslinked PGE or crosslinked OC, wherein the plant-derived human collagen is crosslinked to the crosslinked hyaluronic acid or crosslinked PVA or crosslinked PGE or crosslinked OC; The above-described uses include injecting the above-described polymerizable or non-polymerizable solution into the tissue space below the epidermis, and then molding or shaping the polymerizable or non-polymerizable solution into a desired configuration to reduce lines, folds, fine lines, wrinkles, or scars.

[0049] In related aspects, the crosslinkable 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 thereof, poly(vinyl alcohol) (PVA) or a modified derivative thereof or a photopolymerizable modified derivative thereof, polyethylene glycol (PEG) or a modified derivative thereof or a photopolymerizable modified derivative thereof, polymethylmethacrylate (PMMA) microspheres or a modified derivative thereof or a photopolymerizable modified derivative thereof, tricalcium phosphate (TCP) or a modified derivative thereof or a photopolymerizable modified derivative thereof, calcium hydroxylapatite (CaHA) or a modified derivative thereof or a photopolymerizable modified derivative thereof, carboxymethylcellulose or a modified derivative thereof or a photopolymerizable modified derivative thereof, crystalline nanocellulose (CNC) or a modified derivative thereof or a photopolymerizable modified derivative thereof, or a combination thereof, optionally wherein the derivative comprises a methacrylated or thiolated derivative, or a combination thereof.

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

[0051] In one aspect, a method of inducing a cell growth promoting scaffold into a tissue space below the epidermis comprises introducing a solution into the tissue space, the solution comprising: (a) plant-derived human collagen; (b) at least one growth factor or a source thereof; The method promotes healing or replacement of collagen-containing tissue. Methods are disclosed herein.

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

[0053] In another aspect, the method is non-therapeutic, wherein the cell growth promoting scaffold fills within tissue spaces and reduces lines, folds, fine lines, wrinkles, or scars, or a combination thereof.

[0054] In one aspect, disclosed herein is a solution for use in inducing a cell growth promoting scaffold, the solution comprising plant-derived human collagen and at least one growth factor or source thereof, the use comprising injecting the solution into a tissue space below the epidermis, the use being for promoting healing or replacement of collagen-containing skin tissue due to degradation or damage.

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

[0056] In another related embodiment, the rh collagen includes a methacrylate or thiol derivative thereof.

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

[0058] In a related aspect, the method is non-therapeutic, and the cell growth promoting scaffold fills within tissue spaces to reduce lines, folds, fine lines, wrinkles, or scars, or a combination thereof.

[0059] Disclosed herein is a solution for use in inducing a cell growth promoting scaffold, the solution comprising plant-derived human collagen and at least one growth factor or source thereof, the use comprising injecting the solution into a tissue space below the epidermis, the use being for promoting healing or replacement due to degradation or damage of collagen-containing tissue.

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

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

[0062] In one aspect, disclosed herein is a method for filling a tissue space beneath the epidermis, comprising: (a) introducing into the tissue space a polymerizable solution comprising (i) cross-linkable plant-derived human collagen and (ii) a photoinitiator; and irradiating the surface of the epidermis at the surface of the space with light to induce polymerization.

[0063] In a related aspect, the polymerizable solution further comprises a step of molding or shaping the polymerizable solution into a desired configuration within the tissue space, which step occurs simultaneously with or subsequent to the step of applying light.

[0064] In another related aspect, the method is non-therapeutic and the shaping or shaping step reduces lines, folds, fine lines, wrinkles, or scars, or a combination thereof.

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

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

[0067] [Figure 1A] Figure 1 shows the construction of various expression cassettes and vectors used to transform test plants to date. All coding sequences synthesized as part of the study were optimized for expression in tobacco. Figure 2 shows the cloning scheme for type I collagen αI chain or type II collagen α2 chain in plant expression vectors according to some embodiments of the present invention. [Figure 1B] Figure 1 shows the construction of various expression cassettes and vectors used to transform test plants to date. All coding sequences synthesized as part of the study were optimized for expression in tobacco. Figure 2 shows the cloning scheme for the enzyme prolyl-4-hydroxylase (P4H) in a plant expression vector according to some embodiments of the present invention. [Figure 1C] Figure 1 shows the construction of various expression cassettes and vectors used to transform test plants to date. All coding sequences synthesized as part of the study were optimized for expression in tobacco. Figure 2 shows the cloning scheme for proteinase C or proteinase N in plant expression vectors according to some embodiments of the present invention. [Figure 1D] Figure 1 shows the construction of various expression cassettes and vectors used to transform test plants to date. All coding sequences synthesized as part of the study were optimized for expression in tobacco. Figure 2 shows the cloning scheme for lysyl hydroxylase 3 (LH3) in plant expression vectors according to some embodiments of the present invention. [Figure 2]Various cotransformation approaches that have been used to date are shown. Each expression cassette is represented by the short name of its coding sequence. The coding sequences are specified in Table 1. Each cotransformation was performed with two pBINPLUS binary vectors. Each rectangle represents a single pBINPLUS vector carrying one, two, or three expression cassettes. The promoters and terminators are specified in Example 1. [Figure 3] Conventional multiplex PCR screening of transformants showing plants that were positive for collagen α1 (324 bp fragment) or collagen α2 (537 bp fragment) or both. [Figure 4]Conventional Western blot analysis of transgenic plants generated by cotransformation 2, 3, and 4. Total soluble protein was extracted from tobacco cotransformants 2, 3, and 4 and tested using an anti-collagen type I antibody (Chemicon Inc., no. AB745). The size marker was Fermentas Inc., no. SM0671. WT indicates wild-type tobacco. Positive collagen bands are visible in plants PCR-positive for collagen type I α1 or α2, or both. A positive control band of 500 ng of collagen type I from human placenta (Chemicon Inc., no. CC050, extracted from human placenta by pepsin digestion) represents approximately 0.3% (approximately 150 μg) of the total soluble protein in the transgenic plant samples. The larger band of approximately 140 kDa in the human collagen sample is procollagen with its C-propeptide, as detected by anti-carboxy-terminal propeptide collagen type I antibody (product number MAB1913, manufactured by Chemicon Inc.). The smaller band of approximately 120 kDa in the human collagen sample is collagen without the propeptide. Due to their unusual composition, proline-rich proteins (including collagen) consistently migrate on polyacrylamide gels as bands with higher molecular weights than expected. Thus, propeptide-free collagen chains, with a molecular weight of approximately 95 kDa, migrate as a band of approximately 120 kDa. [Figure 5] Conventional Western blot analysis of transgenic plants generated by cotransformation #8 (carrying an apoplastic signal translationally fused to the collagen chain). Total soluble protein was extracted from transgenic tobacco leaves and tested using an anti-collagen type I antibody (product number AB745, Chemicon Inc.). A positive collagen α2 band was observed in plants #8-141. Type I collagen from human placenta (product number CC050, Chemicon Inc.) was used as a control. [Figure 6A]Collagen triple helix organization and its previously validated thermostability were demonstrated by heat treatment and trypsin or pepsin digestion. Total soluble protein from tobacco strains 2-9 (expressing only Col α1, but not P4H) and 3-5 (expressing Col α1+2 and both human P4H α and β subunits) was heat-treated (38°C or 43°C for 15 min), then trypsin-digested (room temperature [RT] for 20 min), and tested in a Western blot procedure using an anti-collagen type I antibody. The positive control was a 500 ng sample of human collagen type I + wt tobacco total soluble protein. [Figure 6B] Collagen triple helix organization and its thermal stability, previously validated by heat treatment and trypsin or pepsin digestion, are demonstrated. Total soluble protein was extracted from transgenic tobacco 13-6 (expressing collagen type I α1 and α2 chains (indicated by arrows), human P4H α and β subunits, and human LH3), heat-treated (33°C, 38°C, or 42°C for 20 min), immediately chilled on ice to prevent triple helix reorganization, and incubated with pepsin for 30 min at room temperature (approximately 22°C). It was then tested using an anti-collagen type I antibody (Chemicon Inc., no. AB745) in a standard Western blot procedure. A positive control was a sample of 50 ng of human collagen type I (Chemicon Inc., no. CC050, extracted from human placenta by pepsin digestion) spiked into total soluble protein extracted from wild-type (wt) tobacco. [Figure 7]

[0023] Figure 1 shows a conventional Northern blot analysis performed on wild-type tobacco. The blot was probed with tobacco P4H cDNA. [Figure 8] Conventional Western blot analysis of transgenic plants generated by co-transformation. Total soluble proteins were extracted from tobacco co-transformants and tested using anti-human P4H α and β and anti-collagen type I antibodies. [Figure 9]Conventional Western blot analysis (lane 1) of hybrid vacuolar-targeted plant A (2-300 + 20-279) grown under normal light regimen and 13-652 vacuolar-targeted plant grown in the dark for 8 days. All plants express exogenous col1, col2, P4H-α and P4H-β, and LH3 (PCR verified). [Figure 10] Purified collagen from tobacco leaves after trypsin digestion is shown. Collagen was purified from transgenic tobacco plant leaves, line number 13-6, which were ground in 100 mM Tris buffer, centrifuged, proteolyzed, and precipitated in high-salt buffer, as detailed in the Materials and Methods section. After resuspension, the collagen-containing pellet was washed, dialyzed, and concentrated to the final product. This gel shows Coomassie staining analysis of the collected collagen samples; lanes 1 and 2 are collagen obtained after digestion of procollagen with 300 mg / L trypsin. Porcine collagen (0.5 mg / ml) without the propeptide was loaded and run as a positive control for collagen type 1 α1 and α2 chains. [Figure 11] Purified collagen from tobacco leaves after digestion with various concentrations of trypsin is shown. After digestion with 20 mg / L trypsin (lanes 1-7) or 30 mg / L trypsin (lanes 8-10), collagen was extracted and purified as shown in Figure 10. Products were separated on a 10% SDS-PAGE and analyzed with Coomassie staining. Porcine collagen (0.5 mg / ml) without the propeptide was loaded and run as a positive control for collagen type 1 α1 and α2 chains. [Figure 12] Purified collagen from tobacco leaves after digestion with trypsin and pepsin is shown. After digestion with 30 mg / L trypsin and 1 μg / 200 ml pepsin, collagen was extracted and purified as in Figure 10 (lanes 1-2). Products were separated on a 10% SDS-PAGE and analyzed with Coomassie-based staining. Porcine collagen (0.5 mg / ml) without the propeptide was loaded and run as a positive control for collagen type 1 α1 and α2 chains. [Figure 13]Collagen chains obtained by digesting procollagen with subtilisin or bromelain are shown. Collagen was purified from transgenic tobacco plant leaves, line number 13-361, which were crushed in 100 mM Tris buffer, centrifuged, and proteolyzed by incubation with either subtilisin (1–25 mg / L) or bromelain (1–25 mg / L) for 3 or 6 hours. Samples were separated on 10% SDS-PAGE and blotted onto nitrocellulose membranes. Collagen chains were immunodetected using anti-collagen type I. Untreated supernatant collected after homogenization and centrifugation was used as a negative control without collagen (lanes 3–4). Porcine collagen (2.5 μg) without the propeptide was used as a positive control for α1 and α2 chains (lane 1). [Figure 14] Collagen chains obtained by digesting procollagen with papain are shown. Collagen was purified from transgenic tobacco plant leaves, line number 13-361, by crushing in 100 mM Tris buffer, centrifuging, and proteolyzing with papain (1–25 mg / L) for 3 or 6 hours. Samples were separated on a 10% SDS-PAGE and blotted onto a nitrocellulose membrane. Collagen chains were immunodetected using anti-collagen type I. Untreated supernatants collected after homogenization, centrifugation, and incubation without enzyme at 15°C for 3 hours (lane 3) or 6 hours (lane 2) served as negative controls containing no collagen. Porcine collagen (2.5 μg) without the propeptide was used as a positive control for α1 and α2 chains (lane 1). [Figure 15]Collagen chains obtained by digesting procollagen with ficin or savinase are shown. Collagen was purified from transgenic tobacco plant leaves (line number 13-361) by crushing in 100 mM Tris buffer, centrifuging, and proteolyzing with ficin (1-25 mg / L) or savinase (1-25 mg / L) for 3 or 6 hours. Samples were separated on 10% SDS-PAGE and blotted onto nitrocellulose membranes. Collagen chains were immunodetected using anti-collagen type I. Untreated supernatant collected before proteolysis was used as a collagen-free control sample (lane 3). Porcine collagen (2.5 μg) without the propeptide was used as a positive control for α1 and α2 chains (lane 1). [Figure 16] Collagen chains obtained by digesting procollagen with Protamex or Alcalase are shown. Collagen was purified from transgenic tobacco plant leaves (line number 13-361) by crushing in 100 mM Tris buffer, centrifuging, and proteolyzing with Protamex (1-25 mg / L) or Alcalase (1-25 mg / L) for 3 or 6 hours. Samples were separated on 10% SDS-PAGE and blotted onto nitrocellulose membranes. Collagen chains were immunodetected using anti-collagen type I. Untreated supernatant collected before proteolysis was used as a collagen-free control sample (lane 14). Porcine collagen (2.5 μg) without the propeptide was used as a positive control for α1 and α2 chains (lane 1). [Figure 17]Collagen chains obtained by digesting procollagen with Esperase or Neutrase are shown. Collagen was purified from transgenic tobacco plant leaves, line number 13-361, by crushing in 100 mM Tris buffer, centrifuging, and proteolyzing with Esperase (1-25 mg / L) or Neutrase (1-25 mg / L) after 3 or 6 hours of incubation. Samples were separated on a 10% SDS-PAGE and blotted onto a nitrocellulose membrane. Collagen chains were immunodetected using anti-collagen type I. Pig-derived collagen (2.5 μg) without the propeptide was used as a positive control for the α1 and α2 chains (lane 1). [Figure 18] Collagen chains obtained by digesting procollagen with Esperase 8.0L or Alcalase are shown. Collagen was purified from transgenic tobacco plant leaves, line number 13-361, by grinding in 100 mM Tris buffer, centrifuging, and proteolyzing with Esperase (1-25 mg / L) or Neutrase (1-25 mg / L) after 3 or 6 hours of incubation. Samples were separated on 10% SDS-PAGE and blotted onto nitrocellulose membranes. Collagen chains were immunodetected using anti-collagen type I. Untreated supernatants collected after homogenization, centrifugation, and incubation at 15°C for 3 hours (lane 3) or 6 hours (lane 2) without protease were used as negative controls without collagen. Porcine collagen (2.5 μg) without the propeptide was used as a positive control for α1 and α2 chains (lane 1). [Figure 19]Collagen strands obtained at various stages of purification after ficin digestion of procollagen are shown. Collagen was purified from transgenic tobacco plant leaves, line number 13-361, which were ground in 100 mM Tris buffer, centrifuged, and proteolyzed with ficin (5 mg / L) after a 3-hour incubation at 15°C. Samples were separated on a 10% SDS-PAGE and blotted onto a nitrocellulose membrane. Collagen strands were immunodetected using anti-collagen type I. Lane 5 contains a sample collected after grounding, centrifugation, and incubation of the supernatant with ficin. Lanes 6–14 show samples of ficin-treated collagen at various stages of the purification process. Lane 6: Sample incubated with ficin and centrifuged. Lane 7: After salt precipitation and resuspension in 0.5 M acetic acid. Lane 8: The same sample as lane 7, with an additional centrifugation step. Lane 9: The 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: The same sample as lane 10, but with an additional filtration step. Lane 12: The same sample as lane 11, but with an additional 5-fold concentration step. Lane 13: The same sample as lane 11, but with an additional 20-fold concentration step. Lane 14: The same sample as lane 13, but with an additional 5-fold concentration step. An untreated procollagen sample (lanes 3-4) was used as a negative control. Porcine collagen (2.5 μg) without propeptides was used as a positive control for the α1 and α2 chains (lane 1). [Figure 20]Collagen strands obtained at various stages of purification after ficin digestion of procollagen are shown. Collagen was purified from transgenic tobacco plant leaves, line number 13-361, which were ground in 100 mM Tris buffer, centrifuged, and proteolyzed with ficin (5 mg / L) after a 3-hour incubation at 15°C. Samples were separated on a 10% SDS-PAGE and blotted onto a nitrocellulose membrane. Collagen strands were immunodetected using anti-collagen type I. Lane 5 contains a sample collected after grounding, centrifugation, and incubation of the supernatant with ficin. Lanes 6–14 show samples of ficin-treated collagen at various stages of the purification process. Lane 6: Sample incubated with ficin and centrifuged. Lane 7: After salt precipitation and resuspension in 0.5 M acetic acid. Lane 8: The same sample as lane 7, with an additional centrifugation step. Lane 9: The 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: The same sample as lane 10, but with an additional filtration step. Lane 12: The same sample as lane 11, but with an additional 5-fold concentration step. Lane 13: The same sample as lane 11, but with an additional 20-fold concentration step. Lane 14: The same sample as lane 13, but with an additional 5-fold concentration step. An untreated procollagen sample (lanes 3-4) was used as a negative control. Porcine collagen (2.5 μg) without propeptides was used as a positive control for the α1 and α2 chains (lane 1). [Figure 21] Collagen content of ficin-treated samples at various stages of purification is shown. Collagen-containing samples were collected at each extraction and purification stage of the reactor-sized AMS-based purification procedure described in the Materials and Methods section. Samples were treated with ficin (5 mg / L, 15°C, 3 hours) for propeptide removal, separated on 10% SDS-PAGE, and analyzed with a Coomassie-based stain. [Figure 22]Optimization of procollagen cleavage with food-grade ficin (optimization of ficin concentration and reaction time) is shown. AMS-pelleted tobacco leaf extract expressing procollagen was resuspended in extraction buffer and then incubated with increasing concentrations of food-grade ficin (5-15 mg / L). The reaction mixture was then incubated at 15°C for 1-3 hours. Cleavage was terminated by centrifugation, and protein samples were separated on 8% SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted for α-1 and α-2 collagen chains using anti-collagen type I antibodies. The procollagen bands are indicated by white arrows, while the red arrows indicate the cleaved collagen bands. [Figure 23A] Optimization of procollagen cleavage with pharmaceutical-grade ficin (optimization of ficin concentration and reaction time) is shown. AMS-pelleted tobacco leaf extract expressing procollagen was resuspended in extraction buffer and then incubated with increasing concentrations of pharmaceutical-grade ficin (2.5-10 mg / L). The reaction mixture was then incubated at 15°C for 0.5-3 hours. Cleavage was terminated by centrifugation, and protein samples were separated on 8% SDS-PAGE, transferred to a nitrocellulose membrane, and immunoblotted for α-1 and α-2 collagen chains using an anti-collagen type I antibody. Arrows indicate the procollagen and collagen bands. [Figure 23B] Optimization of procollagen cleavage with pharmaceutical-grade ficin (optimization of ficin concentration and reaction time) is shown. AMS-pelleted tobacco leaf extract expressing procollagen was resuspended in extraction buffer and then incubated with increasing concentrations of pharmaceutical-grade ficin (2.5-10 mg / L). The reaction mixture was then incubated at 15°C for 0.5-3 hours. Cleavage was terminated by centrifugation, and protein samples were separated on 8% SDS-PAGE, transferred to a nitrocellulose membrane, and immunoblotted for α-1 and α-2 collagen chains using an anti-collagen type I antibody. Arrows indicate the procollagen and collagen bands. [Figure 23C]Optimization of procollagen cleavage with pharmaceutical-grade ficin (optimization of ficin concentration and reaction time) is shown. AMS-pelleted tobacco leaf extract expressing procollagen was resuspended in extraction buffer and then incubated with increasing concentrations of pharmaceutical-grade ficin (2.5-10 mg / L). The reaction mixture was then incubated at 15°C for 0.5-3 hours. Cleavage was terminated by centrifugation, and protein samples were separated on 8% SDS-PAGE, transferred to a nitrocellulose membrane, and immunoblotted for α-1 and α-2 collagen chains using an anti-collagen type I antibody. Arrows indicate the procollagen and collagen bands. [Figure 24A] Optimization of procollagen cleavage by pharmaceutical-grade ficin (reaction buffer pH and salt concentration optimization) is shown. AMS-pelleted tobacco leaf extract expressing procollagen was resuspended in extraction buffer containing 10 mg / L pharmaceutical-grade ficin at various 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. Cleavage was terminated by centrifugation, and protein samples from both the pellet and supernatant were separated on an 8% SDS-PAGE, transferred to a nitrocellulose membrane, and immunoblotted for α-1 and α-2 collagen chains using an anti-collagen type I antibody. Arrows indicate collagen bands. [Figure 24B] Optimization of procollagen cleavage by pharmaceutical-grade ficin (reaction buffer pH and salt concentration optimization) is shown. AMS-pelleted tobacco leaf extract expressing procollagen was resuspended in extraction buffer containing 10 mg / L pharmaceutical-grade ficin at various 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. Cleavage was terminated by centrifugation, and protein samples from both the pellet and supernatant were separated on an 8% SDS-PAGE, transferred to a nitrocellulose membrane, and immunoblotted for α-1 and α-2 collagen chains using an anti-collagen type I antibody. Arrows indicate collagen bands. [Figure 25] Optimization of procollagen cleavage with pharmaceutical-grade ficin (optimizing the concentrations of EDTA and L-cysteine ​​in the reaction buffer) is shown. AMS-pelleted tobacco leaf extracts expressing procollagen were resuspended in extraction buffer (pH 7.5) containing various concentrations of L-cysteine ​​(10-100 mM, upper panel) and EDTA (8-80 mM, lower panel). The samples were then incubated with 1 mg / L pharmaceutical-grade ficin for 1 hour at 15°C. Cleavage was terminated by centrifugation, and protein samples were separated on 8% SDS-PAGE, transferred to a nitrocellulose membrane, and immunoblotted for α-1 and α-2 collagen chains using anti-collagen type I. [Figure 26] Effective procollagen digestion by recombinant trypsin at pH 7.5 is demonstrated. AMS-pelleted tobacco leaf extracts expressing procollagen were resuspended in extraction buffer (pH 7.5) containing L-cysteine ​​and EDTA. The samples were then incubated with 30–100 mg / L recombinant trypsin at 15°C for 1–3 hours. Cleavage was terminated by centrifugation, and protein samples were separated on 8% SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted for α-1 and α-2 collagen chains using anti-collagen type I. [Figure 27] Viscosity (eta [η], cP) as a function of shear rate. Solid line: 2.7 mg / mL bovine collagen in phosphate buffered saline (PBS). Dashed line: 2.79 mg / mL rh collagen in PBS. ▼: Measurement at 4°C. ▲: Measurement at 37°C. [Figure 28] Viscosity of 3.4 mg / mL bovine collagen in FB as a function of shear rate. ▼: Measurement at 4°C, ▲: Measurement at 37°C. [Figure 29] Viscosity as a function of shear rate for 10 mg / mL rhcollagen-MA in PBS. ▲: Measurement at 4°C, ▼: Measurement at 37°C. [Figure 30]Viscosity measurements of rhcollagen-MA in DMEM with and without the addition of HA / HAMA are shown. [Figure 31] 1 shows the storage and loss moduli and tan phase shift angles of rh collagen-MA formulations at different concentrations before (upper graph) and after (lower graph) photocrosslinking. [Figure 32] Shown are G' and G" values ​​at 37°C recorded in a frequency sweep experiment and plotted at 1 Hz. [Figure 33A]

[0023] Figure 1 provides a flow chart for the processing of rh collagen and rh collagen methacrylate. The upstream isolation and processing of procollagen and collagen is shown (steps A-H). [Figure 33B]

[0023] Figure 1 provides a flow chart for the processing of rh collagen and rh collagen methacrylate. Two stages of downstream processing (Step IM and Steps N-P and Z, respectively) are shown. [Figure 33C]

[0023] Figure 1 provides a flow chart for the processing of rh collagen and rh collagen methacrylate. Two stages of downstream processing (Step IM and Steps N-P and Z, respectively) are shown. [Figure 34] The viscosity (eta [η], cP) of 5 mg / ml rh collagen methacrylate (CollMA) (solid black curve) and 5 mg / ml collagen MA + polyvinyl alcohol methacrylate (PVMA) (light gray curve) is shown at collMA:PVAMA ratios of 5:1 (solid curve), 2:1 (dashed curve), and 1:2 (dotted curve). For comparison, the viscosity of 5 mg / ml rh collagen methacrylate is reported (black curve). [Figure 35]The viscosity of 5 mg / ml CollMA (solid black curve) and 5 mg / ml Collagen MA + Hyaluronic Acid Methacrylate (HAMA) (gray curve) is shown at CollMA:HAMA ratios of 5:1 (solid curve) and 2:1 (dashed curve). For comparison, the viscosity of 5 mg / ml rh-collagen methacrylate is reported (solid black curve). These materials are not yet cross-linked, but will be cross-linked after injection. The viscosity represents the injectability of the material. (HAMA-HA methacrylate; Collagen MA (ColMA)-rh-collagen methacrylate.) [Figure 36] The viscosity of 5 mg / ml CollMA (solid black curve) and 5 mg / ml CollagenMA + oxidized cellulose (OC) (gray curve) is shown for CollagenMA:OC ratios of 5:1 (solid curve), 2:1 (dashed curve), and 1:2 (dotted curve). For comparison, the viscosity of 5 mg / ml rh-collagen methacrylate is reported (solid black curve). [Figure 37] A comparison of the data in Figures 33-35 is provided. The viscosity of 5 mg / ml Collagen MA (solid black curve) and 5 mg / ml Collagen MA + various additives (light to dark gray curves as indicated in the figure) is shown in ratios of 5:1 (solid curve), 2:1 (dashed curve), and 1:2 (dotted curve). For comparison, the viscosity of 5 mg / ml rh collagen methacrylate is reported (solid black curve). [Figure 38] Polymerizable scaffolds of rh collagen methacrylate (ColMA) + additive at a CollMA:additive ratio of 2:1 were shown. ColMA alone was compared with ColMA combined with polyvinyl alcohol methacrylate (PVMA), hyaluronic acid methacrylate (HAMA), or oxidized cellulose (OC). The solution was mixed with the photoinitiator 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (0.1%) and irradiated with ultraviolet (UV) light (365 nm) for 20 seconds. [Figure 39]Viability studies are shown. Top graph: Comparison of viability (and proliferation) of normal human fibroblasts (nHDFs) cultured in the presence of GFs released from rh-collagen-PRP matrix (black), in the presence of GFs released from activated PRP (gray), and under starvation conditions (white). The data are the average of two separate fibroblast proliferation assays performed on PRP extracted from two different donors. *Significant difference (p<0.0002). Bottom inset: Microscopic images of nHDF cells grown in the presence of GFs released from rh-collagen matrix combined with PRP (A), grown in the presence of GFs released from activated PRP (B), and cultured under starvation conditions (C). Images were taken 7 days after seeding. [Figure 40] Scaffold weight as a function of time is shown (each point is the average of 6 scaffolds, 2 rats per time point, 3 injections per rat). [Figure 41A] A study using a subcutaneous rat model (PDGF content as a function of time) is shown. *Significant difference between rh collagen matrix combined with PRP and PRP alone (p<0.038), and between rh collagen matrix alone and PRP alone (p<0.004). **Significant difference between rh collagen matrix alone and PRP alone, and between rh collagen matrix alone and rh collagen matrix combined with PRP (p<0.021). [Figure 41B] 1 shows a study using a subcutaneous rat model (VEGF content as a function of time in a subcutaneous rat model). **Significant differences (p<0.007) between rh collagen matrix combined with PRP and PRP alone, and between rh collagen matrix combined with PRP and rh collagen matrix alone. [Figure 42] Figure 1 shows the integral of nominal PDGF and VEGF content in injected matrices over 45 (or 30) days in a rat model. [Figure 43A]1 shows histopathological scoring of Achilles tendons in a rat model of tendinopathy (mature fibrosis) treated with PRP or rhcollagen / PRP matrix. [Figure 43B] 1 shows histopathological scoring of Achilles tendons in a rat model of tendinopathy (presence of mononuclear inflammatory cells) treated with PRP or rhcollagen / PRP matrix. [Figure 43C] 1 shows histopathological scoring of Achilles tendons in a rat model of tendinopathy (presence of immature granulation tissue) treated with PRP or rhcollagen / PRP matrix. [Figure 44] A comparison of the expressive force (Newtons, N) required to inject cross-linked hyaluronic acid (HA) (black curve - square), cross-linked hyaluronic acid (HA) plus monomeric collagen (▽), or cross-linked hyaluronic acid (HA) plus fibrillated collagen (△) through a 32-gauge needle and 1 ml syringe (Becton Dickinson [BD], reference number 309628). Cross-linked HA plus monomeric collagen and cross-linked HA plus fibrillated collagen are semi-interpenetrating networks, and the collagen is not cross-linked in either case. [Figure 45] A comparison of the exertion force (Newtons, N) required to inject cross-linked hyaluronic acid (HA) (black) or a double-crosslinked network of cross-linked hyaluronic acid (HA) and collagen (gray) from a 32-gauge needle and 1 ml syringe (Becton Dickinson [BD], reference number 309628). [Figure 46] A comparison of the viscosity (eta [η], cP) of cross-linked hyaluronic acid (HA) (black), cross-linked hyaluronic acid (HA) + monomeric collagen (▽), or cross-linked hyaluronic acid (HA) + fibrillated collagen (△) is shown. [Figure 47] A comparison of the viscosity (eta [η], cP) of cross-linked hyaluronic acid (HA) (black) and a double-cross-linked network of cross-linked hyaluronic acid (HA) and collagen (gray) is shown. [Figure 48A] Shown is a photograph of a mouse patch placed on top of a methacrylated collagen (collMA / rhcollagenMA) solution. [Figure 48B]Photographs of methacrylated collagen (collMA / rhcollagenMA) polymerized and incorporated within skin tissue upon illumination with a white light-emitting diode (LED) torch through the skin are shown. [Figure 49] Two examples of dermal filler components are shown. The left side is a schematic diagram of a semi-interpenetrating dermal filler containing cross-linked hyaluronic acid (HA) and rh collagen. The right side is a schematic diagram of a dual-crosslinked dermal filler containing cross-linked hyaluronic acid and rh collagen, where the cross-linked HA is further cross-linked to the rh collagen. The light gray rods represent the HA cross-linker, the black strings represent HA, the rh collagen is represented as light gray strings, and the second cross-linker, which cross-links the cross-linked HA and the rh collagen, is represented as a black circle. [Figure 50] 1 shows a graph illustrating rheological measurements of storage and loss moduli for various dual crosslinked formulations measured using a HAAKE-RHEO STRESS 600™ instrument (THERMO SCIENTIFIC™) using a cone (1°) to plate configuration (C35 / 1). Frequency sweep measurements were performed at a constant deformation of 0.8% and frequencies ranging from 0.02 to 100 Hz. Storage modulus (solid line) and loss modulus (dashed line) of a representative dual crosslinked formulation (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 pointing up—Formulation 3; solid pentagon pointing down—Formulation 1A; solid ○—Formulation 1; dashed △—commercial product G"; dashed ▽—Formulation 2G"; dashed □—Formulation 2A-G"; dashed ○—Formulation 1G"; dashed pentagon pointing down—Formulation 1A G"; dashed pentagon pointing up—Formulation 3"). [Figure 51] Figure 50 shows a graph comparing the storage and loss moduli of the reported formulations at f=1 Hz. (White bars: G' [Pa]; gray bars: G [Pa].) [Figure 52]FIG. 1 shows a graph illustrating the injectability of selected dual-crosslink formulations, as measured using a MULTITEST 1-i MECMESIN™ compression tester with a 1 ml LUER-LOK™ syringe (BECTON-DICKINSON™) and a 30G needle used for Formulations 2, 2A, and 3 (Table 8). Commercially available dermal fillers are included for comparison with the dual-crosslink formulations. The application force as a function of plunger displacement (12 mm / min) of representative dual-crosslink formulations was compared to a commercially available dermal filler. (Black △—commercial dermal filler; gray □—Formulation 3; gray up-pointing pentagon—Formulation 2; gray ▽—Formulation 2A.) [Figure 53] Figure 1 shows a graph depicting rheological measurements of storage and loss moduli for various combinations of highly cross-linked hyaluronic acid (HA), rh-collagen methacrylate (MA), and / or rh-collagen (see Table 10) before (dashed lines) and after (solid lines) photocuring with visible light, compared to highly cross-linked HA (horizontal triangles with black intermittent lines). Prior to photocuring, storage and loss moduli were measured using a HAAKE-RHEO STRESS 600™ instrument (THERMO SCIENTIFIC™) using a cone (1°) to plate configuration (C35 / 1). Frequency sweep measurements were performed at a constant deformation of 0.8%, at frequencies ranging from 0.02 to 100 Hz. After photocuring (6 minutes of visible light irradiation using a white LED flashlight), the storage and loss moduli were measured using a HAAKE-RHEO STRESS 600™ instrument (THERMO SCIENTIFIC™) using a sawtooth plate-to-plate configuration (PP20). Frequency sweep measurements were performed at a constant shear stress of 3 Pa, frequencies ranging from 0.02 to 100 Hz, and a constant normal load of 0.3 N. (Solid △ - after Formulation 4; solid ▽ ​​- after Formulation 5; solid □ - after Formulation 6; dashed up-pointing pentagon - before Formulation 4; dashed down-pointing pentagon - before Formulation 5; dashed ○ - before Formulation 6; dashed horizontal triangle - highly crosslinked HA.) [Figure 54]Figure 1 shows a graph comparing the storage and loss moduli of uncured, highly crosslinked HA before and after photocuring of Formulations 4, 5, and 6 from Table 10 at a frequency of F = 1 Hz. (White bars: G' [Pa]; gray bars: G [Pa].) [Figure 55] Figure 1 shows a graph illustrating the injectability of selected dual-crosslinked formulations measured using a MULTITEST 1-i MECMESIN™ compression tester with a 1 ml LUER-LOK™ syringe (BECTON-DICKINSON™) and 30G needle used for all samples. The applied force as a function of plunger displacement (12 mm / min) of a representative dual-crosslinked formulation was compared to highly crosslinked HA. (Black △ - highly crosslinked HA; gray ▽ - Formulation 4; gray □ - Formulation 5; gray ○ - Formulation 6.) [Figure 56] Representative histology images are shown 7 days after subcutaneous injection of Formulations 2, 2A, and a control (a commercially available dermal filler) into the backs of Sprague-Dawley rats. In each case, arrows indicate an increased (but not severe) inflammatory response in Formulations 2 and 2A, indicating the onset of tissue regeneration. [Figure 57] Shown are histological scores at day 7 for Formulations 2, 2A, and control from tissues analyzed in Figure 56. (Black - control; light gray - Formulation 2; dark gray - Formulation 2A.) [Figure 58] Shown are histological scoring results of light-cured dermal fillers on days 7, 14, and 20. (Black - control highly crosslinked HA; Gray - Formulation 4 highly crosslinked HA and rhColMA.) [Figure 59] Fibrosis scores are shown at 7 and 14 days after injection of Formulation 4 (grey - highly cross-linked HA+rhColMA) and control (black - highly cross-linked HA). DETAILED DESCRIPTION OF THE INVENTION

[0068] Disclosed herein are photoinitiated dermal fillers and dual crosslinking dermal fillers and cell growth promoting scaffolds, and methods of using the same, for example, soft tissue augmentation.

[0069] Collagen-producing plants can be used to produce collagen chains, not just collagen, but such chains are not properly hydroxylated and therefore their self-assembly, whether in planta or not, results in collagen that is inherently unstable, in contrast to the plant-derived human collagen of the present application.

[0070] To reduce the complexity of the polymerizable and dual crosslinking solutions and methods of use of the present invention, practitioners have devised a plant expression approach that ensures proper hydroxylation of collagen, thereby enabling the production in plants of collagen that closely mimics the characteristics (e.g., molecular structure, temperature stability, cellular interactions) of human type I collagen.

[0071] In one aspect, a method of filling a tissue space below the epidermis comprises: (a) introducing a polymerizable solution into the tissue space, the polymerizable solution comprising: (i) cross-linkable plant-derived human collagen, and (ii) the introducing step, which includes a photoinitiator; (b) applying light to a surface of the epidermis at the surface of the space to induce polymerization.

[0072] In certain embodiments, the method further comprises molding or shaping the polymerizable solution into a desired configuration within the tissue space prior to or simultaneously with the light applying step, hi another specific embodiment, the molding or shaping step reduces lines, folds, fine lines, wrinkles, or scars.

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

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

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

[0076] Those skilled in the art will appreciate that photocurable formulations are actually semi-IPNs before curing and become IPNs (interpenetrated networks) after curing. An IPN may include two intertwined networks, each one crosslinked to itself and not to the other.

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

[0078] In some embodiments, HA or MA-HA may be crosslinked to itself using a crosslinking agent, such as, but not limited to, BDDE, as described in Example 23. In some embodiments, the crosslinking agent that crosslinks HA or MA-HA includes divinyl sulfone (DVS) or glutaraldehyde. In certain embodiments, BDDE-crosslinked HA or MA-HA is not further crosslinked to rh collagen or MA-rh collagen, producing a so-called interpenetrating network (left side of Figure 49).

[0079] In yet another specific embodiment, the plant-derived collagen comprises rh collagen. 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. In particular, the genetically modified plant is a tobacco plant.

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

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

[0082] 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 from enzymatic digestion (e.g., with ficin) of procollagen, the product of SEQ ID NO: 1 and SEQ ID NO: 4.

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

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

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

[0086] 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.

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

[0088] In yet another embodiment, the polymerizable solution within the tissue space is essentially non-gelling at room temperature. In another embodiment, the polymerizable solution within the tissue space is essentially non-gelling at 37°C. In yet another embodiment, a polymerizable solution comprising plant-derived human collagen has a lower viscosity at room temperature compared to a similar polymerizable solution comprising human- or animal-derived collagen extracted from tissue, such as, but not limited to, bovine, porcine, or equine collagen, at the same concentration and formulation. In another embodiment, a polymerizable solution comprising plant-derived human collagen has a lower viscosity at 37°C compared to a similar polymerizable solution comprising human- or animal-derived collagen extracted from tissue, at the same concentration and formulation.

[0089] As used throughout, the term "animal-derived collagen" may include bovine or porcine or equine collagen or rat tail collagen, as opposed to human-derived collagen.

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

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

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

[0093] In another aspect, there is provided a method of filling a tissue space below the epidermis, comprising: Disclosed herein are methods comprising the steps of: (a) introducing a polymerizable solution into a tissue space, wherein the polymerizable solution comprises crosslinkable plant-derived human collagen.

[0094] The present technology relates, in part, to collagen-based polymerizable fillers for cosmetic and medical applications that form polymerizable, moldable compositions upon photoactivation using a light source, e.g., a visible light source. The polymerizable fillers include crosslinkable plant-derived human collagen along with a photoinitiator.

[0095] This subject technology has the advantage of allowing for the in situ formation of custom-contoured dermal fillers or implants, typically without invasive surgical intervention or general anesthesia. Generally, a collagen-based polymerizable solution is introduced into the tissue space below the epidermis (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 to the epidermis.

[0096] In situ polymerization methods provide cosmetic and medical corrective and / or augmentation procedures using polymerizable solutions containing polymeric components capable of forming a water-insoluble crosslinked network upon photoactivation with a visible light source.

[0097] In some embodiments, the dermal fillers or cell growth-promoting scaffolds disclosed herein are for cosmetic applications. In some embodiments, the dermal fillers or cell growth-promoting scaffolds disclosed herein are for medical orthodontic applications. In some embodiments, the dermal fillers or cell growth-promoting scaffolds disclosed herein are for use in augmentation procedures, such as, but not limited to, tissue augmentation. In some embodiments, the dual-crosslinked dermal fillers disclosed herein are for cosmetic applications. In some embodiments, the dual-crosslinked dermal fillers disclosed herein are for medical orthodontic applications. In some embodiments, the dual-crosslinked dermal fillers disclosed herein are needed as a result of a medical or dental condition (gum graft / periodontal disease). In some embodiments, the dual-crosslinked dermal fillers disclosed herein are needed as a result of a medical condition requiring dermal augmentation. In some embodiments, the dual-crosslinked dermal fillers disclosed herein are for use in augmentation procedures, such as, but not limited to, tissue augmentation. In some embodiments, the light-curable dermal fillers disclosed herein are for cosmetic applications. In some embodiments, the light-curable dermal fillers disclosed herein are for medical orthodontic applications. In some embodiments, the light-curable dermal fillers disclosed herein are needed as a result of a medical or dental condition (gum graft / periodontal disease). In some embodiments, the light-curable dermal fillers disclosed herein are needed as a result of a medical condition requiring skin augmentation. In some embodiments, the light-curable dermal fillers disclosed herein are for use in augmentation procedures, such as, but not limited to, tissue augmentation. In some embodiments, the cell growth-promoting scaffolds disclosed herein are for cosmetic applications. In some embodiments, the cell growth-promoting scaffolds disclosed herein are for medical orthodontic applications. In some embodiments, the cell growth-promoting scaffold dermal fillers disclosed herein are needed as a result of a medical or dental condition (gum graft / periodontal disease).In some embodiments, the cell growth-promoting scaffold dermal fillers disclosed herein are indicated as a result of a medical condition requiring skin augmentation. In some embodiments, medical orthodontic applications include treating tendonitis. In some embodiments, the cell growth-promoting scaffolds disclosed herein are for use in augmentation procedures, such as, but not limited to, tissue augmentation.

[0098] In some embodiments, the tissue augmentation is tissue augmentation of dermal tissue.

[0099] In some embodiments, the use of a dermal filler comprising a cell growth-promoting scaffold disclosed herein in a human is for the reduction of 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 is for the augmentation of tissue, such as, but not limited to, epidermal or dermal tissue. In some embodiments, the tissue augmentation is for cosmetic purposes. In some embodiments, the tissue augmentation is for medical treatment. In some embodiments, the tissue augmentation is part of an augmentation procedure. In some embodiments, the tissue augmentation is part of a skin augmentation procedure.

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

[0101] In certain embodiments, dermal fillers for use herein comprise an interpenetrating (IPN) network or semi-interpenetrating (semi-IPN) network, in which different components may be crosslinked to themselves but not to each other. In some embodiments, the IPN or semi-IPN dermal filler comprises rh collagen and a filler, such as hyaluronic acid (HA), poly(vinyl alcohol) (PVA), polyethylene glycol (PEG), oxidized cellulose (OC), or a derivative thereof, or a combination thereof. In some embodiments, the IPN or semi-IPN comprises rh collagen and crosslinked HA. In some embodiments, the IPN or semi-IPN comprises a derivative of rh collagen, such as, but not limited to, methacrylated rh collagen or thiolated rh collagen, 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.

[0102] In some embodiments, the IPN or semi-IPN network or double-crosslinked network comprising a dermal filler comprises a filler, such as but not limited to, HA, PVA, PEG, or OC, 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 to rh collagen. In some embodiments, the IPN or semi-IPN network comprising a dermal filler comprises a MA filler, such as but not limited to, HA, PVA, PEG, or OC, 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 to rh collagen. In some embodiments, the IPN or semi-IPN network comprising a dermal filler comprises a filler, such as, but not limited to, HA, PVA, PEG, or OC, 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 to MA-rh collagen. In some embodiments, the IPN or semi-IPN network comprising a dermal filler comprises a MA-filler to MA-rh collagen 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.

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

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

[0105] In some embodiments, an IPN or semi-IPN network comprising a dermal filler or a dual crosslinked dermal filler comprises a cell growth-promoting scaffold.

[0106] In certain embodiments, the dermal filler for use herein comprises a photo-curable dermal filler, wherein at least one of the components, for example, but not limited to, rh collagen, comprises a methacrylate-rh collagen derivative or a thiol-rh collagen derivative. In some embodiments, the photo-curable dermal filler comprises rh collagen and a filler, for example, hyaluronic acid (HA), poly(vinyl alcohol) (PVA), polyethylene glycol (PEG), oxidized cellulose (OC), or a derivative thereof, or a combination thereof. In some embodiments, the photo-curable dermal filler comprises MA-rh collagen and HA or a derivative thereof. In some embodiments, the photo-curable dermal filler comprises a rh collagen derivative, for example, but not limited to, methacrylated rh collagen or thiol rh collagen, and / or a filler derivative, for example, but not limited to, methacrylated HA, PVA, PEG, or OC, or a thiolated HA, PVA, PEG, or OC, or a combination thereof.

[0107] In some embodiments, the light-curable dermal filler comprises a filler, such as but not limited to, HA, PVA, PEG, or OC, or a derivative thereof, to rh collagen in a ratio of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 1:2, 1:3, 1:4, 1:5, 1:6, or 0:1. In some embodiments, the light-curable dermal filler comprises a filler, such as but not limited to, HA, PVA, PEG, or OC, or a derivative thereof, to MA-rh collagen in a ratio of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 1:2, 1:3, 1:4, 1:5, 1:6, or 0:1. In some embodiments, the photo-curable dermal filler comprises a filler, such as but not limited to, HA, PVA, PEG, or OC, 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 to thiol-rh collagen. In some embodiments, the photo-curable dermal filler comprises a MA-filler to MA-rh collagen ratio of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 1:2, 1:3, 1:4, 1:5, 1:6, or 0:1.

[0108] In some embodiments, the light-curable dermal filler comprises a ratio of HA to MA-rh collagen of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 1:2, 1:3, 1:4, 1:5, 1:6, or 0:1, 1:3, 1:4, 1:5, or 0:1. In some embodiments, the light-curable dermal filler comprises a ratio of MA-HA to MA-rh collagen of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 1:2, 1:3, 1:4, 1:5, 1:6, or 0:1. In some embodiments, the light-curable dermal filler comprises a ratio of PVA, PEG, or OC to MA-rh collagen of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 1:2, 1:3, 1:4, 1:5, 1:6, or 0:1. In some embodiments, the light-curable dermal filler comprises a ratio of MA-PVA, MA-HA-, or OC to MA-rh collagen of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 1:2, 1:3, 1:4, 1:5, 1:6, or 0:1. In some embodiments, the HA component of the light-curable dermal filler comprises crosslinked HA or crosslinked MA-HA.

[0109] Throughout this application, various embodiments of the dermal filler and their uses may be presented in a range format. It should be understood that the range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the present invention. Thus, the description of a range should be considered to specifically disclose all possible subranges and individual numerical values ​​within that range. For example, the description of a range such as 1:1 to 6:1 should be considered to specifically disclose subranges such as 1.1:1, 1.2:1, 1.3:1 to 5.9:1, 1:1.1 to 1:1.9, and the individual numerical values ​​within that range and subranges, e.g., 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0110] Whenever a numerical range is given herein, it is meant to include any recited numbers (fractional or integer) within the range given. The expressions "ranging between" a first recited number and a second recited number, and "ranging from" a first recited number to a second recited number, are used interchangeably herein and are meant to include the first recited number and the second recited number, and all fractional and integer numbers therebetween.

[0111] For example, the present disclosure provides dermal fillers for tissue spaces below the epidermis that may comprise crosslinkable plant-derived human collagen, alone or in combination with fillers such as hyaluronic acid (HA), poly(vinyl alcohol) (PVA), polyethylene glycol (PEG), oxidized cellulose (OC), or combinations thereof, which crosslink to form a water-insoluble crosslinked polymer preparation in situ upon visible light activation in the presence of a photoinitiator. In some embodiments, the collagen is methacrylated or thiolated.

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

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

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

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

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

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

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

[0119] Those skilled in the art will understand that dermal fillers that involve dual crosslinking, in which the filler is crosslinked to itself and then to rh collagen, differ from dermal fillers that involve directly crosslinking collagen and HA with a single type of crosslinker in a single reaction. The properties of such dermal fillers will be different.

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

[0121] "Augmentation" means the repair, prevention, or alleviation of defects, particularly defects resulting from the loss or absence of tissue, by providing, augmenting, or replacing such tissue with a polymer or network or object. Augmentation is also meant to include the reinforcement of natural structures or features, i.e., constructs that are added to existing body parts (e.g., lips, nose, breasts, ears, organ parts, chin, cheeks, etc.) to, for example, increase their size. Thus, tissue augmentation can include, for example, filling or reduction of lines, folds, wrinkles, scars, small facial depressions, chapped lips, epidermal creases, etc., in or on the face, neck, hands, feet, fingers and toes; correction of mild deformities due to aging or disease, including in the hands and feet, fingers and toes; augmentation of the vocal cords or glottis to restore speech; filling the skin of muscles and expression lines caused by sleep; replacing skin and subcutaneous tissue lost due to aging; lip augmentation; filling of wrinkles around the eyes and orbital sulci; breast augmentation; chin augmentation; cheek and / or nose augmentation; filling depressions in the skin or subcutaneous soft tissue resulting from, for example, excessive liposuction or other external factors; filling of acne or traumatic scars and wrinkles; filling of nasolabial folds, glabellar lines, and suboral lines;

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

[0123] Once injected into the tissue space, the polymerizable solution may be manipulated, massaged, molded, or shaped within the desired contours within the tissue space, typically after photoinitiation of polymerization has been facilitated. In one embodiment, the manipulation, massaging, molding, or shaping occurs during the gelation process. The polymerizable solution, polymerizing solution, or partially polymerized solution can be shaped by external manipulation, for example, using a shaping means such as a surgical compression device or other tool or instrument using a flat or curved surface, finger, palm, knuckle, etc.

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

[0125] The "delivery force" of the injection (Newtons, N) includes the force required to inject from the needle or cannula.

[0126] "Absolute viscosity" ("dynamic viscosity") is the resistance of a fluid to flow when a force is applied. Absolute viscosity is proportional to the ratio of force to velocity. The Greek letter η (eta) represents absolute viscosity in calculations. It is usually measured in cP, as the viscosities of many common fluids range from 0.5 cP to 1000 cP.

[0127] A "gel" is a semi-rigid slab or cylinder of organic polymer used as a medium for the separation of macromolecules. Gels are substantially thin, cross-linked systems that do not exhibit flow properties at steady state. Gels are primarily liquid by weight, but retain some properties of liquids, such as deformability, and behave partially as solids due to a three-dimensional cross-linked network within the liquid. It is the cross-links within the fluid that give the gel its structure (rigidity) and contribute to its adhesive cohesion (stickiness). As a result, a gel can be viewed as a dispersion of liquid molecules within a solid, i.e., liquid particles dispersed within a solid medium. "Gelation time" is the time it takes for a polymerizable solution to form a gel.

[0128] A "hydrogel" is a network of polymer chains that is hydrophilic and sometimes found as a colloidal gel in which water is the dispersion medium. Hydrogels are highly absorbent (e.g., can contain more than 90% water) polymer networks that, due to their significant water content, have flexibility very similar to that of natural tissue.

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

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

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

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

[0133] Thiols are organosulfur compounds containing carbon-linked sulfhydryl (R-SH) groups, where R represents alkyl or other organic substituents. Thiolation of collagen may improve its cohesive and mucoadhesive properties and affect its swelling capacity.

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

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

[0136] The monomer solution of some embodiments may contain any of a variety of other materials, as known in the pharmaceutical arts, such as inert materials (e.g., preservatives, fillers, excipients, or diluents), pharmacologically active molecules or agents (e.g., small molecules or biological cells, etc.). Accordingly, suitable inert or biologically active agents may be added to the monomer solution. In the latter case, the active agent may exert a pharmacological effect locally at or near the site of the intended polymerized structure or network, or may be released from the formed scaffold, matrix, or network and migrate through adjacent tissue spaces or enter the circulatory system to reduce local effects.

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

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

[0139] In yet another aspect, disclosed herein is a method of inducing a cell growth-promoting scaffold into a tissue space below the epidermis, 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 source thereof.

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

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

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

[0143] In one embodiment, the transgenic 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.

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

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

[0146] In one particular embodiment, the human collagen alpha-1 chain is encoded by the sequence set forth in SEQ ID NO: 1. In another particular embodiment, the human collagen alpha-2 chain is encoded by the sequence set forth in SEQ ID NO: 2.

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

[0148] In one embodiment, the method further comprises targeting human collagen alpha-1 to the vacuole of the plant or transgenic plant and digesting it with ficin. In one embodiment, the method further comprises targeting human collagen alpha-2 to the vacuole of the plant or transgenic plant and digesting it with ficin.

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

[0150] Those skilled in the art will understand that the term "dermal filler," in some embodiments, encompasses a solution comprising plant-derived human collagen, such as type 1 recombinant human collagen (rh collagen) or a derivative thereof. The term "dermal filler," in some embodiments, also encompasses a solution comprising plant-derived human collagen, such as type 1 recombinant human collagen (rh collagen) or a derivative thereof, and a filler or derivative thereof, or a cross-linked filler or derivative thereof, all having the same meaning and qualities, and wherein the dermal filler may be used to enhance tissue structure or to reduce lines, folds, fine lines, wrinkles, or scars, or a combination thereof.

[0151] Those skilled in the art will understand that the dermal fillers described herein include different formulations, such as, but not limited to, the following: rh collagen or its MA or thiol derivatives, an IPN or semi-IPN or double crosslinked network comprising rh collagen or rh collagen-MA or rh collagen-thiol and a filler or a derivative thereof, an IPN or semi-IPN or double crosslinked network comprising rh collagen or rh collagen-MA or rh collagen-thiol and HA or MA-HA or thiol-HA, IPN or semi-IPN or double crosslinked network structures comprising rh collagen or rh collagen-MA or rh collagen-thiol and PVA or MA-PVA or thiol-PVA, an IPN or semi-IPN or double crosslinked network comprising rh collagen or rh collagen-MA or rh collagen-thiol and PEG or MA-PEG or thiol-PEG, an IPN or semi-IPN or double crosslinked network comprising rh collagen or rh collagen-MA or rh collagen-thiol and OC or MA-OC or thiol-OC, an IPN or semi-IPN or double crosslinked network structure or cell growth promoting scaffold comprising rh-collagen and an autologous platelet-rich plasma (PRP) fraction of blood containing a high concentration of platelets, an IPN or semi-IPN or double-crosslinked network structure, or a cell growth-promoting scaffold, each comprising rh collagen and an autologous platelet-rich plasma (PRP) fraction 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 growth factor-β (TGF-β), platelet-derived growth factor (PDGF), platelet-derived epidermal growth factor (PDEGF), fibroblast growth factor (bFGF), epidermal growth factor (EGF), or hepatocyte growth factor (HGF), or a combination thereof; A dual cross-linked dermal filler comprising rhcollagen or rhcollagen-MA or rhcollagen-thiol cross-linked to a cross-linked filler or its derivatives, a dual-crosslinked dermal filler comprising rh collagen or rh collagen-MA or rh collagen-thiol crosslinked to crosslinked HA or crosslinked MA-HA or crosslinked thiol-HA; Dual crosslinked dermal fillers comprising rh collagen or rh collagen-MA or rh collagen-thiol crosslinked to crosslinked PVA or crosslinked MA-PVA or crosslinked thiol-PVA, a dual cross-linked dermal filler comprising rh collagen or rh collagen-MA or rh collagen-thiol cross-linked to cross-linked PEG or cross-linked MA-PEG or cross-linked thiol-PEG, or A dual-crosslinked dermal filler comprising rh collagen or rh collagen-MA or rh collagen-thiol crosslinked to crosslinked OC or crosslinked MA-OC or crosslinked thiol-OC.

[0152] Those skilled in the art will understand that in some embodiments, the term "cell growth-promoting scaffold" encompasses dermal fillers comprising collagen and an autologous platelet-rich plasma (PRP) fraction of blood or a component thereof. In some embodiments, PRP does not contain "cells," but rather comprises (cell-derived) membrane vesicles containing growth factors and plasma components such as fibrinogen and prothrombin. In some embodiments, the term "cell growth-promoting scaffold" encompasses dermal fillers comprising collagen, an autologous platelet-rich plasma (PRP) fraction of blood or a component thereof, and at least one additional filler component.

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

[0154] In some embodiments, the dermal fillers described herein comprise a polymerizable solution. In some embodiments, the dermal fillers described herein comprise a non-polymerizable solution. In some embodiments, polymerization of the dermal filler solution occurs in vivo. In some embodiments, the components of a polymerizable dermal filler solution are injected together and then polymerized to form a hardened dermal filler. In some embodiments, the components of a polymerizable dermal filler solution are injected independently and then polymerized to form a hardened dermal filler. One example of a unique approach to injecting dermal filler components independently may, in some embodiments, include injecting a filler, such as, but not limited to, HA or a derivative thereof, into the dermis of the skin and separately injecting methacrylated or thiol-rh collagen into the skin dermis adjacent to the first injection, where this component is in a semi-liquid phase and may then be crosslinked in situ. This approach, in some embodiments, allows for easier injection and in situ shaping before hardening the dermal filler components together via photopolymerization.

[0155] In some embodiments, the dermal fillers provided herein are used in methods 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 methods of soft tissue augmentation degrade over time. In some embodiments, the dermal fillers provided herein are used in methods of soft tissue augmentation, where the dermal filler fills tissue spaces below the epidermis. In some embodiments, the dermal fillers provided herein are used in methods of soft tissue augmentation, where the use reduces lines, folds, fine lines, wrinkles, or scars.

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

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

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

[0159] In some embodiments, transgenic plants are provided that are capable of expressing at least one collagen alpha chain and accumulating it in a subcellular compartment that lacks endogenous P4H activity.

[0160] As used herein, the phrase "transgenic plant" refers to any lower (e.g., moss) or higher (e.g., vascular) plant, or tissues or isolated cells thereof (e.g., in a cell suspension), that are stably or transiently transformed with an exogenous polynucleotide sequence. Examples of plants include lower plants such as tobacco, corn, alfalfa, rice, potato, soybean, tomato, wheat, barley, canola, cotton, carrot, and moss.

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

[0162] As used herein, the phrase "subcellular compartment lacking endogenous P4H activity" refers to any compartmentalized region of a cell that does not contain a plant P4H or an enzyme with plant-like P4H activity. Examples of such subcellular compartments include the vacuole, apoplast, and cytoplasm, as well as organelles such as chloroplasts and mitochondria.

[0163] Any type of collagen chain can be expressed by the transgenic plants of the invention, including fibril-forming collagens (types I, II, III, V, and XI), meshwork-forming collagens (types IV, VIII, and X), collagens associated with fibril surfaces (types IX, XII, and XIV), collagens occurring as transmembrane proteins (types XIII and XVII), or collagens forming 11 nm periodic spherical filaments (type VI).

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

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

[0166] The processing of procollagen by plant proteolytic activity differs from normal processing in humans, and the propeptide C is removed by plant proteolytic activity, although the cleavage site is unknown. C-propeptide cleavage may occur in procollagen peptides prior to trimer assembly (the association of three C-propeptides is essential for initiating trimer assembly).

[0167] Cleavage of the N-propeptide by plant proteolytic activity occurs in mature plants but not in immature plants, resulting in the removal of two amino acids from the N-telopeptide (2 out of 17).

[0168] The C-propeptide (and, to a lesser extent, the N-propeptide) maintains procollagen solubility during passage through animal cells (Bulleid et al., 2000) and is expected to have a similar effect in plant cells. After or during secretion of procollagen molecules into the extracellular matrix, the propeptide is removed by procollagen N- and C-proteinases, thereby triggering spontaneous self-assembly of collagen molecules into fibrils. Removal of the propeptide by procollagen N- and C-proteinases reduces procollagen solubility by more than 10,000-fold, which is necessary and sufficient to initiate self-assembly of collagen into fibrils. Critical to this assembly process are short, non-triple-helical peptides at the ends of the triple-helical domain, called telopeptides, which ensure the correct positioning of collagen molecules within the fibril structure and reduce the critical concentration for self-assembly. Pepsin can cleave the propeptide during collagen production. However, pepsin damages the telopeptides, so that collagen extracted by pepsin cannot form a regular fibrous structure.

[0169] Protein disulfide isomerase (PDI), which forms the β-subunit of human P4H, was shown to bind to the C-propeptide prior to trimer assembly, thereby also functioning as a molecular chaperone during chain assembly.

[0170] The use of human procollagen type I N-proteinase and procollagen C-proteinase expressed in different plants may produce collagen that is more similar to native human collagen and can form regular fibrillar structures.

[0171] When the N or C propeptide, or both, are included in the expressed collagen chains, the transgenic plants of the present invention can also express the respective proteases (i.e., C or N, or both). Polynucleotide sequences encoding such proteases are exemplified by SEQ ID NOs: 18 (protease C) and 20 (protease N). Such proteases can be expressed such that they accumulate in the same intracellular compartment as the collagen chains.

[0172] Accumulation of expressed collagen chains in intracellular compartments lacking endogenous P4H activity can be brought about via any one of several approaches.

[0173] For example, the expressed collagen chains can include a signal sequence for targeting the expressed protein into 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 Examples section below provides further examples of suitable signal sequences, as well as guidelines for using such signal sequences in the expression of collagen chains in plant cells.

[0174] 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.

[0175] As described hereinabove, the ER of plants contains P4Hs that are unable to properly hydroxylate collagen chains. The collagen α chain naturally contains an ER targeting sequence that directs expressed collagen to the ER where it is post-translationally modified (including incorrect hydroxylation). Therefore, removal of the ER targeting sequence would result in the cytoplasmic accumulation of collagen chains that lack any post-translational modifications, including hydroxylation.

[0176] Example 1 in the Examples section below describes the generation of collagen sequences lacking ER sequences.

[0177] Alternatively, collagen chains can be expressed and accumulated within DNA containing organelles such as chloroplasts or mitochondria. Further description of chloroplast expression is provided herein below.

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

[0179] Such in vitro hydroxylation can result in correctly hydroxylated collagen chains, but can be difficult and expensive to achieve.

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

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

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

[0183] Since P4H needs to accumulate along with the expressed collagen chains, its coding sequence is preferably modified accordingly (addition of a signal sequence, deletions that may prevent ER targeting, etc.).

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

[0185] Therefore, the transgenic plants of the present invention preferably also express a mammalian LH3. An LH3 coding sequence, such as that set forth by SEQ ID NO: 22, can be used for such purposes.

[0186] The collagen chains and modifying enzymes described above may be expressed from stably integrated or transiently expressed nucleic acid constructs containing polynucleotide sequences encoding the α chains and / or modifying enzymes (e.g., P4H and LH3) placed under the transcriptional control of a plant-functional promoter. Such nucleic acid constructs (also referred to herein as expression constructs) may be configured for expression in the whole plant, defined plant tissues or cells, or defined developmental stages of the plant. Such constructs may also include selectable markers (e.g., antibiotic resistance), enhancer elements, and origins of replication for bacterial replication.

[0187] It will be understood that constructs containing two expressible inserts (e.g., two alpha procollagen chains, or an alpha chain and P4H) preferably contain individual promoters for each insert, or alternatively, such constructs may express a single transcript chimera containing both insert sequences from a single promoter. In such cases, the chimeric transcript will contain an IRES sequence between the two insert sequences so that the downstream insert can be translated therefrom.

[0188] A large number of plant functional expression promoters and enhancers, which can be either tissue-specific, developmentally specific, constitutive or inducible, can be utilized by the constructs of the present invention, some examples of which are provided herein below.

[0189] In the section of this specification and claims following the phrase "plant promoter" or "promoter," as used herein, includes a promoter capable of directing gene expression in plant cells (including DNA-containing organelles). Such promoters may be of plant, bacterial, viral, fungal, or animal origin. Such promoters may be constitutive (i.e., capable of directing high levels of gene expression in multiple plant tissues), tissue-specific (i.e., capable of directing gene expression in a particular plant tissue or multiple tissues), inducible (i.e., capable of directing gene expression upon a stimulus), or chimeric (i.e., formed from portions of at least two different promoters).

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

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

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

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

[0194] Preferably, the promoter utilized by the present invention is a strong constitutive promoter such that overexpression of the insert of the construct occurs following plant transformation.

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

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

[0197] Additionally, there are several methods by which nucleic acid constructs can be introduced directly into the DNA of DNA-containing organelles such as chloroplasts.

[0198] There are two primary methods for achieving stable genomic integration of exogenous sequences into a plant genome, such as those contained within the nucleic acid constructs of the present invention in the plant genome. (i) Agrobacterium-mediated gene transfer: Klee et al. (1987) Annu. Rev. Plant Physiol. 38:467-486; Klee and Rogers in Cell Culture and Somatic Cell Genetics of Plants, Vol. 6, edited by Schell, J. and Vasil, LK, Academic Publishers, San Diego, Calif. (1989) pp. 2-25; Gatenby in Plant Biotechnology, edited by Kung, S. and Arntzen, CJ, Butterworth Publishers, Boston, Mass. (1989) pp. 93-112. (ii) Direct DNA uptake: Paszkowski et al., in Cell Culture and Somatic Cell Genetics of Plants, Vol. 6, Molecular Biology of Plant Nuclear Genes (eds.), Schell, J. and Vasil, LK, Academic Publishers, San Diego, Calif. (1989) pp. 52-68, including a method for direct uptake of DNA into protoplasts, Toriyama, K. et al. (1988) Bio / Technology 6:1072-1074. DNA uptake induced by brief electric shock in plant cells, Zhang et al., Plant Cell Rep. (1988) 7:379-384. Fromm et al., Nature (1986) 319:791-793. Injection of DNA into plant cells or tissues by particle bombardment, Klein et al., Bio / Technology (1988) 6:559-563; McCabe et al., Bio / Technology (1988) 6:923-926; Sanford, Physiol. Plant. (1990) 79:206-209. by the use of a micropipette system, Neuhaus et al., Theor. Appl. Genet. (1987) 75:30-36; Neuhaus and Spangenberg, Physiol. Plant. (1990) 79:213-217, or by direct incubation of DNA with germinated pollen, DeWet et al., in Experimental Manipulation of Ovule Tissue (eds.), Chapman, GP and Mantell, SH and Daniels, W. Longman, London, (1985) pp. 197-209, and Ohta, Proc. Natl. Acad. Sci. USA (1986) 83:715-719.

[0199] The Agrobacterium system involves the use of a plasmid vector containing a defined DNA segment that is integrated into the plant's genomic DNA. Methods for inoculating plant tissue vary depending on the plant species and the Agrobacterium delivery system. A widely used approach is the leaf disc procedure, which can be performed on any tissue explant, providing an excellent source for initiating whole plant differentiation. (Horsch et al., Plant Molecular Biology Manual A5, Kluwer Academic Publishers, Dordrecht (1988) pp. 1-9) A complementary approach employs the Agrobacterium delivery system in combination with vacuum infiltration. The Agrobacterium system is particularly viable for creating transgenic dicotyledonous plants.

[0200] There are various 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 particle bombardment, DNA is adsorbed onto microprojectiles such as magnesium sulfate crystals, tungsten particles, or gold particles, and the microprojectiles are physically accelerated into cells or plant tissue.

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

[0202] Transient expression methods that can be used to transiently express the isolated nucleic acids contained in the nucleic acid constructs of the present invention include, but are not limited to, microinjection and bombardment, as described above, but under conditions desirable for transient expression, and viral-mediated expression, in which an encapsulated or unencapsulated recombinant viral vector containing the nucleic acid construct is used to infect plant tissue or cells such that the established propagating recombinant virus expresses the non-viral nucleic acid sequence.

[0203] Viruses that have been shown to be useful for the transformation of plant hosts include CaMV, TMV and BV.The transformation of plants using plant viruses is described in U.S. Patent No. 4,855,237 (BGV), EP-A67,553 (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).Pseudovirus particles for use in expressing foreign DNA in many hosts, including plants, are described in WO87 / 06261.

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

[0205] If the virus is a DNA virus, construction can be performed on the virus itself. Alternatively, to facilitate the construction of the desired viral vector using foreign DNA, the virus can first be cloned into a bacterial plasmid. The virus can then be excised from the plasmid. If the virus is a DNA virus, a bacterial origin of replication may be attached to the viral DNA, which is then replicated by the bacteria. Transcription and translation of this DNA produces a coat protein that encapsidates the viral DNA. If the virus is an RNA virus, the virus is typically cloned as cDNA and inserted into a plasmid. The plasmid is then used to create all of the constructs. The RNA virus is then generated by transcribing the viral sequences from the plasmid and translating the viral genes to produce a coat protein that encapsidates the viral RNA.

[0206] The construction of plant RNA viruses for the introduction and expression in plants of non-viral exogenous nucleic acid sequences, such as those contained in the constructs of the present invention, is set forth in the above references and in US Pat. No. 5,316,931.

[0207] In one embodiment, a plant viral nucleic acid is provided in which the native coat protein coding sequence has been deleted from the viral nucleic acid and a non-native plant viral coat protein coding sequence and a non-native promoter, preferably a subgenomic promoter for the non-native coat protein coding sequence, have been inserted, allowing expression in the plant host, encapsulation of the recombinant plant viral nucleic acid, and ensuring systemic infection of the host by the recombinant plant viral nucleic acid. Alternatively, the coat protein gene can be inactivated by inserting a non-native nucleic acid sequence therein so that the protein is produced. The recombinant plant viral nucleic acid may contain one or more additional non-native subgenomic promoters. Each non-native subgenomic promoter is capable of transcribing or expressing adjacent genes or nucleic acid sequences in the plant host and is incapable of recombining with each other or with the native subgenomic promoter. The non-native (foreign) nucleic acid sequence can be inserted adjacent to the native plant viral subgenomic promoter, or, if multiple nucleic acid sequences are included, the native and non-native plant viral subgenomic promoters. The non-native nucleic acid sequence is transcribed or expressed in the host plant under the control of the subgenomic promoter to produce the desired product.

[0208] In a second embodiment, a recombinant plant viral nucleic acid is provided similar to the first embodiment, except that the native coat protein coding sequence is positioned adjacent to one of the non-native coat protein subgenomic promoters in place of the non-native coat protein coding sequence.

[0209] 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 are 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 incapable of recombining with each other or with the native subgenomic promoter. A non-native nucleic acid sequence may be inserted adjacent to the non-native subgenomic plant viral promoter, such that the sequence is transcribed or expressed in the host plant under the control of the subgenomic promoter to produce a desired product.

[0210] In a fourth embodiment, a recombinant plant viral nucleic acid is provided similar to the third embodiment, except that the native coat protein coding sequence is replaced by a non-native coat protein coding sequence.

[0211] The viral vector is encapsidated by a coat protein encoded by the recombinant plant viral nucleic acid to produce a recombinant plant virus. The recombinant plant viral nucleic acid or recombinant plant virus is used to infect a suitable host plant. The recombinant plant viral nucleic acid is capable of replicating in the host, spreading systemically in the host, and transcribing or expressing a foreign gene (isolated nucleic acid) in the host to produce the desired protein.

[0212] A technique for introducing exogenous nucleic acid sequences into the chloroplast genome is known. This technique involves chemically treating plant cells to reduce the number of chloroplasts to approximately one per cell. Next, an exogenous nucleic acid is introduced into the cells via particle bombardment, with the goal of introducing at least one exogenous nucleic acid molecule into the chloroplast. The exogenous nucleic acid is selected so that it can be integrated into the chloroplast genome via homologous recombination, facilitated by enzymes native to chloroplasts. For this purpose, the exogenous nucleic acid contains, in addition to the gene of interest, at least one nucleic acid extension derived from the chloroplast genome. Additionally, the exogenous nucleic acid contains a selectable marker, which facilitates successive selection procedures to confirm that all or substantially all copies of the chloroplast genome following such selection contain the exogenous nucleic acid. Further details regarding this technique can be found in U.S. Patent Nos. 4,945,050 and 5,693,507, which are incorporated herein by reference. Thus, the polypeptide can be produced by a chloroplast protein expression system and integrated into the inner membrane of the chloroplast.

[0213] The transformation approaches described above can be used to produce collagen chains and / or modifying enzymes, as well as organized collagen (with or without propeptides) in any species of plant, or in plant tissues or isolated plant cells derived therefrom.

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

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

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

[0217] Preferably, a first plant expressing collagen alpha 1 and P4H (and optionally LH3 and protease C and / or N) can be crossed with a second (and preferably isogenic) plant expressing collagen alpha 2, or a first plant expressing both alpha chains can be crossed with a second plant expressing P4H and optionally LH3 and protease C and / or N.

[0218] It should be noted that although the plant breeding approach described above utilizes two independently transformed plants, approaches utilizing three or more independently transformed plants, each expressing one or two components, are also available.

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

[0220] While a plant breeding approach is preferred, it should be noted that a single plant expressing collagen α1 and 2, P4H and LH3 (and optionally protease C and / or N) can be generated through several transformation events, each designed to introduce one or more expressible components into the cell. In such cases, the stability of each transformation event can be verified using specific selectable markers.

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

[0222] Progeny obtained from breeding or multiply transformed plants can be selected by confirming the presence of exogenous mRNA and / or polypeptide by using nucleic acid or protein probes (e.g., antibodies). The latter approach is preferred because it allows for localization of the expressed polypeptide components (e.g., by probing fractionated plant extracts), thus also verifying the likelihood of correct processing and organization. Examples of suitable probes are described in the Examples section that follows.

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

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

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

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

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

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

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

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

[0231] In some embodiments, as shown herein by analysis of proteolytic results by SDS-PAGE, 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 capable of removing the telopeptides from telocollagen derived from animal sources), whereas other proteases (e.g., esperase, savinase) do not effectively cleave the propeptide region from soluble procollagen, thereby preventing effective fibril formation. Through extensive experimentation, the present inventors have demonstrated that only certain plant-derived proteases, such as ficin, and bacterial-derived proteases, such as neutrase and subtilisin, can correctly cleave the propeptide portion (including the telopeptides) from soluble procollagen, producing homogeneous preparations of soluble atelocollagen without digesting the helical region of non-animal procollagens (Figures 13, 15, 17, 19, and 20). In addition, we have shown that recombinant trypsin is also capable of correct cleavage (Figure 26). We have further shown that cleavage by ficin allows the resulting atelocollagen to retain its fibrillogenicity (Table 5 in the Examples section below).

[0232] Thus, according to one aspect, there is provided a method of producing atelocollagen, the method comprising contacting human recombinant telopeptide-containing collagen with a protease selected from the group consisting of neutrase, subtilisin, recombinant trypsin, recombinant pepsin, and ficin, wherein the human recombinant telopeptide-containing collagen is expressed in a non-animal cell, thereby producing atelocollagen.

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

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

[0235] As used herein, the term "telocollagen" refers to collagen molecules that lack both the N- and C-terminal propeptides normally found in procollagen, but still contain telopeptides. As noted in the background section above, the telopeptides of fibrillar collagen are remnants of the N- and C-terminal propeptides following digestion with native N / C proteinases.

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

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

[0238] Any type of atelocollagen can be produced according to the methods disclosed herein. Examples include fibril-forming collagens (types I, II, III, V, and XI), meshwork-forming collagens (types IV, VIII, and X), fibril surface-associated collagens (types IX, XII, and XIV), collagens occurring as transmembrane proteins (types XIII and XVII), or collagens forming 11 nm periodic spherical filaments (type VI). According to one embodiment, atelocollagen contains the α-1 and / or α-2 chains of type I collagen.

[0239] It will be appreciated that in some embodiments, recombinant forms of collagen / atelocollagen are disclosed herein, such as collagenase-resistant collagen.

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

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

[0242] The production of recombinant human procollagen is usually effected by stable or transient transformation with an exogenous polynucleotide sequence encoding human procollagen.

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

[0244] As noted above, production of human telocollagen is typically achieved by stable or transient transformation with an exogenous polynucleotide sequence encoding a human procollagen and at least one exogenous polynucleotide sequence encoding an associated protease.

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

[0246] Thus, in one embodiment, procollagen or telocollagen is expressed in a plant subcellular compartment lacking endogenous P4H activity to avoid its incorrect hydroxylation. As used herein, the phrase "subcellular compartment lacking endogenous P4H activity" refers to any compartmentalized region of a cell that does not contain plant P4H or an enzyme with plant-like P4H activity. In one embodiment, the subcellular compartment is a vacuole.

[0247] Accumulation of expressed procollagen in intracellular compartments lacking endogenous P4H activity can be brought about via any one of several approaches.

[0248] For example, the expressed collagen / telocollagen may contain a signal sequence for targeting the expressed protein into 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).

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

[0250] In some embodiments, genetically engineered cells are disclosed herein that co-express both human procollagen and P4H and are capable of correctly hydroxylating 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 to form the active enzyme, but the β subunit also possesses chaperone function.

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

[0252] Since it is essential that P4H accumulates along with the expressed procollagen chains, its coding sequence is preferably modified accordingly (eg, by addition or deletion of a signal sequence).

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

[0254] Thus, the recombinant cells disclosed herein may also express mammalian LH3, and an LH3 coding sequence, such as that set forth by SEQ ID NO: 38, can be used for such purposes.

[0255] The procollagens and modifying enzymes described above may be expressed from stably integrated or transiently expressed nucleic acid constructs containing polynucleotide sequences encoding the procollagen α-chain and / or modifying enzymes (e.g., P4H and LH3) placed under the transcriptional control of a functional promoter. Such nucleic acid constructs (also referred to herein as expression constructs) may be configured for expression throughout an organism (e.g., a plant, a defined tissue, or a defined cell) and / or at a defined developmental stage of the organism. Such constructs may also include selectable markers (e.g., antibiotic resistance), enhancer elements, and origins of replication for bacterial replication.

[0256] It will be understood that constructs containing two expressible inserts (e.g., two alpha procollagen chains, or a procollagen alpha chain and P4H) preferably contain individual promoters for each insert, or alternatively, such constructs may use a single promoter to express a single transcript chimera containing both inserts. In such cases, the chimeric transcript may contain an internal ribosome entry region (IRES) sequence between the two inserts so that the downstream insert can be translated therefrom.

[0257] A large number of functional expression promoters and enhancers, which can be either tissue-specific, developmentally specific, constitutive or inducible, can be utilized by the constructs of the present invention, some examples of which are provided herein below.

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

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

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

[0261] Exemplary methods for purifying or semi-purifying the telopeptide-containing collagen of the present invention include, but are not limited to, salting out with, for example, ammonium sulfate and / or removing small molecules by ultrafiltration.

[0262] As described in the background section of this specification above, the use of animal-sourced materials for medical purposes carries risks. This risk is also relevant when selecting proteolytic enzymes to be used in processing procollagen expressed in plants into atelocollagen. The application of animal-sourced enzymes, such as trypsin or pepsin, can itself contaminate the final preparation with disease carriers. Therefore, it is desirable to devise a production system in which all components are not of animal origin.

[0263] It is disclosed herein that only certain proteases can properly cleave collagen containing recombinant propeptides or telopeptides. Specific proteases include certain plant-derived proteases, such as ficin (EC 3.4.22.3), and certain bacterial proteases, such as subtilisin (EC 3.4.21.62), neutrase. In some embodiments, the use of recombinant enzymes such as rhtrypsin and rhpepsin is disclosed herein. Such enzymes are commercially available, for example, ficin from fig latex (Sigma, catalog number F4125 and Europe Biochem), subtilisin from Bacillus licheniformis (Sigma, catalog number P5459), neutrase from the bacterium Bacillus amyloliquefaciens (Novozymes, catalog number PW201041) and TrypZean™, recombinant human trypsin expressed in maize (Sigma catalog number T3449).

[0264] The procollagen or telocollagen is preferably contacted with the protease under conditions that allow the protease to cleave the propeptide or telopeptide from the contact point. Typically, the conditions are determined according to the particular 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.

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

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

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

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

[0269] As mentioned in the background chapter, type I collagen appears to be a perfect candidate for use as the primary building block for 3D bioprinting. Despite the significant advantages offered by this natural polymer, several factors hinder its use in 3D bioprinting. The use of tissue-extracted collagen for this purpose is limited due to its sensitivity to temperature and ionic strength, which promotes natural gel formation at temperatures above 20 °C under physiological conditions [see, for example, PureCol, Advanced BioMatrix, Inc.]. The typical temperature-dependent gel formation of tissue-extracted collagen significantly compromises its proper fluidity during printing. Keeping the print medium at a low temperature until application is a possible solution to this phenomenon, but it poses serious technical limitations. Another solution is the use of gelatin, a denatured form of collagen that does not gel under these conditions. However, gelatin lacks the true tissue-cell interactions of natural collagen, thereby missing important biological functions.

[0270] Recent technological developments have led to the development of a system for purifying native human type I collagen (rh collagen) by introducing five human genes encoding heterotrimeric type I collagen (COLLPLANT™, Israel, now also available at SIGMA-ALDRICH®, St. Louis, MO, USA) into tobacco plants. The protein is purified to homogeneity through a cost-effective industrial process that exploits the unique properties of collagen. See also WO2006 / 035442, WO2009 / 053985, and patents and patent applications derived therefrom, all of which are incorporated by reference as if fully set forth herein.

[0271] Thus, according to one aspect, disclosed herein is a transgenic plant capable of expressing and accumulating at least one collagen alpha chain in a subcellular compartment lacking endogenous P4H activity.

[0272] Type I collagen and rh collagen are considered candidates for use as the main components of the build material for 3D bioprinting. Various types of scaffolds have been used in cosmetic and other reconstructive applications.

[0273] In addition, the use of dermal fillers for soft tissue augmentation, such as wrinkle reduction, has been increasing. One possible method for using dermal fillers involves injecting a polymerizable dermal filler material into the desired area, followed by contouring or molding the filler into the desired shape. Polymerization and crosslinking of the material by one of various methods can convert the monomers in the injected material to form polymers and chains, which form a network structure and can retain the desired molded shape. There are several methods for forming polymers and crosslinking polymers. One method involves a photoinduced reaction with a photoreactive reagent that generates reactive species in the monomer solution.

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

[0275] Embodiments of the dermal fillers disclosed herein and their uses include, but are not limited to, the following: 1. A method for filling a tissue space below the epidermis, comprising: a. introducing a polymerizable solution into the tissue space, the polymerizable solution comprising: i. cross-linkable plant-derived human collagen, and ii. the introducing step includes a photoinitiator; b. applying light to the surface of the epidermis at the surface of the space to induce polymerization. 2. A method of filling a tissue space below the epidermis, comprising: (a) A method further comprising the step of molding or shaping the polymerizable solution into a desired configuration within the tissue space, which step occurs simultaneously with or subsequent to the step of applying light. 3. A method of filling tissue spaces below the epidermis, wherein the molding or shaping process reduces lines, folds, fine lines, wrinkles, or scars. 4. A method for filling tissue spaces below the epidermis, wherein cross-linkable plant-derived human collagen is methacrylated or thiolated. 5. A method of filling a tissue space below the epidermis, wherein the polymer solution further comprises hyaluronic acid (HA) or a modified derivative thereof, poly(vinyl alcohol) (PVA) or a modified derivative thereof, polyethylene glycol (PEG) or a modified derivative thereof, oxidized cellulose (OC) or a modified derivative thereof, polymethylmethacrylate (PMMA) microspheres or a modified derivative thereof, tricalcium phosphate (TCP) or a modified derivative thereof, calcium hydroxylapatite (CaHA) or a modified derivative thereof, carboxymethylcellulose or a modified derivative thereof, crystalline nanocellulose (CNC) or a modified derivative thereof, or a combination thereof. 6. A method for filling a tissue space beneath the epidermis, wherein the modified derivatives of hyaluronic acid (HA), poly(vinyl alcohol) (PVA), polyethylene glycol (PEG), oxidized cellulose (OC), polymethyl methacrylate (PMMA) microspheres, tricalcium phosphate (TCP), calcium hydroxylapatite (CaHA), carboxymethylcellulose, or crystalline nanocellulose (CNC) include photopolymerizable modified derivatives. 7. The method of claim 5, wherein the modified derivatives of hyaluronic acid (HA), poly(vinyl alcohol) (PVA), polyethylene glycol (PEG), oxidized cellulose (OC), polymethyl methacrylate (PMMA) microspheres, tricalcium phosphate (TCP), calcium hydroxylapatite (CaHA), carboxymethylcellulose or crystalline nanocellulose (CNC) include methacrylated or thiolated derivatives. 8. A method of filling a tissue space below the epidermis, wherein the plant-derived collagen comprises rh collagen. 9. A method for filling tissue spaces below the epidermis, wherein the plant-derived collagen is obtained from a genetically modified plant. 10. A method for filling a tissue space below the epidermis, 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. 11. A method for filling a tissue space below the epidermis, wherein the transgenic plant is a tobacco plant. 12. A method for filling a tissue space beneath the epidermis, wherein the transgenic 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. 13. A method for filling a tissue space below the epidermis, wherein the plant-derived human collagen comprises at least one modified human collagen alpha-1 chain as set forth in SEQ ID NO:3 and expressed in a transgenic plant, and at least one modified human collagen alpha-2 chain as set forth in SEQ ID NO:6 and expressed in the transgenic plant, wherein the transgenic plant further expresses exogenous prolyl-4-hydroxylase (P4H). 14. A method of filling a tissue space beneath the epidermis, further comprising expressing an exogenous polypeptide selected from the group consisting of lysyl hydroxylase (LH), protease N and protease C. 15. A method for filling a tissue space below the epidermis, wherein the human collagen alpha-1 chain is encoded by the sequence set forth in SEQ ID NO:1. 16. A method for filling a tissue space below the epidermis, wherein the human collagen alpha-2 chain is encoded by the sequence set forth in SEQ ID NO:2. 17. A method for filling a tissue space below the epidermis, wherein the exogenous P4H is mammalian P4H. 18. A method for filling a tissue space below the epidermis, wherein the exogenous P4H is human P4H. 19. A method for filling a tissue space below the epidermis, further comprising targeting human collagen alpha-1 to vacuoles of a plant or transgenic plant and digesting it with ficin. 20. A method for filling a tissue space below the epidermis, further comprising targeting human collagen alpha-2 to vacuoles of a plant or transgenic plant and digesting it with ficin. 21. A method for filling a tissue space below the epidermis, wherein the plant-derived human collagen is atelocollagen. 22. A method of filling a tissue space below the epidermis, wherein the light source comprises a light emitting diode (LED), a laser, or a xenon lamp. 23. A method of filling a tissue space below the epidermis, wherein a photoinitiator induces polymerization of a polymerizable solution in response to visible light. 24. A method for filling a tissue space below the epidermis, wherein the visible light has a wavelength of 390 to 700 nm. 25. A method of filling a tissue space below the epidermis, wherein the photoinitiator is selected from the group consisting of eosin Y plus triethanolamine or riboflavin. 26. A method of filling a tissue space below the epidermis, wherein a photoinitiator induces polymerization of a polymerizable solution in response to ultraviolet (UV) light. 27. A method of filling a tissue space below the epidermis, wherein the photoinitiator is selected from the group consisting of lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) or 1-[4 2-hydroxy-1-[4-(2-hydroxyethoxy)phenyl]-2-methylpropan-1-one (IRGACURE® 2959). 28. A method of filling a tissue space below the epidermis, wherein a photoinitiator induces polymerization of a polymerizable solution in response to infrared light. 29. A method of filling a tissue space below the epidermis, wherein a polymerizable solution is introduced into the tissue space through a hollow needle or cannula ranging from 27 gauge to 33 gauge. 30. A method of filling a tissue space below the epidermis, wherein a polymerizable solution within the tissue space is molded or shaped into a desired configuration by manual massage. 31. A method of filling a tissue space below the epidermis, wherein a polymerizable solution within the tissue space is molded or shaped into a desired configuration using a molding or shaping device. 32. A method of filling a tissue space below the epidermis, wherein the polymerizable solution within the tissue space is essentially non-gelling at room temperature. 33. A method of filling a tissue space below the epidermis, wherein the polymerizable solution within the tissue space is essentially non-gelling at 37°C. 34. A method for filling tissue spaces below the epidermis, wherein a polymerizable solution comprising plant-derived human collagen has a lower viscosity at room temperature compared to a similar polymerizable solution comprising human or bovine collagen extracted from tissue at the same concentration and formulation. 35. A method for filling tissue spaces below the epidermis, wherein a polymerizable solution comprising plant-derived human collagen has a lower viscosity at 37°C compared to a similar polymerizable solution comprising tissue-extracted human or bovine collagen at the same concentration and formulation. 36. A method for filling a tissue space beneath the epidermis, wherein a polymerizable solution containing plant-derived human collagen is introduced into the tissue space at room temperature with reduced force compared to a similar polymerizable solution containing human or bovine collagen extracted from tissue at the same concentration and formulation. 37. A method of filling a tissue space beneath the epidermis, wherein a polymerizable solution containing plant-derived human collagen is introduced into the tissue space at 37°C with lower force compared to a similar polymerizable solution containing human or bovine collagen extracted from tissue at the same concentration and formulation. 38. A method for filling tissue spaces below the epidermis, wherein a polymerizable solution containing plant-derived human collagen results in increased tissue augmentation compared to a similar polymerizable solution containing tissue-extracted human or bovine collagen at the same concentration and formulation. 39. Use of a polymerizable solution injected into a tissue space below the epidermis to reduce lines, folds, fine lines, wrinkles, or scars, the polymerizable solution comprising crosslinkable plant-derived human collagen and a photoinitiator for inducing polymerization prior to and simultaneously with the application of visible light, the use comprising the step of molding or shaping the polymerizable solution into a desired configuration to reduce lines, folds, fine lines, wrinkles, or scars. 40. Use of a polymerizable solution injected into tissue spaces below the epidermis to reduce lines, folds, fine lines, wrinkles, or scars, wherein crosslinkable plant-derived human collagen is methacrylated or thiolated. 41. Use of a polymerizable solution injected into a tissue space below the epidermis to reduce lines, folds, fine lines, wrinkles, or scars, wherein the polymer solution further comprises hyaluronic acid (HA) or a modified derivative thereof, poly(vinyl alcohol) (PVA) or a modified derivative thereof, polyethylene glycol (PEG) or a modified derivative thereof, polymethylmethacrylate (PMMA) microspheres or a modified derivative thereof, tricalcium phosphate (TCP) or a modified derivative thereof, calcium hydroxylapatite (CaHA) or a modified derivative thereof, carboxymethylcellulose or a modified derivative thereof, crystalline nanocellulose (CNC) or a modified derivative thereof, or a combination thereof. 42. Use of a polymerizable solution injected into tissue spaces below the epidermis to reduce lines, folds, fine lines, wrinkles, or scars, wherein the modified derivatives of hyaluronic acid (HA), poly(vinyl alcohol) (PVA), polyethylene glycol (PEG), oxidized cellulose (OC), polymethyl methacrylate (PMMA) microspheres, tricalcium phosphate (TCP), calcium hydroxylapatite (CaHA), carboxymethylcellulose, or crystalline nanocellulose (CNC) include photopolymerizable modified derivatives. 43. Use of a polymerizable solution injected into tissue spaces below the epidermis to reduce lines, folds, fine lines, wrinkles, or scars, wherein the polymerizable solution is a modified derivative of hyaluronic acid (HA), poly(vinyl alcohol) (PVA), polyethylene glycol (PEG), oxidized cellulose (OC), polymethylmethacrylate (PMMA) microspheres, tricalcium phosphate (TCP), calcium hydroxylapatite (CaHA), carboxymethylcellulose, or crystalline nanocellulose (CNC), including methacrylated or thiolated derivatives. 44. A method of filling a tissue space beneath the epidermis, comprising the step of introducing a polymerizable solution into the tissue space, the polymerizable solution comprising crosslinkable plant-derived human collagen. 45. A method of filling a tissue space below the epidermis, comprising: (a) The method further comprising the step of molding or shaping the polymerizable solution into a desired configuration within the tissue space. 46. ​​A method of filling tissue spaces below the epidermis, wherein the molding or shaping process reduces lines, folds, fine lines, wrinkles, or scars. 47. A method for filling a tissue space below the epidermis, wherein the polymerizable solution further comprises hyaluronic acid (HA) or a modified derivative thereof, poly(vinyl alcohol) (PVA) or a modified derivative thereof, polyethylene glycol (PEG) or a modified derivative thereof, 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 hydroxylapatite (CaHA) or a modified derivative thereof, carboxymethylcellulose or a modified derivative thereof, crystalline nanocellulose (CNC) or a modified derivative thereof, or a combination thereof. 48. A method for filling a tissue space below the epidermis, wherein the plant-derived collagen comprises rh collagen. 49. A method for filling a tissue space below the epidermis, wherein the plant-derived collagen is obtained from a genetically modified plant. 50. A method for filling a tissue space below the epidermis, 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. 51. A method for filling a tissue space below the epidermis, wherein the transgenic plant is a tobacco plant. 52. A method for filling a tissue space beneath the epidermis, wherein the transgenic 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. 53. A method for filling a tissue space below the epidermis, wherein the plant-derived human collagen comprises at least one modified human collagen alpha-1 chain as set forth in SEQ ID NO:3 and expressed in a genetically modified plant, and at least one modified human collagen alpha-2 chain as set forth in SEQ ID NO:6 and expressed in the genetically modified plant, wherein the genetically modified plant further expresses exogenous prolyl-4-hydroxylase (P4H). 54. A method of filling a tissue space below the epidermis, further comprising expressing an exogenous polypeptide selected from the group consisting of lysyl hydroxylase (LH), protease N, and protease C. 55. A method for filling a tissue space below the epidermis, wherein the human collagen alpha-1 chain is encoded by the sequence set forth in SEQ ID NO:1. 56. A method for filling a tissue space below the epidermis, wherein the human collagen alpha-2 chain is encoded by the sequence set forth in SEQ ID NO:2. 57. A method for filling a tissue space below the epidermis, wherein the exogenous P4H is mammalian P4H. 58. A method for filling a tissue space below the epidermis, wherein the exogenous P4H is human P4H. 59. A method for filling a tissue space below the epidermis, further comprising targeting human collagen alpha-1 to vacuoles of a plant or transgenic plant and digesting it with ficin. 60. A method for filling a tissue space below the epidermis, further comprising targeting human collagen alpha-2 to vacuoles of a plant or transgenic plant and digesting it with ficin. 61. A method for filling a tissue space below the epidermis, wherein the plant-derived human collagen is atelocollagen. 62. A method of filling a tissue space below the epidermis, wherein a polymerizable solution is introduced into the tissue space through a hollow needle or cannula ranging from 27 gauge to 33 gauge. 63. A method of filling a tissue space below the epidermis, wherein a polymerizable solution within the tissue space is molded or shaped into a desired configuration by manual massage. 64. A method of filling a tissue space below the epidermis, wherein a polymerizable solution within the tissue space is molded or shaped into a desired configuration using a molding or shaping device. 65. A method of filling a tissue space below the epidermis, wherein the polymerizable solution within the tissue space is essentially non-gelling at room temperature. 66. A method of filling a tissue space below the epidermis, wherein the polymerizable solution within the tissue space is essentially non-gelling at 37°C. 67. A method for filling tissue spaces below the epidermis, wherein a polymerizable solution containing plant-derived human collagen has a lower viscosity at room temperature compared to a similar polymerizable solution containing human or bovine collagen extracted from tissue at the same concentration and formulation. 68. A method for filling tissue spaces below the epidermis, wherein a polymerizable solution containing plant-derived human collagen has a lower viscosity at 37°C compared to a similar polymerizable solution containing human or bovine collagen extracted from tissue at the same concentration and formulation. 69. A method for filling a tissue space beneath the epidermis, wherein a polymerizable solution containing plant-derived human collagen is introduced into the tissue space at room temperature with reduced force compared to a similar polymerizable solution containing human or bovine collagen extracted from tissue at the same concentration and formulation. 70. A method for filling a tissue space beneath the epidermis, wherein a polymerizable solution containing plant-derived human collagen is introduced into the tissue space at 37°C with lower force compared to a similar polymerizable solution containing human or bovine collagen extracted from tissue at the same concentration and formulation. 71. A method for filling a tissue space below the epidermis, wherein a polymerizable solution containing plant-derived human collagen results in increased tissue augmentation compared to a similar polymerizable solution containing tissue-extracted human or bovine collagen at the same concentration and formulation. 72. Use of a polymerizable solution injected into a tissue space below the epidermis to reduce lines, folds, fine lines, wrinkles, or scars, the polymerizable solution comprising crosslinkable plant-derived human collagen, and the use comprising molding or shaping the polymerizable solution into a desired configuration to reduce the lines, folds, fine lines, wrinkles, or scars. 73. A method of inducing a cell growth scaffold into a tissue space below the epidermis, comprising introducing a solution into the tissue space, the solution comprising: (a) plant-derived human collagen; (b) at least one growth factor or a source thereof. 74. A method of inducing a cell growth scaffold within a tissue space below the epidermis, wherein the source of at least one growth factor comprises plasma or platelet-rich plasma. 75. A method for inducing a cell growth scaffold into a tissue space below the epidermis, wherein the cell growth scaffold promotes healing or replacement due to degradation or damage of collagen-containing tissue. 76. A method for inducing a cell growth scaffold into a tissue space beneath the epidermis, 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. 77. A method for inducing a cell growth scaffold into a tissue space below the epidermis, wherein the cell growth scaffold promotes healing of tendonitis. 78. A method for inducing a cell growth scaffold within a tissue space below the epidermis, wherein the plant-derived collagen comprises rh collagen. 79. A method for inducing a cell growth scaffold within a tissue space below the epidermis, wherein the plant-derived collagen is obtained from a genetically modified plant. 80. A method for inducing a cell growth scaffold within a tissue space below the epidermis, 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. 81. A method for inducing a cell growth scaffold within a tissue space below the epidermis, wherein the transgenic plant is a tobacco plant. 82. A method for inducing a cell growth scaffold within a tissue space beneath the epidermis, wherein the transgenic 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. 83. A method for inducing a cell growth scaffold within a tissue space below the epidermis, wherein the plant-derived human collagen comprises at least one modified human collagen alpha-1 chain as set forth in SEQ ID NO:3 and expressed in a genetically modified plant, and at least one modified human collagen alpha-2 chain as set forth in SEQ ID NO:6 and expressed in the genetically modified plant, wherein the genetically modified plant further expresses exogenous prolyl-4-hydroxylase (P4H). 84. A method for inducing a cell growth scaffold within a tissue space below the epidermis, the method further comprising expressing an exogenous polypeptide selected from the group consisting of lysyl hydroxylase (LH), protease N, and protease C. 85. A method for inducing a cell growth scaffold within a tissue space below the epidermis, wherein the human collagen alpha-1 chain is encoded by the sequence set forth in SEQ ID NO:1. 86. A method for inducing a cell growth scaffold within a tissue space below the epidermis, wherein the human collagen alpha-2 chain is encoded by the sequence set forth in SEQ ID NO:2. 87. A method for inducing a cell growth scaffold within a tissue space below the epidermis, wherein the exogenous P4H is mammalian P4H. 88. A method for inducing a cell growth scaffold within a tissue space below the epidermis, wherein the exogenous P4H is human P4H. 89. A method for inducing a cell growth scaffold within a tissue space below the epidermis, the method further comprising targeting human collagen alpha-1 to the vacuole of a plant or transgenic plant and digesting it with ficin. 90. A method for inducing a cell growth scaffold within a tissue space below the epidermis, the method further comprising targeting human collagen alpha-2 to the vacuole of a plant or transgenic plant and digesting it with ficin. 91. A method for inducing a cell growth scaffold within a tissue space below the epidermis, wherein the plant-derived human collagen is atelocollagen. 92. A method for inducing a cell growth scaffold within a tissue space below the epidermis, wherein a solution containing plant-derived human collagen has a lower viscosity at room temperature compared to a similar solution containing human or bovine collagen extracted from tissue at the same concentration and formulation. 93. A method for inducing a cell growth scaffold within a tissue space below the epidermis, wherein a solution containing plant-derived human collagen has a lower viscosity at 37°C compared to a similar polymerizable solution containing human or bovine collagen extracted from tissue at the same concentration and formulation. 94. A method for inducing a cell growth scaffold within a tissue space beneath the epidermis, wherein a solution containing plant-derived human collagen is introduced into the tissue space at room temperature with reduced force compared to a similar solution containing human or bovine collagen extracted from tissue at the same concentration and formulation. 95. A method for inducing a cell growth scaffold within a tissue space beneath the epidermis, wherein a solution containing plant-derived human collagen is introduced into the tissue space at 37°C with lower force compared to a similar solution containing human or bovine collagen extracted from tissue at the same concentration and formulation. 96. A method for inducing cell growth scaffolding within tissue spaces below the epidermis, wherein a solution containing plant-derived human collagen increases scaffold formation or promotes an increase in cell proliferation compared to a similar solution containing human or bovine collagen extracted from tissue at the same concentration and formulation. 97. Use of a solution injected into a tissue space below the epidermis to induce a cell growth scaffold, the solution comprising plant-derived human collagen and at least one growth factor or source thereof, to promote healing or replacement of collagen-containing tissue resulting from degradation or damage. 98. Use of a solution injected into a tissue space below the epidermis to induce a cell growth scaffold, wherein the source of at least one growth factor comprises plasma or platelet-rich plasma.

[0276] definition

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

[0278] As used herein, the term "about" refers to ±10% or ±5%.

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

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

[0281] The term "consisting essentially of" means that a composition, method, or structure may include additional ingredients, steps, and / or parts only if the additional ingredients, steps, and / or parts do not materially alter the basic and novel characteristics of the claimed composition, method, or structure.

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

[0283] As used herein, the phrase "transgenic plant" includes any lower (e.g., moss) or higher (e.g., vascular) plant, or tissues or isolated cells thereof (e.g., in a cell suspension), that are stably or transiently transformed with an exogenous polynucleotide sequence. Examples of plants include, but are not limited to, lower plants such as tobacco, corn, alfalfa, rice, potato, soybean, tomato, wheat, barley, canola, cotton, carrot, and moss.

[0284] As used herein, the phrase "collagen chain" encompasses collagen subunits such as the α1 or α2 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 contains two α1 chains and one α2 chain. A collagen fiber is collagen lacking the terminal propeptides C and N.

[0285] As used herein, the phrase "telopeptide-containing collagen" encompasses soluble collagen molecules that contain telopeptides longer than the telopeptide remnants contained in atelocollagen. Thus, telopeptide-containing collagen can be procollagen containing full-length propeptides. Alternatively, telopeptide-containing collagen can be procollagen molecules containing partially digested propeptides. Alternatively, telopeptide-containing collagen can be telocollagen.

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

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

[0288] As used herein, the term "atelocollagen" encompasses collagen molecules lacking both the N- and C-terminal propeptides typically found in procollagen and at least a portion of its telopeptides, but containing sufficient portions of its telopeptides to be capable of forming fibrils under suitable conditions. Any type of atelocollagen can be produced by the methods of the present invention. Examples include fibril-forming collagens (types I, II, III, V, and XI), meshwork-forming collagens (types IV, VIII, and X), collagens associated with fibril surfaces (types IX, XII, and XIV), collagens occurring as transmembrane proteins (types XIII and XVII), or collagens forming periodic 11-nm spherical filaments (type VI). According to one embodiment, atelocollagen contains the α1 and / or α2 chains of type I collagen.

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

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

[0291] As used herein, the phrase "subcellular compartment lacking endogenous P4H activity" refers to any compartmentalized region of a cell that does not contain a plant P4H or an enzyme with plant-like P4H activity. Examples of such subcellular compartments include the vacuole, apoplast, and cytoplasm, as well as organelles such as chloroplasts and mitochondria.

[0292] Throughout this specification, the phrase "build material" encompasses the phrases "uncured build material" or "uncured build material formulation," which generically describes the materials used to sequentially form layers as described herein. This phrase encompasses the uncured materials that form the final object, i.e., one or more uncured modeling material formulations, and, optionally, the uncured materials used to form the support, i.e., uncured support material formulations. The uncured build material may include one or more modeling formulations and can be dispensed such that different portions of the object are created upon curing of different modeling formulations, and thus are made of different cured modeling materials or different mixtures of cured modeling materials.

[0293] As used herein, "bioprinting" means performing an additive manufacturing process utilizing one or more bio-ink formulations that include biological components by a methodology that is compatible with an automated or semi-automated computer-assisted additive manufacturing system (e.g., a bioprinter or bioprinting system) described herein.

[0294] Throughout this specification, in the context of bioprinting, the term "object" describes an additively manufactured end product that includes a biological component at least in part thereof. This term refers to the product obtained by the bioprinting methods described herein after removal of the support material, if used as part of the uncured build material. In some embodiments, the biological component comprises recombinant human collagen, for example, as described in WO2006 / 035442, WO2009 / 053985, and patents and patent applications derived therefrom, all of which are incorporated by reference as if fully set forth herein.

[0295] As used throughout this specification, the term "object" refers to the entire object or a portion thereof.

[0296] Throughout this specification, a "hardenable material" refers to a compound (monomer, oligomer, or polymer compound) that solidifies or hardens upon exposure to the curing conditions described herein to form a hardened modeling material as defined herein. Hardenable materials are typically polymerizable materials that undergo polymerization and / or crosslinking upon exposure to a suitable energy source. Alternatively, hardenable materials are thermoresponsive materials that solidify or harden upon exposure to a temperature change (e.g., heating or cooling). In some cases, hardenable materials are biological materials that undergo a reaction to form a hardened or solidified material upon a biological reaction (e.g., an enzyme-catalyzed reaction).

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

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

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

[0300] As used herein, the term "curable" encompasses the terms "polymerizable" and "crosslinkable."

[0301] As used herein, "aeroponics" is the process of growing plants in an air or mist environment without the use of soil or a flocculating medium (also known as "geoponics").

[0302] As used herein, "hydroponics" is the process of growing plants without soil using mineral nutrient solutions in a water medium ("geoponics").

[0303] As used herein, "endophyte" includes all plant endophytes.

[0304] As used herein, "exudate" is a fluid released by an organism through pores or wounds. "Exudation" is the process of releasing "exudate."

[0305] As used herein, "hydroponics" is the process of growing plants without soil using mineral nutrient solutions in a water medium ("geoponics").

[0306] As used herein, "integration" or "integrative hybridization" is the transfer of genes (i.e., "gene flow") from the gene pool of one species to the gene pool of another species via repeated backcrossing with one of the interspecific hybrids, resulting in a complex mixture of parental genes, unlike simple crossing.

[0307] As used herein, a "metabolome" is the complete set of small molecule chemicals found within a "biological sample" (including, but not limited to, a cell, organelle, organ, tissue, tissue extract, biological fluid, or organism). The small molecule chemicals of the metabolome may be "endogenous metabolites" or "exogenous chemicals." "Endogenous metabolites" are naturally produced by an organism 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 an organism and include, but are not limited to, drugs, environmental pollutants, food additives, toxins, and other xenobiotics. The "endogenous metabolome" is composed of endogenous metabolites, and the "exogenous metabolome" is composed of "exogenous chemicals." The "endogenous metabolome" is composed of the "primary metabolome" and the "secondary metabolome," particularly for plants, fungi, and prokaryotes. The "primary metabolome" is composed of "primary metabolites" (i.e., metabolites directly involved in the normal growth, development, and reproduction of an organism), and the "secondary metabolome" is composed of "secondary metabolites" (i.e., metabolites not directly involved in the normal growth, development, and reproduction of an organism). Secondary metabolites often have important ecological functions.

[0308] As used herein, a "metabolite" is typically a small molecule having a molecular weight of less than 1500 Da. "Metabolites" can include, but are not limited to, glycolipids, polysaccharides, short peptides, small oligonucleotides, organic acids, taxanes, alkaloids, and strigolactones, although very large macromolecules (e.g., proteins, mRNA, rRNA, and DNA) are generally not metabolites or part of the metabolome.

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

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

[0311] Methods for isolating, sequencing, amplifying and / or cloning DNA are known to those skilled in the art.The most commonly used method for DNA amplification is PCR (polymerase chain reaction, see, for example, 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 self-sustained sequence replication, and nucleic acid-based sequence amplification (NASBA).Similarly, methods for isolating, characterizing, etc., RNA and protein, and protein expression are known to those skilled in the art.

[0312] The following examples are presented to more fully illustrate some embodiments of the dermal fillers disclosed herein and their uses. However, they should in no way be construed as limiting the broad scope of the dermal fillers disclosed herein and their uses. Those skilled in the art can easily devise numerous variations and modifications of the principles disclosed herein without departing from the scope of the present invention. [Example]

[0313] Example 1. Constructs and transformation schemes.

[0314] The construction of the expression cassettes and vectors used in this study is shown in Figures 1A-1D (see also U.S. Patent No. 8,455,717). All coding sequences in this study were optimized for expression in tobacco and chemically synthesized with the desired flanking regions (SEQ ID NOS: 1, 4, 7, 12, 14, 16, 18, 20, and 22). Figure 1A: Synthetic genes encoding Col1 and Col2 (SEQ ID NOS: 1 and 4) fused to either a vacuolar signal or an apoplastic signal (encoded by SEQ ID NOS: 7), or no signal, cloned within an expression cassette composed of the Chrysanthemum rbcS1 promoter and 5' UTR (SEQ ID NOS: 10), the Chrysanthemum rbcS1 3' UTR, and a terminator (SEQ ID NOS: 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 either a vacuolar signal, an apoplastic signal (encoded by SEQ ID NO: 7), or no signal were cloned into expression cassettes composed of the CaMV 35S promoter, TMV omega sequence, and Agrobacterium nopaline synthetase (NOS) terminator carried by vector pJD330 (Galili et al., 1987, Nucleic Acids Res 15:3257-3273). The complete expression cassettes were 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 NOs: 18, 20) fused to either a vacuolar or apoplastic signal (encoded by SEQ ID NO: 7) were cloned into expression cassettes composed of the Chrysanthemum rbcS1 promoter and 5'UTR (SEQ ID NO: 10), 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) flanked by the strawberry vein banding virus (SVBV) promoter (NCBI accession AF331666 REGION:623.950 version AF331666.1 GI:13345788) and terminated by the Agrobacterium octopine synthase (OCS) terminator (NCBI accession Z37515 REGION:1344.1538 version Z37515.1 GI:886843) fused to either a vacuolar or apoplastic signal (encoded by SEQ ID NO: 7) or no signal was cloned into the multiple cloning site of the pBINPLUS vector carrying expression cassettes for Col1 and P4Hβ.

[0315] The co-transformation scheme for host plants using the expression cassettes described in Figures 1A-1D is shown in Figure 2. Each expression cassette insert is represented by the short name of the coding sequence. The coding sequences and associated SEQ ID NOs are listed in Table 1. Each co-transformation is carried out with two pBINPLUS binary vectors. Each rectangle represents a single pBINPLUS vector carrying one, two, or three expression cassettes. The promoters and terminators are specified in Figures 1A-1D.

[0316] Example 2. Plant collagen expression.

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

[0318] [Table 1]

[0319] signal peptide

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

[0321] Plasmid construction

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

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

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

[0325] Each of the above coding sequences is translationally fused to a vacuolar transit peptide or an apoplastic transit peptide, or lacks any transit peptide sequence, in which case cytoplasmic accumulation is predicted.

[0326] Plant transformation and PCR screening

[0327] Tobacco plants (Nicotiana tabacum, Samsun NN) were transformed with the expression vectors described above according to the transformation scheme taught in FIG.

[0328] 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 shows the results of one multiplex PCR screen.

[0329] [Table 2]

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

[0331] Total soluble protein was extracted from tobacco transformants 2, 3, and 4 by crushing 500 mg of leaves with a "Complete" protease inhibitor cocktail (product number 1836145 from Roche Diagnostics GmbH, one tablet per 50 ml of buffer) in 0.5 ml of 50 mM Tris-HCl (pH = 7.5). The crude extract was mixed with 250 μl of 4X sample application buffer containing 10% β-mercaptoethanol and 8% SDS. The sample was boiled for 7 minutes and centrifuged at 13,000 rpm for 8 minutes. 20 μl of the supernatant was loaded onto a 10% polyacrylamide gel and tested using an anti-collagen type I (denatured) antibody (product number AB745 from Chemicon Inc.) in a standard Western blot procedure (Figure 4). WT is wild-type tobacco. Positive collagen bands can be seen in plants PCR-positive for collagen type I α1 or α2, or both. A positive control band of 500 ng of collagen type I from human placenta (Chemicon Inc., no. CC050) represents approximately 0.3% (approximately 150 μg) of the total soluble protein in the sample from the transgenic plants.

[0332] When collagen was targeted to the vacuole, plants expressing collagen at the expected molecular weight of approximately 1% of total soluble protein were detected (Figure 4). Intracellular 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, indicating that intracellular targeting of collagen in plants is important for this success.

[0333] In addition to this, 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 this approach, full-length collagen protein with the C- and N-propeptides accumulated to high levels in intracellular compartments.

[0334] This data also clearly demonstrates that crossing two plants, each expressing a different type of collagen chain, is advantageous in that it allows for the selection of plants expressing optimal levels of each type of chain, allowing subsequent plant crossing to achieve the desired collagen-producing plant.

[0335] Collagen produced by the plant of the present invention contains the native propeptide and is therefore expected to form a larger protein than the human control 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, and Col2 without propeptide - 92 kDa.

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

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

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

[0339] In the first experiment, total soluble protein from tobacco plants 2-9 (expressing only col α1, not P4H) and 3-5 (expressing both col α1+2 and human P4H) was extracted by grinding 500 mg of leaves in 0.5 ml of 50 mM Tris-HCl (pH = 7.5), centrifuging at 13,000 rpm for 10 min, and collecting the supernatant. 0 μl of the supernatant was heat-treated (33 °C or 43 °C for 15 min) and immediately placed on ice. Trypsin digestion was initiated by adding 6 μl of each sample to 1 mg / ml trypsin in 50 mM Tris-HCl (pH = 7.5). Samples were incubated at room temperature (approximately 22 °C) for 20 min. Digestion was stopped by adding 20 μl 4X sample application buffer containing 10% β-mercaptoethanol and 8% SDS, and the samples were boiled for 7 minutes and centrifuged at 13,000 rpm for 7 minutes. 50 μl of the supernatant was loaded onto a 10% polyacrylamide gel and tested using anti-collagen type I (denatured) antibody (Chemicon Inc., no. AB745) in a standard Western blot procedure. The positive control was a sample of 500 ng of human collagen type I (Chemicon Inc., no. CC050, extracted from human placenta by pepsin digestion) added to 50 μl of total soluble protein extracted from wt tobacco.

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

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

[0342] In the second experiment, total soluble protein from transgenic tobacco 13-6 (expressing collagen type I α1 and α2 chains (indicated by arrows), human P4H α and β subunits, and human LH3) was extracted by grinding 500 mg of leaves in 0.5 ml of 100 mM Tris-HCl (pH = 7.5) and 300 mM NaCl, centrifuging at 10,000 rpm for 10 min, and collecting the supernatant. Fifty μl of the supernatant was heat-treated (20 min 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. Samples were incubated at room temperature (approximately 22°C) for 30 min. Digestion was terminated by adding 5 μl of unbuffered 1 M Tris. Each sample was mixed with 22 μl of 4X sample application buffer containing 10% β-mercaptoethanol and 8% SDS, boiled for 7 minutes, and centrifuged at 13,000 rpm for 7 minutes. 40 μl of the supernatant was loaded onto a 10% polyacrylamide gel and tested using an anti-collagen type I antibody (Chemicon Inc., no. AB745) in a standard Western blot procedure. The positive control was a sample of approximately 50 ng of human collagen type I (Chemicon Inc., no. CC050, extracted from human placenta by pepsin digestion) spiked into total soluble protein from wt tobacco.

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

[0344] Example 5. Plant P4H expression.

[0345] Induction of native plant P4H

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

[0347] Detection of human P4H α and β subunits and collagen α1 and α2 chains in transgenic tobacco plants.

[0348] Detection of human P4H α and β subunits and collagen type I α1 and α2 chains in transgenic tobacco plants was performed using anti-human P4H α subunit antibody (no. 63-163, ICN Biomedicals Inc.), anti-human P4H β subunit antibody (no. NMAB2701, Chemicon Inc.), and anti-collagen type I antibody (no. AB745, Chemicon Inc.). The results of Western blots probed with these antibodies are shown in Figure 8.

[0349] Expression of P4Hα, P4Hβ, and collagen type 1 α1 and α2 bands was confirmed in plant 13-6 (also transformed with human LH3). The calculated molecular weights of P4Hα and β, including the vacuolar signal peptide, are 65.5 kDa and 53.4 kDa, respectively. The calculated molecular weights of the collagen α1 and α2 chains, including the propeptides, which are not hydroxylated or glycosylated, are 136 kDa and 127 kDa, respectively.

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

[0351] Plants that express collagen:

[0352] The 20-279 parental tobacco plant line was generated by cotransformation with an expression vector expressing P4Hβ+LH3 and another expression vector expressing P4Hα, each preceded by the vacuolar targeting determinant for aleurene, a plant vacuolar thiol protease.

[0353] The 2-300 parental tobacco plant line was generated by cotransformation with an expression vector expressing col1 and another expressing col2, each preceded by the vacuolar targeting determinant for aleurene, a plant vacuolar thiol protease.

[0354] The 13-652 plants were generated by cotransformation of tobacco plants with an expression vector encoding Col1, P4Hβ, and LH3 and a second expression vector encoding Col2 and P4Hα. Each gene is preceded by the vacuolar targeting determinant of alurein, a plant vacuolar thiol protease; the cassette sequences contained in the vectors are described in Example 1 above.

[0355] Light and Dark Trial

[0356] Analysis of six 13-6 / 52 homozygous plants. Samples from leaf number 4+5 / 6, three plants from normal conditions (16 h light, 8 h dark) and three plants grown only in the dark, were taken daily at the same time (12:30) for eight days.

[0357] Total protein extraction and Western blot analysis.

[0358] Ninety milligrams of tobacco leaves were homogenized at 4°C in extraction buffer (100 mM Tris HCl (pH = 7.5), protease inhibitor cocktail available from Roche catalog number 04-693-116-001) using a Mixer Mill Model MM301 (Retsch). After 30 minutes of centrifugation (20,000 x g at 4°C), the supernatant was collected. Protein samples were fractionated by 8% SDS-PAGE (Laemmli 1970) and transferred to nitrocellulose membranes using a BIO-RAD™ Protein TRANS-BLOT™ apparatus. The membranes were blocked in 3% (g / v) skim milk (Difco) at room temperature for 30 minutes and then reacted with one of the commercially available rabbit anti-human collagen type I polyclonal antibodies (Chemicon) overnight at room temperature. The membranes were rinsed 3–5 times with water and then washed with TBS for 30 minutes. After incubation with the secondary antibody [goat anti-rabbit IgG antibody conjugated to alkaline phosphatase (AP) (Chemicon)] for 2 h at room temperature, the membrane was rinsed 3–5 times with water and then washed with TBS for 30 min. Immunodetection was performed with nitrotetrazolium blue chloride (NBT, Sigma) and 5-bromo-4-chloro-3-indolylphosphate p-toluidine salt (BCIP, Sigma) for 2 h to overnight at room temperature.

[0359] result

[0360] As shown in Figure 9, tobacco plants transgenic for vacuolar-targeted collagen express proα-1 and proα-2 (lane 1). Collagen from vacuolar-targeted plants grown in the dark showed similar stability (lane 2), demonstrating the exceptional stability of collagen produced according to the teachings of the present invention.

[0361] Examples 7-13. General Materials and Methods

[0362] Collagen Extraction and Enzyme Reaction: In a blender, 300 g of tobacco leaves were mixed in chilled extraction buffer (600 ml of 100 mM Tris-HCl, pH 7.5, containing 360 mg of potassium metabisulfite, 530 mg of L-cysteine, and 1 g of EDTA) supplemented with 5 g of PVPP and 2 g of activated charcoal (see U.S. Patent No. 8,759,487). Mixing was performed five times at 1-minute intervals to maintain the temperature below 15°C. The crude extract was filtered through a gauze pad and centrifuged at 25,000 g and 5°C for 30 minutes. The supernatant was collected. CaCl2 was added to a final concentration of 10 mM. The supernatant was divided into 10 ml samples. The desired enzymes were added to each 10 ml sample according to the conditions listed in Table 3 below.

[0363] [Table 3]

[0364] Enzyme description: ficin from fig latex (Sigma, catalog number F4125), subtilisin from Bacillus licheniformis (Sigma, catalog number P5459-5gr), bromelain from pineapple stem (Sigma, catalog number B4882-10gr), papain from Carica papaya (Fluka, catalog number 76220-25gr), Savinase 6.0 t type W from the alkaliphilic bacterium Bacillus lentus (Novozymes, catalog number PX92500501), Neutrase 1.5 MG from the bacterium Bacillus amyloliquefaciens (Novozymes, catalog number PW201041), Protamex, a commercial Bacillus proteinase complex (Novozymes, catalog number PW2A1021), Alcalase 3.0 T, Bacillus Subtilis alkaline proteinase (Novozymes, catalog number PJ90000901), Esperase 6.0 T, alkaliphilic bacterium Bacillus lentus (Novozymes, catalog number PE90110401), Alcalase 2.4 L FG, Bacillus subtilis alkaline proteinase (Novozymes, catalog number PLN05330), Esperase 8.0 L, and alkaliphilic bacterium Bacillus lentus (Novozymes, catalog number PE00077) were all kindly provided by Novozymes. Pancreatic trypsin 6.0 S, salt-free form (Novozymes, catalog number P245-D20), a trypsin derived from animal pancreas, was purchased from Sigma Chemical Co. (catalog number T3449). TRYPZEAN™, a recombinant trypsin expressed in maize, was purchased from Sigma Chemical Co. (catalog number T3449).

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

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

[0367] SDS-PAGE Instant Blue Assay: Samples were boiled in SAB buffer (reducing conditions) for 5 minutes, centrifuged at 12,000 rpm for 5 minutes, and then loaded onto SDS-PAGE, 8% acrylamide gels. Gels were run in a Mini Protean 3-unit (BioRad #165-3301, #165-3302). Instant Blue reagent (Novexin #ISBO1L) was applied to the gel until proteins were visualized as blue bands on the gel. The gel was rinsed with water and allowed to dry. The concentration of collagen bands was calculated by densitometry relative to a human standard loaded on the same gel.

[0368] Coomassie analysis: Collagen samples (in 10 mM HCl) were titrated to pH 7.5 with 1 M Tris. A sample application buffer containing 10% β-mercaptoethanol and 8% SDS was added at a 4-fold dilution with 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 Coomassie solution for 1 hour with shaking. The Coomassie dye was removed using a standard destaining solution.

[0369] SDS-PAGE and Western blot analysis of α-1 and α-2 collagen chains: 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 the supernatant was separated on a 10% polyacrylamide gel. After separation, samples were blotted onto nitrocellulose membranes using standard Western blot protocols. After transfer, the membranes were incubated with anti-collagen type I antibody (Chemicon Inc. catalog #AB745) for immunodetection of α-1 and α-2 collagen chains. Molecular weight markers were purchased from Fermentas Inc. (catalog #SM0671).

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

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

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

[0373] The collagen was sterilized by first filtering the solution through a 0.45 μm filter and then through a 0.2 μM filter using a 30 ml syringe. The collagen was further concentrated by ultrafiltration using a Vivaspin PES 20 ml filtration tube (Vivascience, product number VS2041, MWCO 100000). Centrifugation was performed at 5000 g for 45 minutes at 5°C until the volume was reduced to 0.75 ml.

[0374] Optimization of digestion kinetics and conditions for procollagen cleavage with food-grade ficin: Pellets (collected as described in Example 10 and saturated with 25% ammonium sulfate (AMS)) were 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 pellet to 200 mL of ice-cold buffer. The sample was then stirred for 20 min at 15°C. Aliquots of 10 mL per 15 mL tube were then prepared, followed by administration of increasing concentrations of ficin (5–15 mg / L) (food-grade ficin from Figi Latex, Biochem Europe). Samples were incubated at 15°C for 1-3 hours, separated by SDS-PAGE, and then analyzed by Western blot for the presence of collagens that migrate at lower molecular weights than procollagen.

[0375] Pellets from tobacco leaves resuspended in phosphate buffer A (27.2 g:800 mL buffer) at various pH values ​​(5.5, 7.5, or 8.5) were treated with 10 mg / L ficin in the presence of 0 to 3 M NaCl at 15°C. Reactions were terminated by centrifugation of 1 mL samples from each reaction mixture (10 min, 15,000 g, 4°C). Pellets were resuspended in 1 mL of buffer A (pH 7.5), separated by SDS-PAGE, and analyzed by Western blot.

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

[0377] Fibril formation: Fibril formation is considered a collagen functional test. Therefore, the ability of purified collagen digested with ficin to form fibrils is an essential property of the resulting product. Test method: The pH of the collagen-containing solution (multiple samples) was neutralized to pH 6.7 with sodium phosphate (pH 11.2) and then incubated at 27 ± 2 μC for 6 hours. The samples were centrifuged to sediment the formed hydrogel. The protein concentrations of both the pre-neutralization and post-neutralization (supernatant) samples were determined by the Lowry method. PURECOL™ (purchased from NUTACON, catalog number 5409) was used as a positive control, and gelatin was used as a negative control.

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

[0379] The use of mammalian proteins in human cosmetic or medical applications could pose a risk to human health due to their relative evolutionary proximity, so the production of human collagen in plants was initiated to avoid the use of collagen from mammalian sources. The well-known disease Creutzfeldt-Jakob disease (CJD) is an example of a disease caused by the consumption of infected mammalian proteins by humans.

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

[0381] result

[0382] Propeptide digestion during collagen purification was initially performed with the pancreatic enzyme trypsin. Trypsin digested collagen propeptides at 300 mg / L, but collagen yield was very low at the end of the purification process (Figure 10). Lowering the trypsin concentration to 20 mg / L or 30 mg / L resulted in higher yields, but procollagen digestion was only partial and inconsistent between identical samples (Figure 11).

[0383] In an attempt to overcome this problem, various incubation temperatures and times were tried; however, the results did not result in any change in yield (data not shown). Addition of pepsin enzyme after the purification process resolved the problem of partial digestion (Figure 12), resulting in α-1 and α-2 collagens that comigrated with the porcine collagen control sample.

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

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

[0386] Example 9. Extraction and purification of collagen from transgenic plants after digestion with subtilisin or ficin.

[0387] Collagen purification from 450 grams of leaves from transgenic plants (13-361 or 13-6-52) was performed after procollagen digestion with ficin (Figure 19) or subtilisin (Figure 20). Collagen samples at various stages of the purification process were analyzed by Western blot analysis. Propeptide digestion with ficin and subtilisin resulted in the desired degree of collagen 1 and collagen 2 processing. Low molecular weight bands were observed on Western blots throughout the purification process; these bands appeared in the plant extract before incubation with the enzymes (lanes 3-4) and also in the pig-derived collagen control sample (positive control) (Figure 19).

[0388] Example 10. Scaled-up collagen extraction and purification from transgenic plants after digestion with ficin.

[0389] One kilogram of transgenic tobacco leaves was ground in a 4-liter reactor (ESCO Model EL-3) with 2 liters of pre-chilled extraction buffer (100 mM sodium phosphate buffer pH 7.5, 4.5 mM potassium metabisulfite, 12.23 mM L-cysteine, and 7.5 mM EDTA) for 20 minutes at 5°C, 50% scraper speed, and 100% homogenizer blade rpm. 6.68 g of charcoal and 16.67 g of PVPP were added to the extract and stirred continuously for 20 minutes at 5°C and 50% scraper speed. The extract was centrifuged at 11,000 rpm, 5°C, for 0.5 hours, and the supernatant was saturated with 15% ammonium sulfate (1 hour of stirring, 5°C). After 30 minutes at 6,880 rpm and 5°C, the supernatant was saturated with 25% ammonium sulfate and stirred for 1 hour at 5°C. After recentrifugation, the pellet (6880 rpm, 5°C, 30 min) was resuspended (in extraction buffer) in 15% of the volume collected after the first centrifugation step. Propeptide removal was achieved by digestion with 5 mg / L ficin (Biochem Europe) for 3 h at 15°C. The sample was centrifuged (11,000 rpm, 15°C, 30 min), and mature collagen was precipitated using 3 M NaCl (NaCl was added slowly with stirring and left overnight at 4°C). After precipitation (13,000 rpm, 5°C, 2 h), the supernatant was discarded and the pellet was resuspended in 0.5 M acetic acid. After another round of 3 M salting out (overnight) and centrifugation, the pellet was resuspended in 40 ml of 10 mM HCl. The sample was transferred to a dialysis bag (12–14 kDa) and dialyzed against 4 L of 10 mM HCl for 4 h at 4°C. Dialysis was repeated overnight with 4 L of fresh 10 mM HCl. 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. Finally, the sample was concentrated with a Vivaspin (Vivascience) filtration tube (100 kDa).

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

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

[0392] [Table 4]

[0393] Example 12. Ficin-dependent proteolysis of procollagen from tobacco leaves.

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

[0395] Kinetics of procollagen digestion by pharmaceutical-grade ficin: Similar experiments were performed on procollagen-expressing tobacco leaf pellets to determine the conditions suitable for procollagen digestion with 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. Digestion efficiency was determined by immunodetection of collagen chains on Western blot. As shown in Figures 23A–23C, increasing the ficin concentration increased collagen yield and decreased procollagen levels. The most effective digestion of procollagen with pharmaceutical-grade ficin was observed at 10 mg / L after 1 hour of reaction time.

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

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

[0398] Optimization of temperature conditions for ficin-induced procollagen digestion: Tobacco leaf pellets expressing procollagen were incubated with ficin at 15°C for 1.5 h and then transferred to a 30°C bath for an additional 1.5 h. Western blot and fibrillogenesis assays did not confirm any improvement in collagen yield or sample purity associated with increasing reaction temperature.

[0399] Fibrillogenesis of collagen extracted from ficin-induced procollagen cleavage: After ficin-induced digestion, a fibrillogenesis assay was performed to determine the ability of the resulting collagen to form fibrils, the ultimate method for determining collagen functionality. Table 5 below summarizes the fibrillogenesis results determined after ficin cleavage of procollagen using two different protocols. Both protocols, A and B, differed in reaction buffer pH and salt content and resulted in a significant percentage of collagen fibrils. Therefore, the proteolysis reaction parameters developed and optimized herein result in high yields of functional collagen.

[0400] [Table 5]

[0401] Example 13. Determining the effectiveness of TRYPZEAN™ protease in procollagen cleavage.

[0402] Pellets of tobacco leaves expressing procollagen, resuspended in extraction buffer (pH 7.5) enriched with EDTA (7.5 mM) and L-cysteine ​​(12.5 mM), were incubated with TRYPZEAN™ (30–100 mg / L) at 15°C for 1–3 hours. Within 1 hour, doses of 60 and 100 mg / L TRYPZEAN™ efficiently cleaved procollagen, generating two distinct α-collagen chains, without detectable overdigestion (Figure 26). Thus, treatment of procollagen with TRYPZEAN™ at pH 7.5 resulted in effective digestion into collagen chains α-1 and α-2.

[0403] Consideration

[0404] Examples 7-13 above describe the identification of non-mammalian proteases suitable for use in the purification process of plant-derived collagen. In examining proteases from bacterial and plant sources, three enzymes were found to be suitable for digesting collagen propeptides: neutrase, subtilisin, TRYPZEAN™, and ficin.

[0405] Both neutrase and subtilisin are secreted by the bacterium Bacillus sp. Subtilisin is primarily (more than 90%) used in detergents and household cleaning products. Approximately 10% of subtilisin's uses are for industrial applications, such as protein hydrolysis, leather processing, and the textile and beauty industries. The standard use of subtilisin in collagen purification processes at high concentrations has problems due to overdigestion of collagen. Neutrase is primarily used in the beverage and alcohol industry and cheese ripening. As described hereinabove in Examples 7-13, neutrase was only effective at digesting propeptides at high concentrations, and at least 6 hours were required for desirable digestion results.

[0406] Under the experimental conditions described here, recombinant trypsin and ficin proved to be the most suitable of the four because there was no overdigestion of collagen at high enzyme concentrations or after prolonged incubation. Furthermore, these enzymes clearly did not digest the helical region of collagen, as determined by SDS-PAGE analysis. Ficin, a natural enzyme extracted from the fig plant (Ficus carica), is commercially available at low cost in several grades, including pharmaceutical grade, from several sources. It is used in the alcohol and beer industries, protein hydrolysis, meat processing, baking, and food industries, such as the preparation of pet food and health foods. It is also used in the pharmaceutical industry for contact lens cleaners, cancer treatments, anti-arthritis treatments, and digestive aids, as well as in the beauty and textile industries.

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

[0408] material and method

[0409] material

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

[0411] Preparation of buffer and photoinitiator stock solutions

[0412] Fibrillogenesis 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.

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

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

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

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

[0417] Methacrylation of rh collagen.

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

[0419] Fibrillar rh collagen-methacrylamide

[0420] Fibrillar rh collagen-methacrylamide solutions of 3–10 mg / mL were synthesized at room temperature (RT) or 12°C in either wash buffer, fibrillogenesis buffer, or DDW. Briefly, fibrillar rh collagen-MA was synthesized in DDW as follows: A 3–4 mg / mL solution of monomeric rh collagen (COLLAGE™) in 10 mM HCl was mixed with fibrillogenesis buffer at a 9:1 (v:v) ratio and stirred at room temperature for 1 hour to receive fibrils. The solution was centrifuged at 7500 rpm and 4°C for 30 minutes, and the supernatant was discarded. The pellet was resuspended in an equal volume of wash buffer and centrifuged under the same conditions. The deposited fibrils were then resuspended in DDW to 10 mg / mL. The concentration was confirmed by measuring the percent solids. Methacrylic anhydride (MA) was added dropwise at a molar ratio of 10–20 to collagen-lysine at room temperature under a nitrogen stream. The pH of the reaction solution was monitored over time and adjusted to pH 7 using 10 N NaOH. After 24 h of reaction, the mixture was dialyzed against wash buffer (pH 7) at 4°C for 3 days using a 10 kDa cutoff dialysis tubing (Spectrum Laboratories Inc, CA, US). The dialysate (wash buffer in this case) was exchanged at least six times to remove reaction by-products, and finally lyophilized for 3–4 days.

[0421] Monomeric rh collagen-methacrylamide

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

[0423] Methacrylation of hyaluronic acid (HA)

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

[0425] Preparation of solutions for viscosity measurements

[0426] PureCol and Collage™ in PBS: 8 ml of monomeric collagen solution (3 mg / mL in 10 mM HCl) of either rh collagen (COLLAGE™) or bovine collagen (PureColla) was neutralized by adding 1 ml of PBSX10. The solution was then brought to pH 7-7.5 by titration with 0.1 N NaOH. Finally, double-distilled water was added to bring the final volume to 10 ml. Samples were incubated at 37°C for at least 90 min before measurements were taken (either at 37°C or 4°C).

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

[0428] Fibrillar rh collagen-methacrylamide in PBS: Lyophilized fibrillar rh collagen-MA prepared in DDW and wash buffer (as described above with a 10-fold excess of MA) was dissolved in PBS to a concentration of 10 mg / mL. Samples were incubated at 37°C for at least 90 min before measurements were taken (either at 37°C or 4°C).

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

[0430] rhCollagen-Methacrylamide / Hyaluronic Acid in DMEM: Hyaluronic acid was added to the solution of fibrillar rhCollagen-MA to give a final concentration of 10 mg / mL HA and 20 mg / mL rhCollagen-MA in DMEM medium.

[0431] 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.

[0432] Photocrosslinking of rhcollagen-MA for loss and storage modulus measurements

[0433] Two different preparations of rh collagen-MA crosslinked scaffolds were formed in two separate experiments. In the first preparation, 1-2 wt% fibrous rh collagen-MA synthesized with a 10-fold excess of methacrylic acid reagent was dissolved in 0.1M PBS at room temperature. Then, 0.1% Irgacure 2959 was added, and a final volume of 1 mL of the solution was poured into a circular mold. A curing process was then carried out using a mercury light source at an average intensity of 670 mW / cm² for 7 and 10 seconds from a distance of 1.5 cm, ultimately yielding crosslinked scaffolds. The second preparation involved two different batches of fibrous rh collagen-MA synthesized with a 15-fold and 20-fold excess of methacrylic acid reagent. 1-2 wt% was dissolved in 0.1M PBS, and 0.1% Irgacure 2959 was added, resulting in a final volume of 1.5 mL. To obtain a highly crosslinked scaffold, the curing process was carried out for 60 seconds from a distance of 2 cm with an average intensity of 420 mW / cm. 2 It was carried out at.

[0434] TNBS assay

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

[0436] Rheological characterization

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

[0438] Storage and loss moduli of scaffolds: The rheological behavior of rh collagen-crosslinked discs was investigated using a parallel-plate system with a PP20 sawtooth spindle and a 20 mm sawtooth plate setup. A C60 / 1° Ti cone-plate element was used to characterize uncrosslinked rh collagen-MA. Two sets of experiments were performed separately to evaluate the rheological behavior of rh collagen-MA. In the first experiment, a 1 mL sample was subjected to an oscillatory force in controlled stress mode at a frequency of 1 Hz at 37 °C for 300 s, while a shear stress of 5 Pa was applied. The storage modulus (G') and loss modulus (G") were recorded. The gap was adjusted to 90% of the original sample height, and the G' and G" values ​​were averaged over the range of 150 to 300 s. In the second experiment, a 1.5 mL crosslinked disc was tested under frequency sweep oscillation at 37 °C, and G' was recorded at a shear stress of 1 Pa over a frequency range of 0.01 to 100 Hz. To start the measurement, the spindle was lowered to contact the hydrogel surface and then further lowered until the axial force of the instrument was equal to 0.4 N. Before every measurement, the sample was kept on a plate covered with a humidity lid for 1 min to reach temperature equilibrium.

[0439] result

[0440] TNBS assay

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

[0442] [Table 6]

[0443] This result indicates a high modification potential of fibrillar rh collagen and suggests that adding the methacrylic acid reagent at a molar ratio of 10 may be preferable to obtain maximum functionalization of fibrillar collagen.

[0444] Rheology

[0445] 1. Viscosity

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

[0447] Figure 27 shows the viscosity of rh collagen (COLLAGE™) and bovine collagen (PureCol) in PBS 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) exhibits a clear temperature dependence of the zero shear rate viscosity (η). That is, a viscosity plateau at low shear rates, 37 °C (red), has an η value more than an order of magnitude higher than the 4 °C (blue) value. In contrast, rh collagen (dashed line) shows no significant difference between the η values ​​at 4 °C and 37 °C. Rh collagen neutralized with FB (see Methods) exhibits very similar behavior (Figure 28), i.e., the viscosities at 4 °C and 37 °C are nearly identical. Figure 29 shows the viscosity of fibrillar rh collagen-MA at 4 °C (blue line) and 37 °C (red line). Although the profiles are not identical, the zero shear rate value is approximately 1000 cP at both temperatures.

[0448] Viscosity of rh collagen-methacrylamide

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

[0450] Those skilled in the art will recognize that rhcollagen-MA is not cross-linked and requires the addition of a photoinitiator and light to achieve cross-linking.

[0451] 2. Loss and storage moduli of scaffolds

[0452] Rheological analysis of 1 mL discs over time at 37 °C performed in the first experiment is shown in Figure 31. The upper graph reports the loss and storage moduli and tan(delta) 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 rh-collagen-MA increases two-fold upon irradiation in the presence of photoinitiator. Furthermore, the results demonstrate the ability to control scaffold properties by varying the rh-collagen-MA concentration. The large difference between G' and G" values ​​and the near-zero tan(delta) values ​​of the crosslinked discs indicate their elastic-like behavior. (G' - storage modulus; G" - loss modulus; G', the "storage / elastic modulus," represents the fraction of energy in G* stored by the gel during deformation and later used to return to its original shape. G' measures the elastic behavior of the gel, or how well the gel can recover its shape after shear deformation. For example, vulcanized rubber is a purely elastic material because it deforms instantaneously when stressed and completely recovers its shape when the stress is removed (i.e., G*~G') (~ represents an approximation). G'', the "loss / viscous modulus," represents the fraction of energy in G* lost in shear deformation due to internal friction. Because HA fillers are not purely viscous, G" is not directly related to viscosity. Instead, this term reflects the gel's inability to completely recover its shape after the shear stress is removed.)

[0453] In the second experiment, 1.5 mL discs irradiated for 60 seconds exhibit higher G' values, as shown in Figure 32. The data show that G' increases with rhcollagen-MA concentration and degree of methacrylation, demonstrating the ability to control scaffold properties.

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

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

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

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

[0458] Example 16. Viscosity and Polymerization of rh Collagen Methacrylate with Additives

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

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

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

[0462] Injectable rh-collagen / platelet-rich plasma (PRP) scaffolds have been investigated as scaffolds and healing devices for tendon disorders. A slowly degrading rh-collagen matrix combined with a source of growth factors (GFs), such as platelet-rich plasma (PRP), was injected near the injured tendon to provide the necessary support for promoting healing. This treatment used a matrix containing plant-derived recombinant human type I collagen (rh-collagen) mixed with PRP, which supports the sustained release of growth factors at the injury site and promotes healing. The efficacy of the rh-collagen-PRP matrix was compared in vitro and in vivo with PRP in supporting fibroblast proliferation, clot degradation, GF release, and tendon healing in a rat model of collagenase-induced Achilles tendon tendinopathy. rh-collagen-PRP demonstrated superior performance compared to PRP alone both in vitro and in vivo. These results are encouraging for the use of rh-collagen matrix combined with PRP in clinical trials for tendon disorders.

[0463] material and method

[0464] rh collagen matrix

[0465] A monomer solution of rh collagen (CollPlant, Ness Ziona, Israel) in 10 mM HCl was fibrillated by pH neutralization in a phosphoric acid solution and cross-linked with 18 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (Sigma-Aldrich, Israel). The cross-linked collagen was then washed by repeated centrifugation in double-distilled water, and calcium chloride (CaCl) (Merck, Israel) was added to a final concentration of 20 mM. Syringes filled with the rh collagen slurry were lyophilized and finally sterilized with ethylene oxide.

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

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

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

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

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

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

[0472] In vitro cell proliferation assay

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

[0474] In vivo testing

[0475] animal

[0476] Hsd:Sprague-Dawley SD rats weighing 230 g ± 20% were selected for animal experiments. Animals were given unique ear numbers and randomly assigned to specific groups. Animals were housed in individually ventilated (IVC) cages in a dedicated animal facility with heat, ventilation, and air conditioning (HVAC). Temperature and humidity were continuously monitored. Animals were fed commercial rodent chow (Harlan Teklad TRM Ra / Mouse Diet) ad libitum and autoclaved water ad libitum. The facility was not exposed to external light and maintained on an automated 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 detected in any of the animals throughout the study period. No statistically significant differences were observed in mean body weights and gains between groups. All gains were within the range of values ​​normally expected at termination.

[0477] In vivo clot dissolution and growth factor release

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

[0479] Injection sites on the backs of 34 female Sprague-Dawley rats (Harlan Laboratories, Ness Ziona, Israel) were shaved and marked. Each rat was injected with 0.5 ml of the same formulation at four separate sites on the dorsal plane: two sites on the anterior and two sites on the posterior sides of the rat's back. Animals were euthanized at 1, 7, 14, 21, 30, and 45 days after treatment (10 or 12 rats per group, two per time point). At each time point, the injection sites were exposed and evaluated macroscopically. The skin at the injection site was gently separated from the muscle using scissors, and the site was washed with 0.25 ml of DMEM, 1% FBS (Biological Industries, Israel). The clot was extracted and weighed. The washed medium was transferred to an Eppendorf tube (1.5–2 ml) while the extracted clot was transferred to a 6- or 12-well plate. Once weighed, the clots were combined with the respective wash medium, cut with scissors, and minced with a pestle to facilitate the release of GFs from the clot into the surrounding medium. The Eppendorf tubes were then centrifuged for at least 5 minutes to separate the clot pellet from the medium. The supernatants were collected and stored at -80°C until assayed. Controls (TO) containing approximately 0.5 ml of each formulation were formed in vitro following the same procedure as above, but without injection into animals. At the end of the study, the PDGF and VEGF contents in the preserved supernatants were assessed by ELISA (Quantikine ELISA Mouse / Rat PDGF and Quantikine ELISA Rat VEGF, R&D Systems, Israel).

[0480] In vivo tendon damage induced in rats.

[0481] The healing properties of rh collagen 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). Experiments were performed at Harlan Laboratories Israel Ltd. (Ness Ziona, Israel).

[0482] A skin incision was made in the proximal part of the right hind limb of each rat over the common calcaneus tendon. Under appropriate magnification, the central branch of the tendon was identified and isolated. Tendinopathy was induced by injecting 0.3 mg of collagenase (10 mg / ml, Sigma) under the common calcaneus tendon capsule using a 0.5 ml insulin syringe. Finally, the skin was closed with interrupted subcutaneous sutures using 4 / 0 Vicryl. One week after induction of tendinopathy, a stab incision was made in the tendon capsule using an ocular corneal / scleral knife. A tunnel was then created under the capsule using a cannula, and 50 μl of rh collagen combined with PRP or PRP alone was injected into the pre-prepared tube. After 3, 7, and 14 days of treatment, the animals were euthanized. The treated tendons were excised and preserved for histopathological evaluation.

[0483] histology

[0484] Tissues were embedded in paraffin and serially sectioned into 4-5 micron thick samples. Slides were stained with hematoxylin and eosin (H&E) for histopathological examination and blinded evaluation by a pathologist.

[0485] result

[0486] In vitro cell proliferation assay

[0487] In this study, we compared the viability and proliferation of cells seeded near a matrix composed of rh collagen combined with PRP and a clot composed of thrombin-activated PRP. Cells seeded in untreated wells served as a control. A matrix (composed of either rh collagen combined with PRP or thrombin-activated PRP) was placed in a transwell above the seeded wells to allow diffusion of GFs from the matrix into the wells without direct contact with the cell layer. The number of viable cells on days 7 and 10 is reported as the average of two separate experiments (three replicates per experiment) in which PRP was extracted from two different blood donors (Figure 39, top). As shown in Figure 39, cell viability (days 7 and 10) in the presence of GFs released from the rh collagen matrix combined with PRP was significantly higher than that of the thrombin-activated PRP clot or the control. Furthermore, in the presence of rh-collagen matrix combined with PRP, cell numbers increased from days 7 to 10, whereas cell numbers decreased in the presence of thrombin-activated PRP and in the control group. Both cell viability and proliferation were significantly better in the presence of rh-collagen matrix. The data were confirmed by microscopic analysis (Figure 39, bottom). Cells cultured in the presence of rh-collagen matrix combined with PRP (Figure 39, bottom, panel A) exhibited elongated shapes and reached full confluence already 7 days after seeding. On the other hand, cells cultured in the presence of thrombin-activated PRP were barely viable, which may indicate a toxic effect of thrombin in this experimental setting (Figure 39, bottom, panel B). Cells cultured in the presence of medium alone showed very limited viability (Figure 39, bottom, panel C).

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

[0489] Matrix Decomposition Profile

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

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

[0492] Growth factor content

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

[0494] The increase in GFs seen from days 1 to 14 for PDGF analysis and from days 0 to 7 for VEGF analysis demonstrates the ability of the rh-collagen scaffold to allow for the accumulation of GFs, likely reflecting cells migrating and proliferating within the scaffold. The integral of the nominal PDGF and VEGF content across the study for each formulation is summarized in Figure 42. It is clear that injection of rh-collagen matrix alone or in combination with PRP results in a much higher content of GFs at the injection site compared to activated PRP alone.

[0495] Induced in vivo tendon damage in rats

[0496] The healing properties of rh-collagen matrix combined with PRP compared to PRP alone were evaluated in a rat model of 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 (scores 0-5, as listed in Table 7).

[0497] [Table 7]

[0498] The cumulative histopathological scores associated with each treatment are shown in Figures 43A-43C. The group treated with rh collagen matrix combined with PRP exhibited slightly more mature fibrosis compared to the PRP-treated group, particularly on days 3 and 14. This is consistent and correlates with the lower level of immature granulation tissue exhibited by the group treated with rh collagen matrix combined with PRP on day 14 (Figure 43C). Furthermore, in Figure 43B, the group treated with rh collagen matrix combined with PRP exhibits reduced inflammation, as indicated by the presence of fewer mononuclear inflammatory cells at all time points, particularly on days 3 and 14. Overall, this data demonstrates that treating injured tendons with rh collagen matrix combined with PRP promotes faster healing, as indicated by the higher levels of mature fibrosis and lower levels of immature granulation tissue, accompanied by a significant reduction in inflammatory mononuclear cells, when compared to standard PRP injection treatment.

[0499] Consideration

[0500] Injuries to soft tissues, including tendon and ligament injuries, are very common and cause significant clinical burden. While several treatments are available, their clinical benefit remains limited. This has prompted the search for new options aimed at improving healing and shortening recovery time. An injectable matrix composed of human recombinant type I collagen has been developed. When mixed with PRP, this matrix forms a collagen-fibrin-PRP composite. This composite slowly degrades, attracting cell migration and proliferation to the collagen scaffold and allowing the sustained release of growth factors at the injury site, thereby better supporting the healing process. In vitro experiments (Figure 39) demonstrated significantly superior nHDF viability and proliferation around rh-collagen matrices combined with PRP compared with thrombin-activated PRP. These results indicated that sustained growth factors released from the collagen matrix promote and enhance cell proliferation. When combined with PRP, type I rh-collagen provides a supportive environment that promotes and enhances cell proliferation, even when not in direct contact with the cell layer. Subcutaneous injection into rats demonstrated for the first time that GFs, including fibrin clots formed in situ upon PRP injection, degraded within 24 hours, resulting in GF content below the ELISA detection limit (Figures 41A-41B). When platelets complexed with the rh-collagen matrix, GFs were released over a 45-day period, a time period consistent with scaffold degradation (Figures 40 and 41A-41B). It is interesting to note that the GF content profile was not monotonic, as expected by a standard release profile. PDGF profiles for PRP-treated rh-collagen matrices (Figures 41A-41B) showed a sharp initial decrease on the first day, very similar to that observed with PRP alone, followed by a gradual increase through day 21, eventually decreasing to undetectable levels as the scaffold degraded. Interestingly, rh-collagen alone showed a similar pattern, with PDGF content gradual...

Claims

1. 1. A kit comprising a set of components of a polymerizable solution for tissue augmentation, comprising: The set of components is (i) methacrylated plant-derived recombinant human collagen type I; (ii) hyaluronic acid (HA) or a modified derivative thereof, poly(vinyl alcohol) (PVA) or a modified derivative thereof, polyethylene glycol (PEG) or a modified derivative thereof, oxidized cellulose (OC) or a modified derivative thereof, polymethyl methacrylate (PMMA) microspheres or a modified derivative thereof, tricalcium phosphate (TCP) or a modified derivative thereof, calcium hydroxylapatite (CaHA) or a modified derivative thereof, carboxymethylcellulose or a modified derivative thereof, crystalline nanocellulose (CNC) or a modified derivative thereof, or a combination thereof; (iii) a photoinitiator; wherein the tissue augmentation (a) introducing components (i)-(iii) into a tissue space below the epidermis; (b) applying light to the surface of the epidermis that is superficial to the tissue space to induce polymerization; 1. A kit, wherein the modified derivatives of the hyaluronic acid (HA), the poly(vinyl alcohol) (PVA), the polyethylene glycol (PEG), the oxidized cellulose (OC), the polymethyl methacrylate (PMMA) microspheres, the tricalcium phosphate (TCP), the calcium hydroxylapatite (CaHA), the carboxymethyl cellulose, or the crystalline nanocellulose (CNC) comprise methacrylated or thiolated derivatives.

2. 10. The kit of claim 1, each component of the polymerizable solution is introduced independently at approximately the same location and approximately simultaneously; (a) the methacrylated plant-derived type I recombinant human collagen and the photoinitiator are: (b) the hyaluronic acid (HA) or a modified derivative thereof, the poly(vinyl alcohol) (PVA) or a modified derivative thereof, the polyethylene glycol (PEG) or a modified derivative thereof, the oxidized cellulose (OC) or a modified derivative thereof, the polymethyl methacrylate (PMMA) microspheres or a modified derivative thereof, the tricalcium phosphate (TCP) or a modified derivative thereof, the calcium hydroxylapatite (CaHA) or a modified derivative thereof, the carboxymethylcellulose or a modified derivative thereof, the crystalline nanocellulose (CNC) or a modified derivative thereof, or a combination thereof; The kit according to claim 1, wherein the kit is introduced under the epidermis independently of the step (a).

3. 10. The kit of claim 1, The set of components are introduced together as a mixture into the tissue space below the epidermis.

4. The kit according to any one of claims 1 to 3, wherein the tissue augmentation The kit further comprising molding or shaping the polymerizable solution or components of the polymerizable solution into a desired configuration within the tissue space, wherein the molding or shaping occurs simultaneously with or subsequent to the application of light, and wherein the molding or shaping reduces lines, folds, fine lines, wrinkles, or scars, or a combination thereof.

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

6. 1. A polymerizable solution for tissue augmentation, comprising: (i) methacrylated plant-derived recombinant human collagen type I; (ii) a photoinitiator; wherein the tissue augmentation (a) introducing the polymerizable solution into a tissue space below the epidermis; (b) applying light to the surface of the epidermis that is adjacent to the tissue space to induce polymerization.

7. 7. The polymerizable solution of claim 6, wherein said tissue augmentation further comprises molding or shaping said polymerizable solution into a desired configuration within said tissue space, said molding or shaping occurring simultaneously with or subsequent to said applying light; A polymerizable solution wherein said shaping or shaping reduces lines, folds, fine lines, wrinkles, or scars, or a combination thereof.

8. 7. The polymerizable solution of claim 6, hyaluronic acid (HA) or a modified derivative thereof, poly(vinyl alcohol) (PVA) or a modified derivative thereof, polyethylene glycol (PEG) or a modified derivative thereof, oxidized cellulose (OC) or a modified derivative thereof, polymethyl methacrylate (PMMA) microspheres or a modified derivative thereof, tricalcium phosphate (TCP) or a modified derivative thereof, calcium hydroxylapatite (CaHA) or a modified derivative thereof, carboxymethylcellulose or a modified derivative thereof, crystalline nanocellulose (CNC) or a modified derivative thereof, or a combination thereof; A polymerizable solution, wherein the modified derivatives of the hyaluronic acid (HA), the poly(vinyl alcohol) (PVA), the polyethylene glycol (PEG), the oxidized cellulose (OC), the polymethyl methacrylate (PMMA) microspheres, the tricalcium phosphate (TCP), the calcium hydroxylapatite (CaHA), the carboxymethyl cellulose, or the crystalline nanocellulose (CNC) comprise methacrylated or thiolated derivatives.

9. A polymerizable solution for inducing a cell growth promoting scaffold within a tissue space below the epidermis, comprising: (a) methacrylated plant-derived recombinant human collagen type I; (b) hyaluronic acid (HA) or a modified derivative thereof, poly(vinyl alcohol) (PVA) or a modified derivative thereof, polyethylene glycol (PEG) or a modified derivative thereof, oxidized cellulose (OC) or a modified derivative thereof, polymethyl methacrylate (PMMA) microspheres or a modified derivative thereof, tricalcium phosphate (TCP) or a modified derivative thereof, calcium hydroxylapatite (CaHA) or a modified derivative thereof, carboxymethylcellulose or a modified derivative thereof, crystalline nanocellulose (CNC) or a modified derivative thereof, or a combination thereof; (c) a photoinitiator; and (d) at least one growth factor or a source thereof; inducing the cell growth-promoting scaffold includes introducing the solution into the tissue space to promote healing or replacement of collagen-containing tissue, wherein the cell growth-promoting scaffold fills within the tissue space and reduces lines, folds, fine lines, wrinkles, or scars, or a combination thereof; A polymerizable solution, wherein the modified derivatives of the hyaluronic acid (HA), the poly(vinyl alcohol) (PVA), the polyethylene glycol (PEG), the oxidized cellulose (OC), the polymethyl methacrylate (PMMA) microspheres, the tricalcium phosphate (TCP), the calcium hydroxylapatite (CaHA), the carboxymethyl cellulose, or the crystalline nanocellulose (CNC) comprise methacrylated or thiolated derivatives.

10. 10. The polymerizable solution of claim 9, The polymerizable solution, wherein the source of the at least one growth factor comprises plasma or platelet-rich plasma.

11. 11. The polymerizable solution according to claim 9 or 10, The polymerizable solution, wherein the collagen-containing tissue comprises skin.

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

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